**Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench to the Bedside Approach**

Ana C. Calvo, Pilar Zaragoza and Rosario Osta

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52896

#### **1. Introduction**

[179] Sakurai Y, Hatakeyama H, Sato Y, Akita H, Takayama K, Kobayashi S, et al. Endoso‐ mal escape and the knockdown efficiency of liposomal-siRNA by the fusogenic pep‐

[180] Hatakeyama H, Ito E, Akita H, Oishi M, Nagasaki Y, Futaki S, et al. A pH-sensitive fusogenic peptide facilitates endosomal escape and greatly enhances the gene silenc‐ ing of siRNA-containing nanoparticles in vitro and in vivo. J Control Release 2009

[181] Khalil IA, Kogure K, Futaki S, Hama S, Akita H, Ueno M, et al. Octaarginine-modi‐ fied multifunctional envelope-type nanoparticles for gene delivery. Gene Ther 2007

[182] Suzuki R, Yamada Y, Harashima H. Efficient cytoplasmic protein delivery by means of a multifunctional envelope-type nano device. Biol Pharm Bull 2007 Apr;30(4):

[183] Shaheen SM, Akita H, Nakamura T, Takayama S, Futaki S, Yamashita A, et al. KA‐ LA-modified multi-layered nanoparticles as gene carriers for MHC class-I mediated antigen presentation for a DNA vaccine. Biomaterials 2011 Sep;32(26):6342-6350.

tide shGALA. Biomaterials 2011 Aug;32(24):5733-5742.

Oct 15;139(2):127-132.

248 Gene Therapy - Tools and Potential Applications

Apr;14(8):682-689.

758-762.

Neurodegenerative diseases cover a wide range of neurogenetic disorders including Amyo‐ trophic Lateral Sclerosis (ALS), Alzheimer's disease (AD), Huntington's disease (HD), the spinocerebellar ataxias, inherited prion diseases, the inherited neuropathies, and muscular dystrophies among others.

In particular, ALS belongs to the group of motor neuron diseases, involving the loss of cortex, brainstem, and spinal cord motor neurons that result in muscle paralysis [1]. Motor neurons, which are localized in the brain, brainstem and spinal cord, behave as a crucial links between the nervous system and the voluntary muscles of the body, as they let synaptic signals travel from upper motor neurons in the brain to lower motor neurons in the spinal cord and finally to muscles. In accordance with the revised El Escorial criteria [2], both the upper motor neu‐ rons and the lower motor neurons degenerate or die in ALS, and as a consequence the com‐ munication between neuron and muscle is lost, prompting the progressive muscle weakening and the appearance of fasciculations. In the later stages of the disease, patients become para‐ lyzed although the disease usually does not impair a person's mind or intelligence.

Nowadays, the cause of ALS and its early manifestations still remain to be elucidated. The pathophysiological mechanisms that prompt the neurodegenerative process in both familial (FALS) and sporadic (SALS) ALS are unknown. However, there is growing evidence that the pathogenic process involved in ALS are multifactorial and include oxidative stress, glu‐ tamate excitotoxicity, mitochondrial dysfunction, axonal transport systems and dysfunction of glial cells, yielding the damage of critical proteins and organelles in the motor neuron triggering the neurodegeneration [3]. Due to the fact that FALS and SALS share clinical and

© 2013 Calvo et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

pathological signs, the understanding of the pathophysiological process in FALS would pro‐ vide a better understanding of the neurodegenerative mechanisms in SALS.

(TTC) of tetanus toxin. Tetanus toxin is a neurotoxin produced by *Clostridium tetani*, an anaero‐ bic bacterium whose spores are commonly found in soil and animal waste. This toxin affects the nervous system and causes generalized muscle contractions, called titanic spasms [16, 17].

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

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251

Tetanus toxin is a single peptide of approximately 150 kDa, which consists of 1315 amino-acid residues. The toxin forms a two-chain activated molecule composed of a heavy chain (HC) and a light chain (LC) linked by a disulfide bond. The catalytic domain of the toxin resides in the LC, while the translocation and receptor-binding domains are present in HC [18–21] (Figure 1). Tetanus and botulinum toxins are zinc metalloproteases that cleave SNARE (soluble NSF at‐ tachment receptor) proteins, which interfere with the fusion of synaptic vesicles to the plasma

The nature of the action of tetanus toxin has been widely described in different animal mod‐ els [23–28], exploring its effect not only in the spinal cord but also in the cerebral cortex [29]. One of the unique characteristics of tetanus toxin is that it can be transported retrogradely to the central nervous system and shows remarkable affinity and specificity to neuronal termi‐ nals. The ganglioside-recognition domain in the C-terminal region of HC allows the toxin to be internalized into the neuron at the neuromuscular junction where it enters the axonal ret‐ rograde transport pathway and is subsequently transported to the neuronal soma in the CNS [30,31]. Once the toxin reaches the cytoplasm, it specifically cleaves neuronal proteins integral to vesicular trafficking and neurotransmitter release. In particular, the synaptic vesi‐ cle protein synaptobrevin (VAMP) is the target of tetanus toxin. This protein belongs to a family of proteins that facilitate exocytosis in neurons known as SNARE proteins. The other members of this family are syntaxin and SNAP-25, which are the main molecular targets of botulinum toxin. SNARE proteins are formed by coiled-coil interactions of the alpha-helices

**Figure 1.** Diagram of the tetanus toxin molecule. The targeting and the translocation domains are located in the heavy-chain (HC), whereas the catalytic domain is located in the light-chain (LC) of the molecule. Its proteolytic activity is Zn2+-dependent, and heavy-metal chelators generate inactive apo-neurotoxins. TTC is approximately 50 KDa and re‐ sides in the HC of the toxin. The ganglioside-recognition domain in TTC allows the toxin to be internalized into the

membrane and ultimately blocks neurotransmitter release in nerve cells [22].

of its members, which is required for membrane fusion [32–35].

neuron [35].

FALS follows a predominantly autosomal dominant pattern, while in SALS genetic factors that take place sporadically contribute to its pathogenesis. The majority of ALS cases are sporadic and 5-10% of cases correspond to FALS. Although the ages of onset of FALS, which follow a normal Gaussian distribution, correspond to a decade earlier than for SALS cases which have an age dependent incidence, males and females are affected equally in FALS [4].

The most significant candidate genes for SALS include *VEGF* (vascular endothelial growth factor), *angiogenin* (*ALS9*), *paraoxonoase*, *neurofilaments*, *peripherin* and *SMN* (spinal muscular atrophy). Although *ALS9*, *paraoxonase*, *neurofilaments*, *peripherin* and *SMN* mutations have been found in ALS patients, except for *VEGF* mutations, these genes may play a small role in the pathogenesis of ALS and previous studies are conflicting [5].

Regarding FALS candidate genes, the mutations in the copper/zinc superoxide-dismutase-1 gene (*SOD1*), Tar DNA-binding protein gene (*TARDBP*) and in the most recent discovered DNA/RNA-binding protein called *FUS* (fused in sarcoma) or *TLS* (translocation in liposarco‐ ma) produce the typical adult onset ALS phenotype. Other candidate genes that have been described in genome association studies of FALS include *dynactin*, *senataxin (ALS4)* and *VAPB (ALS8)* (VAMP/synaptobrevin-associated membrane protein B gene) [5,6].

The pathophysiology of *SOD1* mutations is probably the most studied one. Many hypothe‐ ses have been suggested and reinforced in transgenic mouse models that overexpress the mutated *SOD1* gene and therefore develop an ALS-like syndrome. Among the proposed mechanisms that support these hypotheses are the toxic gain of function of the mutated SOD1 enzyme, which mainly increases the production of hydroxyl and free radicals, yield‐ ing improper binding metal properties, oxidative stress and inflammation induced by upre‐ gulation of proinflammatory cytokines [7,8]. Alternative hypothesis also suggested a conformational instability and misfolding of the SOD1 peptide, forming intracellular aggre‐ gates which have been reported in motor neuron and glial cells [9].

Neurotrophic factors have been initially identified as potential therapeutic agents in the treatment of ALS, opening the door to a new tool for the treatment of motor neuron diseases [10]. Based on previous studies ciliary and glial derived neurotrophic factors, insulin-like growth factor (IGF-1) and erythropoietin improved motor behaviour and reduce motor neu‐ ron loss, astrocyte and microglia activation in preclinical animal models [11], albeit clinical trials in ALS patients showed lack of therapeutic efficacy [12].

The failure of standard treatments in ALS could rely on the inappropriate route of adminis‐ tration and/or the poor bioavailability of molecules to the target cell [13]. The subcutaneous and intrathecal delivery of neurotrophic factors can cause adverse side effects such as weight loss, fever, cough, fatigue and behavioral changes [14], whereas viral gene therapy based on the use of an adeno-associated virus or lentivirus vectors is more efficient than the neurotrophic factor delivery but can induce several inherent hazards [15].

An alternative strategy that effectively reaches motor neurons, can exert neuroprotective prop‐ erties and does not show such adverse side effects implies the use of the nontoxic fragment C (TTC) of tetanus toxin. Tetanus toxin is a neurotoxin produced by *Clostridium tetani*, an anaero‐ bic bacterium whose spores are commonly found in soil and animal waste. This toxin affects the nervous system and causes generalized muscle contractions, called titanic spasms [16, 17].

pathological signs, the understanding of the pathophysiological process in FALS would pro‐

FALS follows a predominantly autosomal dominant pattern, while in SALS genetic factors that take place sporadically contribute to its pathogenesis. The majority of ALS cases are sporadic and 5-10% of cases correspond to FALS. Although the ages of onset of FALS, which follow a normal Gaussian distribution, correspond to a decade earlier than for SALS cases which have an age dependent incidence, males and females are affected equally in FALS [4]. The most significant candidate genes for SALS include *VEGF* (vascular endothelial growth factor), *angiogenin* (*ALS9*), *paraoxonoase*, *neurofilaments*, *peripherin* and *SMN* (spinal muscular atrophy). Although *ALS9*, *paraoxonase*, *neurofilaments*, *peripherin* and *SMN* mutations have been found in ALS patients, except for *VEGF* mutations, these genes may play a small role in

Regarding FALS candidate genes, the mutations in the copper/zinc superoxide-dismutase-1 gene (*SOD1*), Tar DNA-binding protein gene (*TARDBP*) and in the most recent discovered DNA/RNA-binding protein called *FUS* (fused in sarcoma) or *TLS* (translocation in liposarco‐ ma) produce the typical adult onset ALS phenotype. Other candidate genes that have been described in genome association studies of FALS include *dynactin*, *senataxin (ALS4)* and

The pathophysiology of *SOD1* mutations is probably the most studied one. Many hypothe‐ ses have been suggested and reinforced in transgenic mouse models that overexpress the mutated *SOD1* gene and therefore develop an ALS-like syndrome. Among the proposed mechanisms that support these hypotheses are the toxic gain of function of the mutated SOD1 enzyme, which mainly increases the production of hydroxyl and free radicals, yield‐ ing improper binding metal properties, oxidative stress and inflammation induced by upre‐ gulation of proinflammatory cytokines [7,8]. Alternative hypothesis also suggested a conformational instability and misfolding of the SOD1 peptide, forming intracellular aggre‐

Neurotrophic factors have been initially identified as potential therapeutic agents in the treatment of ALS, opening the door to a new tool for the treatment of motor neuron diseases [10]. Based on previous studies ciliary and glial derived neurotrophic factors, insulin-like growth factor (IGF-1) and erythropoietin improved motor behaviour and reduce motor neu‐ ron loss, astrocyte and microglia activation in preclinical animal models [11], albeit clinical

The failure of standard treatments in ALS could rely on the inappropriate route of adminis‐ tration and/or the poor bioavailability of molecules to the target cell [13]. The subcutaneous and intrathecal delivery of neurotrophic factors can cause adverse side effects such as weight loss, fever, cough, fatigue and behavioral changes [14], whereas viral gene therapy based on the use of an adeno-associated virus or lentivirus vectors is more efficient than the

An alternative strategy that effectively reaches motor neurons, can exert neuroprotective prop‐ erties and does not show such adverse side effects implies the use of the nontoxic fragment C

*VAPB (ALS8)* (VAMP/synaptobrevin-associated membrane protein B gene) [5,6].

vide a better understanding of the neurodegenerative mechanisms in SALS.

250 Gene Therapy - Tools and Potential Applications

the pathogenesis of ALS and previous studies are conflicting [5].

gates which have been reported in motor neuron and glial cells [9].

trials in ALS patients showed lack of therapeutic efficacy [12].

neurotrophic factor delivery but can induce several inherent hazards [15].

Tetanus toxin is a single peptide of approximately 150 kDa, which consists of 1315 amino-acid residues. The toxin forms a two-chain activated molecule composed of a heavy chain (HC) and a light chain (LC) linked by a disulfide bond. The catalytic domain of the toxin resides in the LC, while the translocation and receptor-binding domains are present in HC [18–21] (Figure 1). Tetanus and botulinum toxins are zinc metalloproteases that cleave SNARE (soluble NSF at‐ tachment receptor) proteins, which interfere with the fusion of synaptic vesicles to the plasma membrane and ultimately blocks neurotransmitter release in nerve cells [22].

The nature of the action of tetanus toxin has been widely described in different animal mod‐ els [23–28], exploring its effect not only in the spinal cord but also in the cerebral cortex [29]. One of the unique characteristics of tetanus toxin is that it can be transported retrogradely to the central nervous system and shows remarkable affinity and specificity to neuronal termi‐ nals. The ganglioside-recognition domain in the C-terminal region of HC allows the toxin to be internalized into the neuron at the neuromuscular junction where it enters the axonal ret‐ rograde transport pathway and is subsequently transported to the neuronal soma in the CNS [30,31]. Once the toxin reaches the cytoplasm, it specifically cleaves neuronal proteins integral to vesicular trafficking and neurotransmitter release. In particular, the synaptic vesi‐ cle protein synaptobrevin (VAMP) is the target of tetanus toxin. This protein belongs to a family of proteins that facilitate exocytosis in neurons known as SNARE proteins. The other members of this family are syntaxin and SNAP-25, which are the main molecular targets of botulinum toxin. SNARE proteins are formed by coiled-coil interactions of the alpha-helices of its members, which is required for membrane fusion [32–35].

**Figure 1.** Diagram of the tetanus toxin molecule. The targeting and the translocation domains are located in the heavy-chain (HC), whereas the catalytic domain is located in the light-chain (LC) of the molecule. Its proteolytic activity is Zn2+-dependent, and heavy-metal chelators generate inactive apo-neurotoxins. TTC is approximately 50 KDa and re‐ sides in the HC of the toxin. The ganglioside-recognition domain in TTC allows the toxin to be internalized into the neuron [35].

From the gene therapy point of view, the most interesting part of the toxin that must be out‐ standing is TTC. This fragment of the toxin is located in the HC of tetanus toxin molecule and it plays an important role in the neuronal internalization (Figure 1). In fact, TTC main‐ tains transport properties of the native tetanus toxin without causing toxic effects, in such a way that in the absence of TTC, the toxin retains little ability to paralyze neuromuscular transmission [35,36].

**2. Neuroprotective nature of TTC**

NGF and BDNF or neurotrophin-4/5 [57,58].

procaspase-3 and chromatin condensation [60,61].

numbers of surviving motor neurons (Figure 2).

Many authors have suggested that the trans-synaptic transcytosis pathway used by tetanus toxin was most likely "designed" for the trafficking of trophic factors through a chain of connected neurons [50]. Furthermore, two trophic factors, GDNF and BDNF, have been re‐ ported to possess similar trans-synaptic transcytotic properties to those of tetanus toxin [51].

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

http://dx.doi.org/10.5772/52896

253

Tetanus toxin can induce an increase in serotonin synthesis in the central nervous system, suggesting that the toxin-affected serotonergic innervation in the perinatal rat brain can trig‐ ger the translocation of calcium phosphatidylserine-dependent protein kinase C (PKC) [52]. In particular, tetanus toxin is able to alter a component involving inositol phospholipid hy‐ drolysis, which is associated with PKC activity translocation [53,54]. In addition to this translocation, an enhancement of the tyrosine phosphorylation of the tyrosine receptor TrkA, phospholipase C (PLCγ-1) and ERK-1/2 can be also observed [55]. Due to the fact that TTC can stimulate the PLC-mediated hydrolysis of phosphoinositides in rat brain neurons, TTC seems to modulate some signaling pathways involving the transport of serotonin [56].

Moreover, the activation of intracellular pathways related to the PLCγ-1 phosphorylation and activation of PKC isoforms and the kinases Akt (at Ser 473 and Thr 308) and ERK-1/2 (at Thr 202/Tyr 204) is induced by TTC in rat brain synaptosomes and cultured cortical neurons. This signal pathway activation is dependent on time and concentration, therefore TTC can exert neuroprotective effects, activating TrkA and TrkB receptors in a similar manner as do

The neuroprotective role of TTC is also supported by the fact that it can also protect cerebel‐ lar granular cells against potassium deprivation-induced apoptotic death [59] and act as a neuroprotector in a model of 1-methyl-4-phenylpyridinium (MPP+)-triggered apoptosis, en‐ hancing the survival pathways in rats with a dopaminergic lesion and improving different motor behaviors. Particularly, TTC is able to induce Ser 112 and Ser 136 BAD phosphoryla‐ tion, activate the transcription factor NF-κB, which prevents neuronal death, and induce a decrease in the release of cytochrome c and, consequently, a reduction in the activation of

More recently, the nature of TTC described by Longstreth and colleagues [62] and Larsen and colleagues [63], based on its stability to reach motor neurons specifically through the retrograde axonal transport system, has been reinforced as a potential neuroprotective agent in previous *in vivo* studies of gene and protein expression after injection of plasmid-DNA in transgenic SOD1G93A mice, which carries the mutation G93A in human superoxide dismutase 1 (SOD1) [64]. These studies suggested that intramuscular naked-DNA TTC gene therapy administered into neurodegenerative mouse model delayed the onset of symptoms (by ap‐ proximately 5 days), prolonged survival (by approximately 13 days) and improved the mo‐ tor function activity in TTC-treated mice throughout disease progression, by increasing

The trans-synaptic transport of TTC was intensively studied in one of the best-characterized systems, the primary visual pathway [37, 38], confirming its capacity as a carrier once it was injected intramuscularly [39-41]. Furthermore, the possibility of constructing recombinant molecules with TTC has opened the door to an interesting research field, the discovery of neuro-anatomical tracers, whose main purpose is to map synaptic connections between neu‐ ronal cells.

One of the most well-known recombinant proteins that have been used for this purpose is the protein encoded by *lacZ*-TTC. This protein has been tested *in vitro* and *in vivo* to deter‐ mine its activity in the hypoglossal system, and the detection of the labeled motor neurons was dependent on time post-injection [40-42]. Since neuronal integrity is crucial for TTC in‐ ternalization, the transneuronal molecular pathway at neuromuscular junctions was inten‐ sively studied using this recombinant protein [43]. The protein was detected not only in the neuromuscular junction postsynaptic side but also the soma of the motor neuron, away from the active zones in large uncoated vesicles.

The advances in the understanding of these recombinant proteins have paved the way for new therapeutic approaches using TTC as a carrier of molecules to ameliorate the disease process of motor neuron diseases, neuropathies and pain. As an example, several proteins conjugated to TTC that have been used to study neuronal internalization *in vitro* and *in vivo* are horseradish peroxidise (HPRT), glucose oxidase (GO), green fluorescent protein (GFP), β -Nacetylhexosaminidase-A (HEXA), superoxide dismutase 1 (SOD1), survival motor neuron 1 (SMN1), cardiotrophin-1 (CT1), B-cell lymphomaextra large (Bcl-xL), IGF-1, glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) [17]. More re‐ cently, a novel multi-component nanoparticle system using polyethylene imine (PEI) has been evaluated to elicit the expression of BDNF in neuronal cell lines [44].

Apart from the carrier properties of TTC, the neuroprotective nature of TTC was one of the best kept properties to discover.

The neurotrophin family has been shown to regulate survival, development and functional aspects of neurons in the central and peripheral nervous systems through the activation of one or more of the three members of the receptor tyrosine kinases (TrkA, TrkB, and TrkC) in cooperation with p75NTR [45-48]. Nerve growth factor (NGF) can bind to the TrkA receptor or a complex of TrkA and p75NTR [45], BDNF and neurotrophin-4/5 can bind to TrkB, and neuro‐ trophin-3 binds to TrkC. Interestingly, the retrograde pathway of TTC is shared by p75NTR, TrkB and BDNF, which is strongly dependent on the activities of the small GTPases Rab5 and Rab7 [49], therefore TTC alone might have a neuroprotective role and therefore it can be a valuable non-viral therapeutic agent in ALS.

#### **2. Neuroprotective nature of TTC**

From the gene therapy point of view, the most interesting part of the toxin that must be out‐ standing is TTC. This fragment of the toxin is located in the HC of tetanus toxin molecule and it plays an important role in the neuronal internalization (Figure 1). In fact, TTC main‐ tains transport properties of the native tetanus toxin without causing toxic effects, in such a way that in the absence of TTC, the toxin retains little ability to paralyze neuromuscular

The trans-synaptic transport of TTC was intensively studied in one of the best-characterized systems, the primary visual pathway [37, 38], confirming its capacity as a carrier once it was injected intramuscularly [39-41]. Furthermore, the possibility of constructing recombinant molecules with TTC has opened the door to an interesting research field, the discovery of neuro-anatomical tracers, whose main purpose is to map synaptic connections between neu‐

One of the most well-known recombinant proteins that have been used for this purpose is the protein encoded by *lacZ*-TTC. This protein has been tested *in vitro* and *in vivo* to deter‐ mine its activity in the hypoglossal system, and the detection of the labeled motor neurons was dependent on time post-injection [40-42]. Since neuronal integrity is crucial for TTC in‐ ternalization, the transneuronal molecular pathway at neuromuscular junctions was inten‐ sively studied using this recombinant protein [43]. The protein was detected not only in the neuromuscular junction postsynaptic side but also the soma of the motor neuron, away

The advances in the understanding of these recombinant proteins have paved the way for new therapeutic approaches using TTC as a carrier of molecules to ameliorate the disease process of motor neuron diseases, neuropathies and pain. As an example, several proteins conjugated to TTC that have been used to study neuronal internalization *in vitro* and *in vivo* are horseradish peroxidise (HPRT), glucose oxidase (GO), green fluorescent protein (GFP), β -Nacetylhexosaminidase-A (HEXA), superoxide dismutase 1 (SOD1), survival motor neuron 1 (SMN1), cardiotrophin-1 (CT1), B-cell lymphomaextra large (Bcl-xL), IGF-1, glial derived neurotrophic factor (GDNF) and brain derived neurotrophic factor (BDNF) [17]. More re‐ cently, a novel multi-component nanoparticle system using polyethylene imine (PEI) has

Apart from the carrier properties of TTC, the neuroprotective nature of TTC was one of the

The neurotrophin family has been shown to regulate survival, development and functional aspects of neurons in the central and peripheral nervous systems through the activation of one or more of the three members of the receptor tyrosine kinases (TrkA, TrkB, and TrkC) in cooperation with p75NTR [45-48]. Nerve growth factor (NGF) can bind to the TrkA receptor or a complex of TrkA and p75NTR [45], BDNF and neurotrophin-4/5 can bind to TrkB, and neuro‐ trophin-3 binds to TrkC. Interestingly, the retrograde pathway of TTC is shared by p75NTR, TrkB and BDNF, which is strongly dependent on the activities of the small GTPases Rab5 and Rab7 [49], therefore TTC alone might have a neuroprotective role and therefore it can be

been evaluated to elicit the expression of BDNF in neuronal cell lines [44].

transmission [35,36].

252 Gene Therapy - Tools and Potential Applications

from the active zones in large uncoated vesicles.

best kept properties to discover.

a valuable non-viral therapeutic agent in ALS.

ronal cells.

Many authors have suggested that the trans-synaptic transcytosis pathway used by tetanus toxin was most likely "designed" for the trafficking of trophic factors through a chain of connected neurons [50]. Furthermore, two trophic factors, GDNF and BDNF, have been re‐ ported to possess similar trans-synaptic transcytotic properties to those of tetanus toxin [51].

Tetanus toxin can induce an increase in serotonin synthesis in the central nervous system, suggesting that the toxin-affected serotonergic innervation in the perinatal rat brain can trig‐ ger the translocation of calcium phosphatidylserine-dependent protein kinase C (PKC) [52]. In particular, tetanus toxin is able to alter a component involving inositol phospholipid hy‐ drolysis, which is associated with PKC activity translocation [53,54]. In addition to this translocation, an enhancement of the tyrosine phosphorylation of the tyrosine receptor TrkA, phospholipase C (PLCγ-1) and ERK-1/2 can be also observed [55]. Due to the fact that TTC can stimulate the PLC-mediated hydrolysis of phosphoinositides in rat brain neurons, TTC seems to modulate some signaling pathways involving the transport of serotonin [56].

Moreover, the activation of intracellular pathways related to the PLCγ-1 phosphorylation and activation of PKC isoforms and the kinases Akt (at Ser 473 and Thr 308) and ERK-1/2 (at Thr 202/Tyr 204) is induced by TTC in rat brain synaptosomes and cultured cortical neurons. This signal pathway activation is dependent on time and concentration, therefore TTC can exert neuroprotective effects, activating TrkA and TrkB receptors in a similar manner as do NGF and BDNF or neurotrophin-4/5 [57,58].

The neuroprotective role of TTC is also supported by the fact that it can also protect cerebel‐ lar granular cells against potassium deprivation-induced apoptotic death [59] and act as a neuroprotector in a model of 1-methyl-4-phenylpyridinium (MPP+)-triggered apoptosis, en‐ hancing the survival pathways in rats with a dopaminergic lesion and improving different motor behaviors. Particularly, TTC is able to induce Ser 112 and Ser 136 BAD phosphoryla‐ tion, activate the transcription factor NF-κB, which prevents neuronal death, and induce a decrease in the release of cytochrome c and, consequently, a reduction in the activation of procaspase-3 and chromatin condensation [60,61].

More recently, the nature of TTC described by Longstreth and colleagues [62] and Larsen and colleagues [63], based on its stability to reach motor neurons specifically through the retrograde axonal transport system, has been reinforced as a potential neuroprotective agent in previous *in vivo* studies of gene and protein expression after injection of plasmid-DNA in transgenic SOD1G93A mice, which carries the mutation G93A in human superoxide dismutase 1 (SOD1) [64]. These studies suggested that intramuscular naked-DNA TTC gene therapy administered into neurodegenerative mouse model delayed the onset of symptoms (by ap‐ proximately 5 days), prolonged survival (by approximately 13 days) and improved the mo‐ tor function activity in TTC-treated mice throughout disease progression, by increasing numbers of surviving motor neurons (Figure 2).

**Figure 2.** Functional and survival effect under TTC treatment. Intramuscular injection of TTC-encoding plasmid in SOD1G93A mice (grey bars) delays significantly disease onset and mortality compared to the control group (\*p<0,05, error barrs indicate SEM) (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permission from BioMed Central).

**Figure 3.** Motor neuron survival and neurophysiological study in gastrocnemius and plantar muscles in SOD1G93A mice. (a) Presence of TTC in the grey matter of the ventral horn of (a) positive control (SOD1G93A transgenic mice injected with empty plasmid) and (b) SOD1G93A-TTC treated mice. Arrows point to some of the neurons positively stained for TTC. Bar = 200 μm. (b) Electrophysiological study of compound muscle action potential (CMAP) in gastrocnemius and plantar muscles in wild-type mice (WT), control SOD1G93A mice, and SOD1G93A mice treated with naked DNA encoding for TTC. Values are the mean ± SEM. CMAP, compound muscle action potential; n, number of mice. \*P < 0.05 vs. WT group at the same age (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011),

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

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255

**3. Neuroprotective properties of recombinant molecules of TTC in a**

It has been very well described the specificity of a trophic factor for motoneurons and pre‐ cisely this specificity could be increased by genetically fusing it to TTC, while the trophic factor could contribute to enhance the benefits observed for TTC. Therefore the next inevita‐ ble approach is to test naked-DNA gene delivery to encode for a chimeric molecule, to study

As previously mentioned, BDNF belongs to the family of neurotrophins and binds specifi‐ cally to TrkB receptors to activate the intracellular signaling pathways that promote neuro‐ nal survival and the differentiation of neurons. The neurotrophic effects of BDNF on motoneuronal degeneration have been widely studied *in vitro* and *in vivo* [66,67]. This neu‐ rotrophin has also been proposed as a potential therapeutic agent for the treatment of hu‐ man ALS [68], although no successful results have been achieved. This failure in the clinical

[65] with permission from BioMed Central).

**mouse model of ALS**

the potential synergistic effect.

Apart from functional and survival results obtained *in vivo* in transgenic SOD1G93A mice, the electrophysiological studies showed that, from three to four months of age, TTC treatment played a partial protective effect as demonstrated by the lower decline in amplitudes of the M waves, improvement in motor behavioral tests, and increased survival of motor neurons in the TTC-treated animals' lumbar spinal cord [64] (Figure 3).

Interestingly, TTC administration can also affect antiapoptic pathways by means of calciumrelated mechanisms [64]. The positive effects on motor neuron preservation, animal motor function, and survival were confirmed with studies of anti-apoptotic effects and survival signals in the spinal cords of treated animals. Transcriptional caspase-1 and caspase-3 levels were downregulated in the spinal cord of TTC-treated animals as well as significant varia‐ tions in calcium-related gene expression were found [64]. Furthermore, a downregulation of the caspase-3 activation protein levels in the spinal cord of TTC-treated animals indicated that TTC might act through an anti-apoptotic pathway. Actually, Bax, Bcl2, phospho-Akt and phospho-ERK 1/2 protein expression levels in TTC-treated animals were statistically significant and close to those of wild-type animals, suggesting a decrease of apoptosis and a lower degree of motor neuron neurodegeneration due to TTC treatment [64].

Taking all these results obtained *in vitro* and *in vivo* as a whole, non-viral gene therapy treat‐ ment based on TTC could be a safe and promising neuroprotective strategy for neurodege‐ nerative diseases, especially in ALS. However, the next question to be tackled is whether a recombinant molecule of TTC may have a synergistic effect and enhance the neuroprotective properties of TTC alone.

**Figure 2.** Functional and survival effect under TTC treatment. Intramuscular injection of TTC-encoding plasmid in SOD1G93A mice (grey bars) delays significantly disease onset and mortality compared to the control group (\*p<0,05, error barrs indicate SEM) (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright

Apart from functional and survival results obtained *in vivo* in transgenic SOD1G93A mice, the electrophysiological studies showed that, from three to four months of age, TTC treatment played a partial protective effect as demonstrated by the lower decline in amplitudes of the M waves, improvement in motor behavioral tests, and increased survival of motor neurons

Interestingly, TTC administration can also affect antiapoptic pathways by means of calciumrelated mechanisms [64]. The positive effects on motor neuron preservation, animal motor function, and survival were confirmed with studies of anti-apoptotic effects and survival signals in the spinal cords of treated animals. Transcriptional caspase-1 and caspase-3 levels were downregulated in the spinal cord of TTC-treated animals as well as significant varia‐ tions in calcium-related gene expression were found [64]. Furthermore, a downregulation of the caspase-3 activation protein levels in the spinal cord of TTC-treated animals indicated that TTC might act through an anti-apoptotic pathway. Actually, Bax, Bcl2, phospho-Akt and phospho-ERK 1/2 protein expression levels in TTC-treated animals were statistically significant and close to those of wild-type animals, suggesting a decrease of apoptosis and a

Taking all these results obtained *in vitro* and *in vivo* as a whole, non-viral gene therapy treat‐ ment based on TTC could be a safe and promising neuroprotective strategy for neurodege‐ nerative diseases, especially in ALS. However, the next question to be tackled is whether a recombinant molecule of TTC may have a synergistic effect and enhance the neuroprotective

lower degree of motor neuron neurodegeneration due to TTC treatment [64].

(2011), [65] with permission from BioMed Central).

254 Gene Therapy - Tools and Potential Applications

properties of TTC alone.

in the TTC-treated animals' lumbar spinal cord [64] (Figure 3).


**Figure 3.** Motor neuron survival and neurophysiological study in gastrocnemius and plantar muscles in SOD1G93A mice. (a) Presence of TTC in the grey matter of the ventral horn of (a) positive control (SOD1G93A transgenic mice injected with empty plasmid) and (b) SOD1G93A-TTC treated mice. Arrows point to some of the neurons positively stained for TTC. Bar = 200 μm. (b) Electrophysiological study of compound muscle action potential (CMAP) in gastrocnemius and plantar muscles in wild-type mice (WT), control SOD1G93A mice, and SOD1G93A mice treated with naked DNA encoding for TTC. Values are the mean ± SEM. CMAP, compound muscle action potential; n, number of mice. \*P < 0.05 vs. WT group at the same age (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permission from BioMed Central).

#### **3. Neuroprotective properties of recombinant molecules of TTC in a mouse model of ALS**

It has been very well described the specificity of a trophic factor for motoneurons and pre‐ cisely this specificity could be increased by genetically fusing it to TTC, while the trophic factor could contribute to enhance the benefits observed for TTC. Therefore the next inevita‐ ble approach is to test naked-DNA gene delivery to encode for a chimeric molecule, to study the potential synergistic effect.

As previously mentioned, BDNF belongs to the family of neurotrophins and binds specifi‐ cally to TrkB receptors to activate the intracellular signaling pathways that promote neuro‐ nal survival and the differentiation of neurons. The neurotrophic effects of BDNF on motoneuronal degeneration have been widely studied *in vitro* and *in vivo* [66,67]. This neu‐ rotrophin has also been proposed as a potential therapeutic agent for the treatment of hu‐ man ALS [68], although no successful results have been achieved. This failure in the clinical application of BDNF may be due to the low efficacy of targeting the neurotrophic factor to motoneurons. Alternatively, TTC possesses a high affinity for motoneurons [40], and the fu‐ sion of BDNF to the TTC protein might increase its accessibility. A previous study reported that some neurotrophic factors, in particular BDNF, facilitate the internalization of TTC re‐ combinant molecules in motor nerve terminals [69]. In addition, TTC and the recombinant protein BDNF-TTC can inhibit apoptosis in cultured neurons, with the quimeric molecule being more effective than TTC alone [70]. Interestingly, BDNF may cause a relocalization of membrane domains containing TTC receptors by activating Trk receptors, thereby facilitat‐ ing the neuronal internalization of TTC. This observation is supported by other authors who state that TTC activates intracellular pathways involving Trk receptors [58]. Therefore, the hypothesis of a synergistic positive effect based on the fusion of the mature form of BDNF genes to TTC in a mouse model of ALS needs to be pointed out for the bench to the bedside approach.

Similarly to the results observed in transgenic SOD1G93A mice [64], an amelioration of the de‐ cline in hindlimb muscle innervation was observed in the animals that were injected with either naked DNA encoding TTC or naked DNA encoding the recombinant molecule TTC and BDNF (BDNF-TTC) [65] (Figures 4,5), in addition to a significant delay in the onset of symptoms and functional deficits (Figure 6), an improvement in the spinal motor neuron survival (Figure 7) (down-regulation of caspase-1 and caspase-3 levels and a significant phosphorylation of serine/threonine protein kinase Akt) (Figure 8) and a prolonged lifespan under both treatments [64,65].

**Figure 5.** Neurophysiological study in gastrocnemius and plantar muscles. (a) Results of wild-type mice (WT), control SOD1G93A mice, and SOD1G93A mice treated with naked DNA encoding for BDNF, TTC, and BDNF-TTC are shown. Values are the mean ± SEM. CMAP, compound muscle action potential; n, number of mice. \*p < 0.05 vs. WT group at the same age. (b) Histogram representation of the decrement in the amplitude of the compound muscle action potential. CMAP was compared at 4 months with respect to values at 3 months of age in SOD1G93A mice, untreated and treated with naked DNA encoding for BDNF, TTC or BDNF-TTC. For each group, the left bar corresponds to the gastrocnemius muscle and the right bar to the plantar muscle (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion mole‐

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**Figure 6.** Improvement in disease clinical outcomes in transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treat‐ ments. Cumulative probability of the onset of disease symptoms (hanging-wire test) and survival in SOD1G93A mice injected

cule, Copyright (2011), [65] with permission from BioMed Central).

**Figure 4.** Motoneuronal preservation in transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treatments. Immu‐ nohistochemical labeling for BDNF expression in the grey matter of the ventral horn of (a) positive control (SOD1G93A transgenic mice injected with empty plasmid), (b) SOD1G93A-BDNF and (c) L2 and (d) L4 spinal segments of SOD1G93A-BDNF-TTC mice. (e, f) Detail of BDNF immunolabeling of the sections shown in c and d, at higher magnification. Pres‐ ence of TTC in the grey matter of the ventral horn of (g) SOD1G93A-BDNF-TTC and (h) SOD1G93A-TTC treated mice. Arrows point to some of the neurons positively stained for TTC. Bar = 200 μm in a, b, c, d, g and h; bar = 100 μm in e and f (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permis‐ sion from BioMed Central).

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench… http://dx.doi.org/10.5772/52896 257

application of BDNF may be due to the low efficacy of targeting the neurotrophic factor to motoneurons. Alternatively, TTC possesses a high affinity for motoneurons [40], and the fu‐ sion of BDNF to the TTC protein might increase its accessibility. A previous study reported that some neurotrophic factors, in particular BDNF, facilitate the internalization of TTC re‐ combinant molecules in motor nerve terminals [69]. In addition, TTC and the recombinant protein BDNF-TTC can inhibit apoptosis in cultured neurons, with the quimeric molecule being more effective than TTC alone [70]. Interestingly, BDNF may cause a relocalization of membrane domains containing TTC receptors by activating Trk receptors, thereby facilitat‐ ing the neuronal internalization of TTC. This observation is supported by other authors who state that TTC activates intracellular pathways involving Trk receptors [58]. Therefore, the hypothesis of a synergistic positive effect based on the fusion of the mature form of BDNF genes to TTC in a mouse model of ALS needs to be pointed out for the bench to the bedside

Similarly to the results observed in transgenic SOD1G93A mice [64], an amelioration of the de‐ cline in hindlimb muscle innervation was observed in the animals that were injected with either naked DNA encoding TTC or naked DNA encoding the recombinant molecule TTC and BDNF (BDNF-TTC) [65] (Figures 4,5), in addition to a significant delay in the onset of symptoms and functional deficits (Figure 6), an improvement in the spinal motor neuron survival (Figure 7) (down-regulation of caspase-1 and caspase-3 levels and a significant phosphorylation of serine/threonine protein kinase Akt) (Figure 8) and a prolonged lifespan

**Figure 4.** Motoneuronal preservation in transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treatments. Immu‐ nohistochemical labeling for BDNF expression in the grey matter of the ventral horn of (a) positive control (SOD1G93A transgenic mice injected with empty plasmid), (b) SOD1G93A-BDNF and (c) L2 and (d) L4 spinal segments of SOD1G93A-BDNF-TTC mice. (e, f) Detail of BDNF immunolabeling of the sections shown in c and d, at higher magnification. Pres‐ ence of TTC in the grey matter of the ventral horn of (g) SOD1G93A-BDNF-TTC and (h) SOD1G93A-TTC treated mice. Arrows point to some of the neurons positively stained for TTC. Bar = 200 μm in a, b, c, d, g and h; bar = 100 μm in e and f (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permis‐

approach.

under both treatments [64,65].

256 Gene Therapy - Tools and Potential Applications

sion from BioMed Central).


**Figure 5.** Neurophysiological study in gastrocnemius and plantar muscles. (a) Results of wild-type mice (WT), control SOD1G93A mice, and SOD1G93A mice treated with naked DNA encoding for BDNF, TTC, and BDNF-TTC are shown. Values are the mean ± SEM. CMAP, compound muscle action potential; n, number of mice. \*p < 0.05 vs. WT group at the same age. (b) Histogram representation of the decrement in the amplitude of the compound muscle action potential. CMAP was compared at 4 months with respect to values at 3 months of age in SOD1G93A mice, untreated and treated with naked DNA encoding for BDNF, TTC or BDNF-TTC. For each group, the left bar corresponds to the gastrocnemius muscle and the right bar to the plantar muscle (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion mole‐ cule, Copyright (2011), [65] with permission from BioMed Central).

**Figure 6.** Improvement in disease clinical outcomes in transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treat‐ ments. Cumulative probability of the onset of disease symptoms (hanging-wire test) and survival in SOD1G93A mice injected

at 60 days of age with TTC, BDNF-TTC, BDNF or empty (positive control) plasmids. Strength and motor function were tested using the rotarod at 15 rpm. Mice were given up to 180 s for the test performance and the time at which mice fell was re‐ corded (\*, #, +, P < 0.05; \*\*, ##, P < 0.01; error bars indicate SEM); \* for BDNF-TTC vs. positive control comparisons; # for TTC vs. control comparisons; + for BDNF vs. positive control comparisons (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fu‐ sion molecule, Copyright (2011), [65] with permission from BioMed Central).

mals treated with TTC (grey), BDNF-TTC (blue, BTTC) and BDNF (soft blue). Western blot quantities are shown as the ratios to b-tubulin and then related to age-matched wild-type (black) mice data (\*P < 0.05 and \*\*P < 0.01 vs. control SOD1G93A mice; \*\*\*p < 0.001; error bars indicate SEM) (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion

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259

Additionally, GDNF is another candidate neurotrophic factor for ALS therapy. This factor has been described to show potent trophic effects on proliferation, differentiation and sur‐ vival of motor neurons *in vitro* and *in vivo* [63,71-76]. Furthermore, after the retrograde transport of GDNF to the cell bodies, a fraction of this trophic factor avoided degradation and was sorted to dendrites [51], similar to the known movement of the TTC [39]. It was also suggested that the transsynaptic and transcytotic pathway used by GDNF was similar to that of TTC, but not identical, and that GDNF protein degradation was lower than that of TTC protein. Furthermore, the combination of TTC and GDNF has been evaluated in a neo‐ natal rat axotomy model [63] and in the ALS mouse model [77]. The combination of TTC with insulin growth factor (IGF-1) has also been assayed in transgenic SOD1G93A mice [78], although the effect of TTC alone has not been compared in any of these studies. When the effect of TTC was compared to the recombinant molecule *in vitro*, a significant increase in the survival capacity of neuronal cells was found [77]. However *in vivo*, no significant differ‐ ences were observed, which is probably due to the possibility that the recombinant molecule

might follow a GDNF route and not the TTC route under axotomy conditions [63].

When focusing the study *in vivo* in a mouse model of ALS, the recombinant molecule TTC and GDNF (GDNF-TTC), GDNF and TTC treatments prompted a delay in disease onset, an improvement in motor function and a longer lifespan in transgenic SOD1G93A mice, compar‐

**Figure 9.** Improvement in disease clinical outcomes in transgenic SOD1G93A mice under TTC, GDNF and GDNF-TTC treatments. Cumulative probability of the onset of disease symptoms (hanging-wire test) and survival in SOD1G93A mice injected at 60 days of age with TTC, GDNF-TTC, GDNF or empty (positive control) plasmids. Strength and motor

molecule, Copyright (2011), [65] with permission from BioMed Central).

ing to empty-plasmid injected control mice [79] (Figure 9).

**Figure 7.** Spinal motor neuron survival of transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treatments. Rep‐ resentative micrographs showing cross-sections of lumbar spinal cords stained with cresyl violet from wild-type, (a) SOD1G93A control (positive control), (b) BDNF-treated, (c) and BDNF-TTC-treated, (d) mice at 16 weeks of age. Bar = 500 μm (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic ef‐ fect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permission from BioMed Central).

**Figure 8.** Apoptotic and survival pathways under TTC, BDNF and BDNF-TTC treatments. (a) Histogram representation of the average number of stained motoneurons per section in L2 and L4 spinal cord segments of wild-type littermates, control SOD1G93A and treated mice (n = 4-5 mice per group). \* p < 0.05 vs. wild type; # p < 0.05 vs. SODG93A control mice. (b) Fold-changes in the expression of pro-Casp3 and active Casp3 proteins, (c) Bax and Bcl2 proteins and (d) phosphorylated states of Akt and ERK1/2 proteins in spinal cord lysates of control SOD1G93A animals (white) and ani‐

mals treated with TTC (grey), BDNF-TTC (blue, BTTC) and BDNF (soft blue). Western blot quantities are shown as the ratios to b-tubulin and then related to age-matched wild-type (black) mice data (\*P < 0.05 and \*\*P < 0.01 vs. control SOD1G93A mice; \*\*\*p < 0.001; error bars indicate SEM) (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permission from BioMed Central).

at 60 days of age with TTC, BDNF-TTC, BDNF or empty (positive control) plasmids. Strength and motor function were tested using the rotarod at 15 rpm. Mice were given up to 180 s for the test performance and the time at which mice fell was re‐ corded (\*, #, +, P < 0.05; \*\*, ##, P < 0.01; error bars indicate SEM); \* for BDNF-TTC vs. positive control comparisons; # for TTC vs. control comparisons; + for BDNF vs. positive control comparisons (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fu‐

**Figure 7.** Spinal motor neuron survival of transgenic SOD1G93A mice under TTC, BDNF and BDNF-TTC treatments. Rep‐ resentative micrographs showing cross-sections of lumbar spinal cords stained with cresyl violet from wild-type, (a) SOD1G93A control (positive control), (b) BDNF-treated, (c) and BDNF-TTC-treated, (d) mice at 16 weeks of age. Bar = 500 μm (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic ef‐ fect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permission

**Figure 8.** Apoptotic and survival pathways under TTC, BDNF and BDNF-TTC treatments. (a) Histogram representation of the average number of stained motoneurons per section in L2 and L4 spinal cord segments of wild-type littermates, control SOD1G93A and treated mice (n = 4-5 mice per group). \* p < 0.05 vs. wild type; # p < 0.05 vs. SODG93A control mice. (b) Fold-changes in the expression of pro-Casp3 and active Casp3 proteins, (c) Bax and Bcl2 proteins and (d) phosphorylated states of Akt and ERK1/2 proteins in spinal cord lysates of control SOD1G93A animals (white) and ani‐

sion molecule, Copyright (2011), [65] with permission from BioMed Central).

258 Gene Therapy - Tools and Potential Applications

from BioMed Central).

Additionally, GDNF is another candidate neurotrophic factor for ALS therapy. This factor has been described to show potent trophic effects on proliferation, differentiation and sur‐ vival of motor neurons *in vitro* and *in vivo* [63,71-76]. Furthermore, after the retrograde transport of GDNF to the cell bodies, a fraction of this trophic factor avoided degradation and was sorted to dendrites [51], similar to the known movement of the TTC [39]. It was also suggested that the transsynaptic and transcytotic pathway used by GDNF was similar to that of TTC, but not identical, and that GDNF protein degradation was lower than that of TTC protein. Furthermore, the combination of TTC and GDNF has been evaluated in a neo‐ natal rat axotomy model [63] and in the ALS mouse model [77]. The combination of TTC with insulin growth factor (IGF-1) has also been assayed in transgenic SOD1G93A mice [78], although the effect of TTC alone has not been compared in any of these studies. When the effect of TTC was compared to the recombinant molecule *in vitro*, a significant increase in the survival capacity of neuronal cells was found [77]. However *in vivo*, no significant differ‐ ences were observed, which is probably due to the possibility that the recombinant molecule might follow a GDNF route and not the TTC route under axotomy conditions [63].

When focusing the study *in vivo* in a mouse model of ALS, the recombinant molecule TTC and GDNF (GDNF-TTC), GDNF and TTC treatments prompted a delay in disease onset, an improvement in motor function and a longer lifespan in transgenic SOD1G93A mice, compar‐ ing to empty-plasmid injected control mice [79] (Figure 9).

**Figure 9.** Improvement in disease clinical outcomes in transgenic SOD1G93A mice under TTC, GDNF and GDNF-TTC treatments. Cumulative probability of the onset of disease symptoms (hanging-wire test) and survival in SOD1G93A mice injected at 60 days of age with TTC, GDNF-TTC, GDNF or empty (positive control) plasmids. Strength and motor

function were tested using the rotarod at 15 rpm. Mice were given up to 180 s for the test performance and the time at which mice fell was recorded (\*, #, +, P < 0.05; \*\*, ##, P < 0.01; error bars indicate SEM); \*GDNF-TTC vs. control com‐ parisons; # TTC vs. control comparisons; + GDNF vs. control comparisons (\*,#, +, *P*<0.05; \*\*, ##, *P*<0.01; error bars indi‐ cate SEM) (Reprinted from Restor. Neurol. Neurosci, 30, Moreno-Igoa M, Calvo AC, Ciriza J. et al. Non-viral gene delivery of the GDNF, either alone or fused to the C-fragment of tetanus toxin protein, prolongs survival in a mouse ALS model, p. 69-80, Copyright (2012), [79] with permission from IOS Press).

As a final point, the active state of the neurotrophic factors BDNF and GDNF in the recombi‐ nant molecule could suggest that either BDNF or GDNF could exert an autocrine and neuro‐ protective role together with TTC to a similar extent as TTC alone; however this effect could not be sufficient enough to prompt a synergistic effect. As a consequence, the recombinant molecules could mainly use the same pathway that mimics a neurotrophic secretion route, prompting survival signals in the spinal cord of transgenic SOD1G93A mice [65,79]. Despite all these contributions to the understanding of the neuroprotective properties of recombi‐ nant molecules, it is undoubtedly that TTC has open the door to an alternative therapeutic strategy for more neurodegenerative diseases although its molecular pathways is not yet

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

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261

**Figure 11.** TTC and BDNF detection in skeletal muscle and spinal cord of ALS transgenic SOD1G93A mice. Western blot detection of TTC in spinal cord and skeletal muscle tissues of wild-type (C-, negative control), SOD1G93A transgenic mice injected with empty plasmid (C+, positive control), TTC- and BDNFTTC (BTTC)-treated mice. In TTC and BDNF-TTC treat‐ ed groups, the detected band was approximately of 50 and ~ 70 KDa respectively (\*), using both anti-TTC and anti-BDNF antibodies. In the BDNF group, the dimeric conformation, indicated by arrows, was observed at approximately 40 KDa (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permis‐

At present, gene and stem cell therapies are holding the hope for an efficient treatment in ALS. Regarding gene therapy, the possibility of delivering therapeutic molecules to dam‐ aged tissues crossing the blood-brain barrier has made possible the study of viral (adenovi‐ rus, adeno-associated and lentivirus) and non-viral (fragment C of tetanus toxin) vectors, which are retrogradely transported to motor neurons, in preclinical animal models showing

Although therapeutic strategies, which tend to stop or slow down the progression of ALS, are one of the main goals in this field of research, the new property of TTC has opened the door to new non-viral therapeutic strategies in this disease. The fact that TTC as well as the recombinant molecules BDNF-TTC and GDNF-TTC can be transported through motoneur‐ ons to induce a later onset of symptoms, improve motoneuron survival and extend the sur‐ vival of SOD1G93A mice support the fact that the naked DNA-mediated intramuscular

well characterized.

sion from BioMed Central).

**4. Conclusions**

promising neuroprotective effects.

Moreover, the recombinant molecule GDNF-TTC and full-length GDNF inhibited apoptotic pathways in spinal cords of SOD1G93A mice by reducing the activation of caspase-3, as well as Bax and Bcl2 protein levels reached a profile expression similar than the one observed in wild type mice, highlighting the fact that treated mice biochemically resemble non-transgen‐ ic mice (Figure 10). In addition, all treatment molecules activated the PI3K survival pathway by phosphorylating Akt and ERK1/2, resembling again the wild type levels [79] (Figure 10).

**Figure 10.** Apoptotic and survival pathways under TTC, GDNF and GDNF-TTC treatments. (a) Fold-changes in the ex‐ pression of GABA(A) receptor subunit-4 (*Gabra4*) mRNA levels in total spinal cord of wild type, control transgenic mice and treated transgenic mice (n=5 per group). (\**P*<0.05, \*\**P*<0.01; error bars indicate SEM). Fold changes in the expres‐ sion of (b) pro-Casp3 and active Casp3 proteins, (c) Bax and Bcl2 proteins, and (d) phosphorylated states of Akt and ERK1/2 proteins. Western blots from spinal cord lysates of control animals (white) and treated with TTC (gray), GDNFTTC (hatched-columns) and GDNF (dotted-columns). Western blot quantities are shown as the ratio to β-tubulin and then related to age-matched wild type (black) mice data (Reprinted from Restor. Neurol. Neurosci, 30, Moreno-Igoa M, Calvo AC, Ciriza J. et al. Non-viral gene delivery of the GDNF, either alone or fused to the C-fragment of teta‐ nus toxin protein, prolongs survival in a mouse ALS model, p. 69-80, Copyright (2012), [79] with permission from IOS Press).

Summarizing, albeit a significant improvement in behavioral assays together with an activa‐ tion of anti-apoptotic and survival pathways under BDNF and GDNF treatments was ob‐ served in transgenic SOD1G93A mice, no synergistic effect was found neither using the BDNF-TTC nor GDNF-TTC recombinant molecules. Interestingly, recombinant plasmids BDNF-TTC and GDNF-TTC were detected in skeletal muscle and the corresponding recombinant protein reached the spinal cord tissue of transgenic SOD1G93A mice (Figure 11), reinforcing the carrier properties of TTC.

As a final point, the active state of the neurotrophic factors BDNF and GDNF in the recombi‐ nant molecule could suggest that either BDNF or GDNF could exert an autocrine and neuro‐ protective role together with TTC to a similar extent as TTC alone; however this effect could not be sufficient enough to prompt a synergistic effect. As a consequence, the recombinant molecules could mainly use the same pathway that mimics a neurotrophic secretion route, prompting survival signals in the spinal cord of transgenic SOD1G93A mice [65,79]. Despite all these contributions to the understanding of the neuroprotective properties of recombi‐ nant molecules, it is undoubtedly that TTC has open the door to an alternative therapeutic strategy for more neurodegenerative diseases although its molecular pathways is not yet well characterized.

**Figure 11.** TTC and BDNF detection in skeletal muscle and spinal cord of ALS transgenic SOD1G93A mice. Western blot detection of TTC in spinal cord and skeletal muscle tissues of wild-type (C-, negative control), SOD1G93A transgenic mice injected with empty plasmid (C+, positive control), TTC- and BDNFTTC (BTTC)-treated mice. In TTC and BDNF-TTC treat‐ ed groups, the detected band was approximately of 50 and ~ 70 KDa respectively (\*), using both anti-TTC and anti-BDNF antibodies. In the BDNF group, the dimeric conformation, indicated by arrows, was observed at approximately 40 KDa (Reprinted from Orphanet J. Rare Dis., 6: 10, Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule, Copyright (2011), [65] with permis‐ sion from BioMed Central).

#### **4. Conclusions**

function were tested using the rotarod at 15 rpm. Mice were given up to 180 s for the test performance and the time at which mice fell was recorded (\*, #, +, P < 0.05; \*\*, ##, P < 0.01; error bars indicate SEM); \*GDNF-TTC vs. control com‐ parisons; # TTC vs. control comparisons; + GDNF vs. control comparisons (\*,#, +, *P*<0.05; \*\*, ##, *P*<0.01; error bars indi‐ cate SEM) (Reprinted from Restor. Neurol. Neurosci, 30, Moreno-Igoa M, Calvo AC, Ciriza J. et al. Non-viral gene delivery of the GDNF, either alone or fused to the C-fragment of tetanus toxin protein, prolongs survival in a mouse

Moreover, the recombinant molecule GDNF-TTC and full-length GDNF inhibited apoptotic pathways in spinal cords of SOD1G93A mice by reducing the activation of caspase-3, as well as Bax and Bcl2 protein levels reached a profile expression similar than the one observed in wild type mice, highlighting the fact that treated mice biochemically resemble non-transgen‐ ic mice (Figure 10). In addition, all treatment molecules activated the PI3K survival pathway by phosphorylating Akt and ERK1/2, resembling again the wild type levels [79] (Figure 10).

**Figure 10.** Apoptotic and survival pathways under TTC, GDNF and GDNF-TTC treatments. (a) Fold-changes in the ex‐ pression of GABA(A) receptor subunit-4 (*Gabra4*) mRNA levels in total spinal cord of wild type, control transgenic mice and treated transgenic mice (n=5 per group). (\**P*<0.05, \*\**P*<0.01; error bars indicate SEM). Fold changes in the expres‐ sion of (b) pro-Casp3 and active Casp3 proteins, (c) Bax and Bcl2 proteins, and (d) phosphorylated states of Akt and ERK1/2 proteins. Western blots from spinal cord lysates of control animals (white) and treated with TTC (gray), GDNFTTC (hatched-columns) and GDNF (dotted-columns). Western blot quantities are shown as the ratio to β-tubulin and then related to age-matched wild type (black) mice data (Reprinted from Restor. Neurol. Neurosci, 30, Moreno-Igoa M, Calvo AC, Ciriza J. et al. Non-viral gene delivery of the GDNF, either alone or fused to the C-fragment of teta‐ nus toxin protein, prolongs survival in a mouse ALS model, p. 69-80, Copyright (2012), [79] with permission from IOS

Summarizing, albeit a significant improvement in behavioral assays together with an activa‐ tion of anti-apoptotic and survival pathways under BDNF and GDNF treatments was ob‐ served in transgenic SOD1G93A mice, no synergistic effect was found neither using the BDNF-TTC nor GDNF-TTC recombinant molecules. Interestingly, recombinant plasmids BDNF-TTC and GDNF-TTC were detected in skeletal muscle and the corresponding recombinant protein reached the spinal cord tissue of transgenic SOD1G93A mice (Figure 11), reinforcing

ALS model, p. 69-80, Copyright (2012), [79] with permission from IOS Press).

260 Gene Therapy - Tools and Potential Applications

Press).

the carrier properties of TTC.

At present, gene and stem cell therapies are holding the hope for an efficient treatment in ALS. Regarding gene therapy, the possibility of delivering therapeutic molecules to dam‐ aged tissues crossing the blood-brain barrier has made possible the study of viral (adenovi‐ rus, adeno-associated and lentivirus) and non-viral (fragment C of tetanus toxin) vectors, which are retrogradely transported to motor neurons, in preclinical animal models showing promising neuroprotective effects.

Although therapeutic strategies, which tend to stop or slow down the progression of ALS, are one of the main goals in this field of research, the new property of TTC has opened the door to new non-viral therapeutic strategies in this disease. The fact that TTC as well as the recombinant molecules BDNF-TTC and GDNF-TTC can be transported through motoneur‐ ons to induce a later onset of symptoms, improve motoneuron survival and extend the sur‐ vival of SOD1G93A mice support the fact that the naked DNA-mediated intramuscular delivery of TTC and fusion molecules can promote neuroprotective effects in the SOD1G93A murine model of ALS. The active states of BDNF and GDNF in the recombinant molecules also confirm that these neurotrophic factors could exert an autocrine and neuroprotective role together with TTC to a similar extent as TTC alone, but this effect was not sufficient to enhance the survival signals observed under TTC treatment alone.

[5] Sreedharan J, Shaw C. The genetics of Amyotrophic Lateral Sclerosis. ACNR 2009;9:

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

http://dx.doi.org/10.5772/52896

263

[6] Van Blitterswijk M, van Es MA, Koppers M, van Rheenen W et al. VAPB and C9orf72 mutations in 1 familial amyotrophic lateral sclerosis patient. Neurobiol. Aging,

[7] Cluskey S, Ramsden DB. Mechanism of neurodegeneration in amyotrophic lateral

[8] Hensley K, Mhatre M, Mou S, Pye QN. et al. On the relation of oxidative stress to neuroinflammation: lessons learned from the G93A-SOD1 mouse model of amyotro‐

[9] Bruijn LI, Houseweart MK, Kato S, Anderson KL. et al. Aggregation and motor neu‐ ron toxicity of an ALS-linked SOD1 mutant independent from wild-type SOD1. Sci‐

[10] Jiasheng Zhang EJH. Dynamic expression of neurotrophic factor receptors in postna‐ tal spinal motoneurons and in mouse model of ALS. J Neurobiol 2006;66: 882–895. [11] Goodall EF, Morrison KE. Amyotrophic lateral sclerosis (motor neuron disease): pro‐ posed mechanisms and pathways to treatment. Expert Rev. Mol. Med. 2006;8: 1-22.

[12] Kalra S, Genge A, Arnold DL. A prospective, randomized, placebo-controlled evalu‐ ation of corticoneuronal response to intrathecal BDNF therapy in ALS using magnet‐ ic resonance spectroscopy: feasibility and results. Amyotroph. Lateral Scler. Other

[13] Thorne RG; Frey WH. Delivery of neurotrophic factors to the central nervous system:

[14] Borasio GD, Robberecht W, Leigh PN. et al. A placebo-controlled trial of insulin-like growth factor-I in amyotrophic lateral sclerosis. European ALS/IGF-I Study Group.

[15] Check, E. Harmful potential of viral vectors fuels doubts over gene therapy. Nature

[16] Farrar JJ, Yen L.M, Cook T, Fairweather N. et al. Tetanus. J. Neurol. Neurosurg. Psy‐

[17] Toivonen JM, Oliván S, Osta R. Tetanus toxin-C fragment: the curier and the cure?.

[18] Johnson EA. Clostridial toxins as therapeutic agents: Benefits of nature's most toxic

[19] Pellizari R, Rossetto O, Schiavo G, Montecucco C. Tetanus and botulinum neurotox‐ ins: mechanism of action and therapeutic uses. Phil. Trans. R. Soc. Lond. B 1999;354:

Toxins 2010;2: 2622-2644. doi:10.3390/toxins2112622.

proteins. Ann. Rev. Microbiol. 1999;53: 551-575.

pharmacokinetic considerations. Clin Pharmacokinet 2001;40: 907–946.

phic lateral sclerosis. Antioxid. Redox. Signal. 2006;8: 2075-2087.

10-16.

2012;Aug 7.

ence 1998;281: 1851-1854.

Motor Neuron Disord. 2003;4: 22-26.

Neurology 1998;51: 583–586.

2003;423: 573–574.

259-268.

chiatr. 2000;69: 292–301.

sclerosis. J. Clin. Pathol. 2001;54(6):386-92.

Definitively, the neuroprotective role of fragment C has shed light on the understanding of the disease neurodegeneration processes and the study of this promising property of TTC can be extended to other neurodegenerative diseases, such as Parkinson's disease, Alzheim‐ er's disease and Spinal Muscular Atrophy (SMN). Essentially, a better understanding of these neurodegenerative diseases will facilitate the translation from animal model to pa‐ tients to find a definitive therapeutic approach.

#### **Acknowledgements**

This work was supported by from Caja Navarra: "Tú eliges, tú decides"; PI10/0178 from the Fondo de Investigación Sanitaria of Spain; ALS association Nº S54406 and Ministerio de Ciencia e Innovacion INNPACTO IPT-2011-1091-900000.

#### **Author details**

Ana C. Calvo, Pilar Zaragoza and Rosario Osta\*

\*Address all correspondence to: osta@unizar.es

Laboratory of Genetics and Biochemistry (LAGENBIO-I3A), Aragon's Institute of Health Sciences (IACS), Faculty of Veterinary School, University of Zaragoza, Spain

#### **References**


[5] Sreedharan J, Shaw C. The genetics of Amyotrophic Lateral Sclerosis. ACNR 2009;9: 10-16.

delivery of TTC and fusion molecules can promote neuroprotective effects in the SOD1G93A murine model of ALS. The active states of BDNF and GDNF in the recombinant molecules also confirm that these neurotrophic factors could exert an autocrine and neuroprotective role together with TTC to a similar extent as TTC alone, but this effect was not sufficient to

Definitively, the neuroprotective role of fragment C has shed light on the understanding of the disease neurodegeneration processes and the study of this promising property of TTC can be extended to other neurodegenerative diseases, such as Parkinson's disease, Alzheim‐ er's disease and Spinal Muscular Atrophy (SMN). Essentially, a better understanding of these neurodegenerative diseases will facilitate the translation from animal model to pa‐

This work was supported by from Caja Navarra: "Tú eliges, tú decides"; PI10/0178 from the Fondo de Investigación Sanitaria of Spain; ALS association Nº S54406 and Ministerio de

Laboratory of Genetics and Biochemistry (LAGENBIO-I3A), Aragon's Institute of Health

[1] Jiasheng Zhang EJH. Dynamic expression of neurotrophic factor receptors in postna‐ tal spinal motoneurons and in mouse model of ALS. J Neurobiol 2006;66: 882–895. [2] Brooks BR, Miller RG, Swash M, Munsat TL. El Escorial revisited: revised criteria for the diagnosis of amyotrophic lateral sclerosis. Amyotroph. Lateral Scler. Other Motor

[3] Vucic S, Kiernan MC. Pathophysiology of neurodegeneration in familial amyotrophic

[4] Wijesekera LC, Leigh PN. Amyotrophic lateral sclerosis. Orphanet Journal of Rare

Sciences (IACS), Faculty of Veterinary School, University of Zaragoza, Spain

enhance the survival signals observed under TTC treatment alone.

tients to find a definitive therapeutic approach.

Ciencia e Innovacion INNPACTO IPT-2011-1091-900000.

Ana C. Calvo, Pilar Zaragoza and Rosario Osta\*

\*Address all correspondence to: osta@unizar.es

Neuron Disord. 2000;1: 293-299.

lateral sclerosis. Curr. Mol. Medicine 2009;9: 255-272.

Diseases 2009; 4:3, doi:10.1186/1750-1172-1184-1183.

**Acknowledgements**

262 Gene Therapy - Tools and Potential Applications

**Author details**

**References**


[38] Manning KA, Erichsen JT, Evinger C. Retrograde transneuronal transport properties

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

http://dx.doi.org/10.5772/52896

265

[39] Fishman PS, Carrigan DR. Retrograde transneuronal transfer of the fragment C of

[40] Coen L, Osta R, Maury M, Brulet P. Construction of recombinant proteins that mi‐ grate retrogradely and transynaptically into the central nervous system. Proc Natl

[41] Miana-Mena FJ, Munoz MJ, Roux S. et al. A non-viral vector for targeting gene thera‐

[42] Miana-Mena FJ, Muñoz MJ, Ciriza J. et al. Fragment C tetanus toxin: A putative ac‐ tivity-dependent neuroanatomical tracer. Acta Neurobiol. Exp. 2003;63: 211-218.

[43] Miana-Mena FJ, Roux S, Benichou JC. et al. Neuronal activity-dependent membrane traffic at the neuromuscular junction. Proc. Nat. Acad. Sci. 2002;99: 3234-3239.

[44] Oliveira H, Fernandez R, Pires LR. et al. Targeted gene delivery into peripheral sen‐ sorial neurons mediated by self-assembled vectors composed of poly (ethylene

[45] Skeldal S, Matusica D, Nykjaer A, Coulson EJ. Proteolytic processing of the p75 neu‐ rotrophin receptor: A prerequisite for signalling? Neuronal life, growth and death signaling are crucially regulated by intra-membrane proteolysis and trafficking of

[46] Skaper SD. The biology of neurotrophins, signalling pathways, and functional pep‐ tide mimetics of neurotrophins and their receptors. CNS Neurol. Disord. Drug Tar‐

[47] Deinhardt K, Berninghausen O, Willison HJ. et al. Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and

[48] Deinhardt K, Reversi A, Berninghausen O. et al. Neurotrophins redirect p75NTR from a clathrin-independent to a clathrin-dependent endocytic pathway coupled to axonal

[49] Deinhardt K, Salinas K, Verastigui C, Watson R. et al. Rab5 and Rab7 control endo‐ cytic sorting along the axonal retrograde transport pathway. Neuron 2006;52:

[50] Schwab M, Thoenen H. Selective trans-synaptic migration of tetanus toxin after retro‐ grade axonal transport in peripheral sympathetic nerves: a comparison with nerve

[51] Rind HB, Butowt R, von Bartheld CS. Synaptic targeting of retrogradely transported trophic factors in motoneurons: comparison of glial cell line-derived neurotrophic

independent of epsin (eosin?) 1. J. Cell Biol. 2006;174: 459–471.

imine) and tetanus toxin fragment C. J. Control Release 2010;143: 350-358.

py to motoneurons in the CNS. Neurodegener Dis. 2004;1: 101–108.

of fragment C of tetanus toxin. Neuroscience 1990;34: 251-263.

tetanus toxin. Brain Res. 1987;406: 275-279.

Acad Sci USA 1997;94: 9400–9405.

p75(NTR). Bioessays 2011;33: 614–625.

transport. Traffic 2007;8: 1736–1749.

growth factor. Brain Res. 1977;122: 459–474.

gets 2008;7: 46–62.

293-305.


[38] Manning KA, Erichsen JT, Evinger C. Retrograde transneuronal transport properties of fragment C of tetanus toxin. Neuroscience 1990;34: 251-263.

[20] Montal M. Botulinum neurotoxin. Annu. Rev. Biochem. 2010;79: 591-617.

and molecular level. Curr. Top Microbiol. Immunol. 1986;129: 93-179.

of dogs. Ann. Surgery 1938;108: 941-957.

264 Gene Therapy - Tools and Potential Applications

toxin. J. Immunol. 1951;66: 687-694.

Sci. 2003;116: 4639–4650

2000;36: 175-182.

Biol. 2003;19: 493–517.

383-388.

ijms13066883 (accessed 7 June 2012).

tem of rabbits. J. Immunol. 1953;71: 41-44.

by tetanus toxin. J Physiol. 1906;34: 315-31.

grade intraaxonal transport. Science 1975;188: 945–947.

ins block neurotransmitter release. Biochimie 2000;82:427-446.

tetanus toxin. J. Pharmacol. Exp. Ther. 1985;232: 223-227.

ity of the muscles of rats. J. Physiol. 1939; 96: 168-171.

[21] Habermann E, Dreyer F. Clostridial neurotoxins: handling and action at the cellular

[22] Chen S, Karalewitz APA, Barbieri JT. Insights into the different catalytic activities of Clostridium neurotoxins. Biochemistry 2012;Apr 24, Epub ahead of print.

[23] Firor WM, Lamont A. The apparent alteration of tetanus toxin within the spinal cord

[24] Martini E, Torda C, Zironi A. The effect of tetanus toxin on the choline esterase activ‐

[27] Ipsen, J. The effect of environmental temperature on the reaction of mice to tetanus

[28] Wright, E.A. The effect of the injection of tetanus toxin into the central nervous sys‐

[29] Roaf, M.D.; Sherrington, C.S. Experiments in examination of the locked jaw induced

[30] Lalli G, Gschmeissner S, Schiavo G. Myosin Va and microtubule-based motors are re‐ quired for fast axonal retrograde transport of tetanus toxin in motor neurons. J. Cell

[31] Price DL, Griffin J, Young A, Peck K. et al. Tetanus toxin: Direct evidence for retro‐

[32] Mochida S. Protein-protein interactions in neurotransmitter release. Neurosci. Res.

[33] Humeau Y, Doussau F, Grant NJ, Poulain B. How botulinum and tetanus neurotox‐

[34] Ungar D, Hughson FM. SNARE protein structure and function. Annu. Rev. Cell Dev.

[35] Calvo AC, Oliván S, Manzano R. et al. Fragment C of tetanus toxin: new insights into its neuronal signalling pathway. Int. J. Mol. Sci. 2012;13(6): 6883-6901. doi:10.3390/

[36] Simpson LL, Hoch DH. Neuropharmacological characterization of fragment B from

[37] Evinger C, Erichsen JT. Transsynaptic retrograde transport of fragment C of tetanus toxin demonstrated by immunohistochemical localization. Brain Res. 1986;380:

[25] Harvey AM. The peripheral action of tetanus toxin. J. Physiol. 1939;96: 348-365.

[26] Manwaring WH. Types of tetanus toxin. Cal West Med. 1943;59: 306-307.


factor, brainderived neurotrophic factor, and cardiotrophin-1 with tetanus toxin. J Neurosci. 2005;25: 539–549.

[64] Moreno-Igoa M, Calvo AC, Penas C. et al. Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy. Journal of Molecular

Gene Therapy Based on Fragment C of Tetanus Toxin in ALS: A Promising Neuroprotective Strategy for the Bench…

http://dx.doi.org/10.5772/52896

267

[65] Calvo AC, Moreno-Igoa M, Mancuso R. et al. Lack of a synergistic effect of a nonviral ALS gene therapy based on BDNF and a TTC fusion molecule. Orphanet J. Rare

[66] Ikeda K, Klinkosz B, Greene T. et al. Effects of brain-derived neurotrophic factor on motor dysfunction in wobbler mouse motor neuron disease. Ann Neurol. 1995;37:

[67] Lohof AM, Ip NY, Poo MM. Potentiation of developing neuromuscular synapses by

[68] A controlled trial of recombinant methionyl human BDNF in ALS: The BDNF Study

[69] Roux S, Saint Cloment C, Curie T. et al. Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in

[70] Ciriza J, Garcia-Ojeda M, Martín-Burriel I. et al. Antiapoptotic activity maintenance of brain derived neurotrophic factorand the C fragment of the tetanus toxin genetic

[71] Henderson CE, Phillips HS, Pollock RA. et al. GDNF: a potent survival factor for mo‐ toneurons present in peripheral nerve and muscle. Science 1994;266: 1062-1064.

[72] Oppenheim RW, Houenou LJ, Johnson JE. et al. Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 1995;373:

[73] Mohajeri MH, Figlewicz DA, Bohn MC. Intramuscular grafts of myoblasts genetical‐ ly modified to secrete glial cell line-derived neurotrophic factor prevent motoneuron loss and disease progression in a mouse model of familial amyotrophic lateral sclero‐

[74] Acsadi G, Anguelov RA, Yang H, et al. Increased survival and function of SOD1 mice after glial cell-derived neurotrophic factor gene therapy. Hum. Gene Ther. 2002;13:

[75] Wang LJ, Lu YY, Muramatsu S. et al. Neuroprotective effects of glial cell line-derived neurotrophic factor mediated by an adenoassociated virus vector in a transgenic ani‐

[76] Manabe Y, Nagano I, Gazi MS. et al. Glial cell line-derived neurotrophic factor pro‐ tein prevents motor neuron loss of transgenic model mice for amyotrophic lateral

mal model of amyotrophic lateral sclerosis. J Neurosci. 2002;22: 6920-6928.

mature mouse motor nerve terminals. Eur. J. Neurosci. 2006;24: 1546-1554.

Medicine 2010;88: 297-308.

505-511.

344-346.

1047-1059.

Dis. 2011;6: 10. http://www.ojrd.com/content/6/1/10.

Group (Phase III). Neurology 1999;52: 1427-1433.

fusion protein. Cent Eur J Biol 2008;3: 105-112.

sis. Hum. Gene Ther. 1999;10: 1853-1866.

sclerosis. Neurol. Res. 2003;25; 195-200.

the neurotrophins NT-3 and BDNF. Nature 1993;363: 350-353.


[64] Moreno-Igoa M, Calvo AC, Penas C. et al. Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy. Journal of Molecular Medicine 2010;88: 297-308.

factor, brainderived neurotrophic factor, and cardiotrophin-1 with tetanus toxin. J

[52] Aguilera J, Lopez LA, Yavin E. Tetanus toxin-induced protein kinase C activation and elevated serotonin levels in the perinatal rat brain. FEBS 1990;263: 61–65.

[53] Gil C, Ruiz-Meana M, Álava M. et al. Tetanus toxin enhances protein kinase C activi‐ ty translocation and increases polyphosphoinositide hydrolysis in rat cerebral cortex

[54] Inserte J, Najib A, Pelliccioni P. et al. Inhibition by tetanus toxin of sodium-depend‐ ent, high-affinity [3H]5-hydroxitryptamine uptake in rat synaptosomes. Biochem.

[55] Gil C, Chaib I, Pelliccioni P, Aguilera J. Activation of signal transduction pathways involving TrkA, PLCγ-1, PKC isoforms and ERK-1/2 by tetanus toxin. FEBS Lett.

[56] Pelliccioni P, Gil C, Najib A. et al. Tetanus toxin modulates serotonin transport in rat-

[57] Gil C, Chaib-Oukadour I, Blasi J, Aguilera J. HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphopro‐

[58] Gil, C.; Chaib-Oukadour, I.; Aguilera, J. C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent

[59] Chaib-Oukadour I, Gil C, Aguilera J. The C-terminal domain of heavy chain of teta‐ nus toxin rescues cerebellar granule neurons from apoptotic death: Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. J.

[60] Mendieta L, Venegas B, Moreno N. et al. The carboxyl-terminal domain of the heavy chain of tetanus toxin prevents dopaminergic degeneration and improves motor be‐

[61] Chaib-Oukadour I, Gil C, Rodríguez-Álvarez J. et al. Tetanus toxin HC fragment re‐

[62] Longstreth WTJr, Meschke JS, Davidson SK. et al. Hypothesis: a motor neuron toxin produced by a clostridial species residing in gut causes ALS. Med Hypotheses

[63] Larsen KE, Benn SC, Ay I. et al. A glial cell line-derived neurotrophic factor (GDNF): tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal

haviour in rats with striatal MPP+-lesions. Neurosci. Res. 2009;65: 98–106.

duces neuronal MPP+ toxicity. Mol. Cell. Neurosci. 2009;41: 297–303.

cord motor neurons in mice. Brain Res. 2006;1120: 1–12.

brain neuronal cultures. J. Mol. Neurosci. 2001;17: 303–310.

teins involved in signal transduction. Biochem. J. 2001;356: 97–103.

manner in cultured cortical neurons. Biochem. J. 2003;373: 613–620.

Neurosci. 2005;25: 539–549.

266 Gene Therapy - Tools and Potential Applications

Pharmacol. 1999;57: 111–120.

Neurochem. 2004;90: 1227–1236.

2005;64: 1153–1156.

2000;481: 177–182.

preparations. J. Neurochem. 1998;70: 1636–1643.


[77] Ciriza J, Moreno-Igoa M, Calvo AC. et al. A genetic fusion GDNF-C fragment of teta‐ nus toxin prolongs survival in a symptomatic mouse ALS model. Restorative Neurol‐ ogy and Neuroscience 2008;26: 459-465.

**Chapter 11**

**Transposons for Non-Viral Gene Transfer**

DNA based transposon vectors offer a mechanism for non-viral gene delivery into mamma‐ lian and human cells. These vectors work via a cut-and-paste mechanim whereby transpo‐ son DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a transposase enzyme. The first DNA based transposon system which worked efficienty in human cells was *sleeping beauty*. This was followed a few years later by the use of the *piggy‐ Bac* transposon system in mammalian and human cells. The advantages of transposon vec‐ tors include lower cost, less innate immunogenicity, and the ability to easily co-deliver multiple genes when compared to viral vectors. However, when compared to viral vectors, non-viral transposon systems are limited by delivery to cells, they are possibly still immuno‐ genic, and they can be less efficient depending on the cell type of interest. Nonetheless, transposons have shown promise in genetic modification of clinical grade cell types such as human T lymphocytes, induced pluripotent stem cells, and stem cells. Recently generated hyperactive transposon elements have improved gene delivery to levels similar to that ob‐ tained with viral vectors. In addition, current research is focused on manipulating transpo‐ son systems to achieve user-selected and site-directed genomic integration of transposon DNA cargo to improve safety and efficacy of transgene delivery. DNA based transposon systems represent a powerful tool for gene therapy and genome engineering applications.

Transposons or mobile genetic elements were first described by Barbara McClintock as "jumping genes" responsible for mosaicism in maize [1]. Transposons are found in the ge‐ nome of all eukaryotes and in humans at least 45% of the genome is derived from such ele‐

> © 2013 Saha and Wilson; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Sunandan Saha and Matthew H. Wilson

http://dx.doi.org/10.5772/52527

**1. Introduction**

Additional information is available at the end of the chapter

**2. Transposons as gene delivery systems**


**Chapter 11**

### **Transposons for Non-Viral Gene Transfer**

Sunandan Saha and Matthew H. Wilson

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52527

#### **1. Introduction**

[77] Ciriza J, Moreno-Igoa M, Calvo AC. et al. A genetic fusion GDNF-C fragment of teta‐ nus toxin prolongs survival in a symptomatic mouse ALS model. Restorative Neurol‐

[78] Chian RJ, Li J, Ay I. et al. IGF-1: Tetanus toxin fragment C fusion protein improves delivery of IGF-1 to spinal cord but fails to prolong survival of ALS mice. Brain Re‐

[79] Moreno-Igoa M, Calvo AC, Ciriza J. et al. Non-viral gene delivery of the GDNF, ei‐ ther alone or fused to the C-fragment of tetanus toxin protein, prolongs survival in a

mouse ALS model. Restor. Neurol. Neurosci. 2012;30: 69-80.

ogy and Neuroscience 2008;26: 459-465.

search 2009;1287: 1-19.

268 Gene Therapy - Tools and Potential Applications

DNA based transposon vectors offer a mechanism for non-viral gene delivery into mamma‐ lian and human cells. These vectors work via a cut-and-paste mechanim whereby transpo‐ son DNA containing a transgene(s) of interest is integrated into chromosomal DNA by a transposase enzyme. The first DNA based transposon system which worked efficienty in human cells was *sleeping beauty*. This was followed a few years later by the use of the *piggy‐ Bac* transposon system in mammalian and human cells. The advantages of transposon vec‐ tors include lower cost, less innate immunogenicity, and the ability to easily co-deliver multiple genes when compared to viral vectors. However, when compared to viral vectors, non-viral transposon systems are limited by delivery to cells, they are possibly still immuno‐ genic, and they can be less efficient depending on the cell type of interest. Nonetheless, transposons have shown promise in genetic modification of clinical grade cell types such as human T lymphocytes, induced pluripotent stem cells, and stem cells. Recently generated hyperactive transposon elements have improved gene delivery to levels similar to that ob‐ tained with viral vectors. In addition, current research is focused on manipulating transpo‐ son systems to achieve user-selected and site-directed genomic integration of transposon DNA cargo to improve safety and efficacy of transgene delivery. DNA based transposon systems represent a powerful tool for gene therapy and genome engineering applications.

#### **2. Transposons as gene delivery systems**

Transposons or mobile genetic elements were first described by Barbara McClintock as "jumping genes" responsible for mosaicism in maize [1]. Transposons are found in the ge‐ nome of all eukaryotes and in humans at least 45% of the genome is derived from such ele‐

© 2013 Saha and Wilson; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ments [2]. Transposons active in eukaryotes can work either by a "copy and paste" (Class I) or "cut and paste" (Class II) mechanism (Figure 1).

nor site without leaving behind any footprints [5], making it an attractive feature for cellular reprogramming. Excision of the transposon from the donor site, creates complimentary TTAA overhangs which undergo simple ligation to regenerate the donor site bypassing

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 271

In "cis" delivery the transposase is carried by the same plasmid backbone as the transposn. In "trans" delivery it is delivered by a separate circular plasmid. For gene therapy purposes transposase and transposon are delivered either in "cis" or in "trans" (Figure 2). In "cis" de‐ livery the transposase is carried on the same vector backbone as the transposon carrying the gene of interest (GOI). In the "trans" configuration, the transposase is delivered by a sepa‐ rate non integrating plasmid. The "cis" configuration has been shown to improve transposi‐ tion efficiency [7], but there is a question of whether the linearized backbone carrying the transposase may also get integrated and lead to residual transposase expression. A compari‐

**Figure 2.** "Cis" and "Trans" transposon mediated gene delivery. GOI, gene of interest; 5'TR, 5' terminal repeat; 3'TR, 3'

In spite of viral vectors having been successfully used in gene therapy clinical trials (e.g. generation of clinical grade T cells for immunotherapy [8], their use in extensive gene thera‐ py regimens is constrained. Clinical grade viral vectors are very expensive to manufacture given the stringent regulatory oversight and limited number of GMP certified production fa‐

terminal repeat; the yellow and beige arrows indicate promoters to drive gene expression.

**3. Advantages of transposon as gene delivery system**

**3.1. Lower cost compared to viral vectors**

son of the properties of *sleeping beauty* and *piggyBac* is described in Table 1.

DNA synthesis during transposition [6].

In the "copy and paste" mechanism, the transposon first makes a copy of itself via an RNA in‐ termediate (hence also known as retrotransposons).Class II DNA-transposons work by a "cut and paste" mechanism in which the transposon is excised by the transposase upon expression and then relocates to a new locus by creating double strand breaks *in situ*. Most transposon sys‐ tems used for gene delivery use a modified "cut and paste" system consisting of a transposon carrying the transgene of interest and a helper plasmid expressing the transposase (Figure 2). The "cut and paste" transposition mechanism involves recognition of the inverted terminal re‐ peat sequences (IRs) by the transposase and excision of the transposon from the donor loci, usually a supplied plasmid. The two most commonly used transposon system for genetic mod‐ ification of mammalian and human cells are *sleeping beauty* and *piggyBac*.

**Figure 1.** Class I and II transposons and mechanisms of integration.

The *sleeping beauty* (SB) transposon was reconstructed from the genome of salmonid fish us‐ ing molecular phylogenetic data [3] and belongs to the Tc1/mariner superfamily of transpo‐ sons. The sleeping beauty transposon is flanked by 230bp IRs which conatin within them non identical direct repeats (DRs).

The *piggyBac* transposon was isolated from cabbage looper moth *Trichoplusia ni*[4].One de‐ sirable feature of the *piggyBac* system is the precise excision of the transposon from the do‐ nor site without leaving behind any footprints [5], making it an attractive feature for cellular reprogramming. Excision of the transposon from the donor site, creates complimentary TTAA overhangs which undergo simple ligation to regenerate the donor site bypassing DNA synthesis during transposition [6].

In "cis" delivery the transposase is carried by the same plasmid backbone as the transposn. In "trans" delivery it is delivered by a separate circular plasmid. For gene therapy purposes transposase and transposon are delivered either in "cis" or in "trans" (Figure 2). In "cis" de‐ livery the transposase is carried on the same vector backbone as the transposon carrying the gene of interest (GOI). In the "trans" configuration, the transposase is delivered by a sepa‐ rate non integrating plasmid. The "cis" configuration has been shown to improve transposi‐ tion efficiency [7], but there is a question of whether the linearized backbone carrying the transposase may also get integrated and lead to residual transposase expression. A compari‐ son of the properties of *sleeping beauty* and *piggyBac* is described in Table 1.

**Figure 2.** "Cis" and "Trans" transposon mediated gene delivery. GOI, gene of interest; 5'TR, 5' terminal repeat; 3'TR, 3' terminal repeat; the yellow and beige arrows indicate promoters to drive gene expression.

#### **3. Advantages of transposon as gene delivery system**

#### **3.1. Lower cost compared to viral vectors**

ments [2]. Transposons active in eukaryotes can work either by a "copy and paste" (Class I)

In the "copy and paste" mechanism, the transposon first makes a copy of itself via an RNA in‐ termediate (hence also known as retrotransposons).Class II DNA-transposons work by a "cut and paste" mechanism in which the transposon is excised by the transposase upon expression and then relocates to a new locus by creating double strand breaks *in situ*. Most transposon sys‐ tems used for gene delivery use a modified "cut and paste" system consisting of a transposon carrying the transgene of interest and a helper plasmid expressing the transposase (Figure 2). The "cut and paste" transposition mechanism involves recognition of the inverted terminal re‐ peat sequences (IRs) by the transposase and excision of the transposon from the donor loci, usually a supplied plasmid. The two most commonly used transposon system for genetic mod‐

ification of mammalian and human cells are *sleeping beauty* and *piggyBac*.

or "cut and paste" (Class II) mechanism (Figure 1).

270 Gene Therapy - Tools and Potential Applications

**Figure 1.** Class I and II transposons and mechanisms of integration.

non identical direct repeats (DRs).

The *sleeping beauty* (SB) transposon was reconstructed from the genome of salmonid fish us‐ ing molecular phylogenetic data [3] and belongs to the Tc1/mariner superfamily of transpo‐ sons. The sleeping beauty transposon is flanked by 230bp IRs which conatin within them

The *piggyBac* transposon was isolated from cabbage looper moth *Trichoplusia ni*[4].One de‐ sirable feature of the *piggyBac* system is the precise excision of the transposon from the do‐ In spite of viral vectors having been successfully used in gene therapy clinical trials (e.g. generation of clinical grade T cells for immunotherapy [8], their use in extensive gene thera‐ py regimens is constrained. Clinical grade viral vectors are very expensive to manufacture given the stringent regulatory oversight and limited number of GMP certified production fa‐ cilities. A batch of clinical grade retroviral supernatant for treating patients costs between \$400,000 to \$500,000 (personal communciation, GMP facility director, Baylor College of Medicine). The production of clinical GMP (cGMP) grade viral supernatant is extremely time intensive as, in addition to optimization of culture conditions, the supernatant needs extensive testing for microbial contamination, presence of replication competent viral parti‐ cles as well as validation of sequence and functionality. The entire production run and asso‐ ciated testing may require up to six months. These viral stocks also have limited shelf life. Upon release the desired cell type is transduced, selected and expanded which is then fol‐ lowed by quality assurance checks. This also requires extensive training of the personnel in‐ volved in production and testing and scaling up production as would be required for future gene therapy regimens will not be economical. In contrast, cGMP grade transposon plas‐ mids can be manufactured more quickly. The production can be scaled up quickly and exist‐ ing facilities can be upgraded and certified in a shorter time frame. The cost of manufacturing and release of cGMP grade plasmid DNA is between \$20,000 and \$ 40,000 [9]. The use of transposons drastically reduces both the time and cost of production of the gene delivery system. In the first clinical trial approved by the FDA for infusion of autolo‐ gous *ex vivo sleeping beauty* modified T cells [10], the most time intensive step was the test for fungal and bacterial contamination (14 days).

ly utilized to modify primary human lymphocytes with 15 kb transposon with an initial transfection efficiency of 20% which increased up to 90% upon selection and expansion [15]. The *piggyBac* system has been successfully used for mobilizing transposons as large as 100 kb in mouse embryonic stem (ES) cells [16]. An increased cargo capacity also imparts the ability to deliver multiple transgenes to the same cell. For example, using the *piggyBac* sys‐ tem, human cells were efficiently modified to express a three subunit functional sodium channel which retained its electro-physiological properties even after 35 passages [17].

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 273

One of the major concerns for viral gene delivery system is the associated immunogenicity as evidenced by the death of a patient receiving liver targeted adenoviral gene therapy for partial ornithine transcarbamylase deficiency in 1999 [13].The systemic delivery of the viral particles initiated a cytokine storm leading to multiple organ failure within four days of ad‐ ministration of the vector [18]. Attempts have been made to reduce the immunogenicity of viral vectors by stripping them of all endogenous viral genes ('gutted' or 'helper-dependent' vectors) [19], but even the use of modified viral delivery systems are potentially immuno‐ genic as evidenced by long term inflammation of rat brains injected with replication defi‐

Transposons are circular plasmid DNA molecules and do not contain a viral shell or viral anti‐ gens. The host response to non-viral vectors has not been well characterized. Toll-like receptor (TLR)-9 is known to recognize DNA with unmethylated CpG dinucleotides in the endosome‐ which can lead to signalling via MyD88 and production of inflammatory mediators such as TNF and IFN-α [21]. Other mechanisms of innate immune sensing of naked DNA include DNA-dependent activator of interferon (IFN)-regulatory factors (DAI) (also called Z-DNAbinding protein 1, ZBP1), RNA polymerase III (Pol III), absent in melanoma 2 (AIM2), leucinerich repeat (in Flightless I) interacting protein-1 (Lrrfip1), DExD/H box helicases (DHX9 and DHX36), and most recently, the IFN-inducible protein IFI16 [22]. These molecules use inde‐ pendent and sometimes overlapping signalling pathways to elicit immune response to deliv‐ ered DNA. Nonetheless, much remains to be discovered about host immune response to

delivered DNA and how to overcome such an obstacle for effective gene therapy.

Human immunodeficiency virus (HIV) has been shown to prefer genes for integration in SupT1 and Jurkat cells [23]. Murine leukemia virus (MLV) derived vectors have been used for stable gene transfer for therapy but they have been shown to prefer transcriptional start sites (TSS) for integration [24]. Integrations near the promoter of the LMO2 proto-oncogene has been associated with leukemia in the French X-SCID gene therapy trial [25]. The genome wide mapping of *sleeping beauty* transposons in mammals have revealed a modest bias to‐ wards transcriptional units and upstream regulatory sequences which varies between cell types [26]. The integration site profiling of both *piggyBac* in primary human cells and cell lines have revealed no preferred chromosomal hotspots [7,27]. It also has no preference for genomic repeat elements and known proto-oncogenes. *PiggyBac* has a preference for inte‐

**3.3. Less immunogenicity**

cient adenoviral vectors [20].

**3.4. Less propensity for oncogenic mutations**


**Table 1.** Comparison of *sleeping beauty* and *piggyBac*properties. TSS, transcriptional start sites.

#### **3.2. Delivery of large and multiple transgenes**

Although retroviral and lentiviral vectors have been successfully used for delivering multi‐ ple transgenes, they are limited by their cargo capacity[11,12]. Both these vector systems can carry a limited cargo of up to 8kb which is limited by the packaging capacity of their capsid envelop [13]. Early reports demontrated the *sleeping beauty* system to have reduced efficien‐ cy beyond transposon size of 10kb [14]. In contrast the *piggyBac* system has been successful‐ ly utilized to modify primary human lymphocytes with 15 kb transposon with an initial transfection efficiency of 20% which increased up to 90% upon selection and expansion [15]. The *piggyBac* system has been successfully used for mobilizing transposons as large as 100 kb in mouse embryonic stem (ES) cells [16]. An increased cargo capacity also imparts the ability to deliver multiple transgenes to the same cell. For example, using the *piggyBac* sys‐ tem, human cells were efficiently modified to express a three subunit functional sodium channel which retained its electro-physiological properties even after 35 passages [17].

#### **3.3. Less immunogenicity**

cilities. A batch of clinical grade retroviral supernatant for treating patients costs between \$400,000 to \$500,000 (personal communciation, GMP facility director, Baylor College of Medicine). The production of clinical GMP (cGMP) grade viral supernatant is extremely time intensive as, in addition to optimization of culture conditions, the supernatant needs extensive testing for microbial contamination, presence of replication competent viral parti‐ cles as well as validation of sequence and functionality. The entire production run and asso‐ ciated testing may require up to six months. These viral stocks also have limited shelf life. Upon release the desired cell type is transduced, selected and expanded which is then fol‐ lowed by quality assurance checks. This also requires extensive training of the personnel in‐ volved in production and testing and scaling up production as would be required for future gene therapy regimens will not be economical. In contrast, cGMP grade transposon plas‐ mids can be manufactured more quickly. The production can be scaled up quickly and exist‐ ing facilities can be upgraded and certified in a shorter time frame. The cost of manufacturing and release of cGMP grade plasmid DNA is between \$20,000 and \$ 40,000 [9]. The use of transposons drastically reduces both the time and cost of production of the gene delivery system. In the first clinical trial approved by the FDA for infusion of autolo‐ gous *ex vivo sleeping beauty* modified T cells [10], the most time intensive step was the test for

**Cargo Capacity** ~10 kb >100 kb

**Needs titration for optimal activity** Yes Yes

**Can be engineerd to bias integration sites** Yes Yes

**Table 1.** Comparison of *sleeping beauty* and *piggyBac*properties. TSS, transcriptional start sites.

**Integration site preference** More random Slight increased preference

Although retroviral and lentiviral vectors have been successfully used for delivering multi‐ ple transgenes, they are limited by their cargo capacity[11,12]. Both these vector systems can carry a limited cargo of up to 8kb which is limited by the packaging capacity of their capsid envelop [13]. Early reports demontrated the *sleeping beauty* system to have reduced efficien‐ cy beyond transposon size of 10kb [14]. In contrast the *piggyBac* system has been successful‐

**Foot Print** Insertion site mutated upon

**Hyper Active Versions** SB100X (most active SB

**Effect of 'N' and 'C' terminal modifications** 50% or more reduction in

*sleeping beauty piggyBac*

No "foot print" mutation

hyPBase

No apparent reduction in efficiency

for genes and TSS

excision

version)

efficacy

fungal and bacterial contamination (14 days).

272 Gene Therapy - Tools and Potential Applications

**3.2. Delivery of large and multiple transgenes**

One of the major concerns for viral gene delivery system is the associated immunogenicity as evidenced by the death of a patient receiving liver targeted adenoviral gene therapy for partial ornithine transcarbamylase deficiency in 1999 [13].The systemic delivery of the viral particles initiated a cytokine storm leading to multiple organ failure within four days of ad‐ ministration of the vector [18]. Attempts have been made to reduce the immunogenicity of viral vectors by stripping them of all endogenous viral genes ('gutted' or 'helper-dependent' vectors) [19], but even the use of modified viral delivery systems are potentially immuno‐ genic as evidenced by long term inflammation of rat brains injected with replication defi‐ cient adenoviral vectors [20].

Transposons are circular plasmid DNA molecules and do not contain a viral shell or viral anti‐ gens. The host response to non-viral vectors has not been well characterized. Toll-like receptor (TLR)-9 is known to recognize DNA with unmethylated CpG dinucleotides in the endosome‐ which can lead to signalling via MyD88 and production of inflammatory mediators such as TNF and IFN-α [21]. Other mechanisms of innate immune sensing of naked DNA include DNA-dependent activator of interferon (IFN)-regulatory factors (DAI) (also called Z-DNAbinding protein 1, ZBP1), RNA polymerase III (Pol III), absent in melanoma 2 (AIM2), leucinerich repeat (in Flightless I) interacting protein-1 (Lrrfip1), DExD/H box helicases (DHX9 and DHX36), and most recently, the IFN-inducible protein IFI16 [22]. These molecules use inde‐ pendent and sometimes overlapping signalling pathways to elicit immune response to deliv‐ ered DNA. Nonetheless, much remains to be discovered about host immune response to delivered DNA and how to overcome such an obstacle for effective gene therapy.

#### **3.4. Less propensity for oncogenic mutations**

Human immunodeficiency virus (HIV) has been shown to prefer genes for integration in SupT1 and Jurkat cells [23]. Murine leukemia virus (MLV) derived vectors have been used for stable gene transfer for therapy but they have been shown to prefer transcriptional start sites (TSS) for integration [24]. Integrations near the promoter of the LMO2 proto-oncogene has been associated with leukemia in the French X-SCID gene therapy trial [25]. The genome wide mapping of *sleeping beauty* transposons in mammals have revealed a modest bias to‐ wards transcriptional units and upstream regulatory sequences which varies between cell types [26]. The integration site profiling of both *piggyBac* in primary human cells and cell lines have revealed no preferred chromosomal hotspots [7,27]. It also has no preference for genomic repeat elements and known proto-oncogenes. *PiggyBac* has a preference for inte‐ grating into RefSeq genes and near TSS and CpG enriched motifs although this may be in‐ fluenced by the state of the cell or type of the cell. Both *sleeping beauty* and *piggyBac* are being engineered for site-directed gene delivery to improve the safety of gene transfer. True geno‐ toxic risk for viral vectors was not discovered until they were used in humans. Transposons have not yet been used in humans, though one clinical trial has be approved.

**Disease Transposon system Reference Hemophilia B** SB [34,36] **Hemophilia A** SB [37,38] **Tyrosinemia Type I** SB [39] **JunctionalEpidermolysisBullosa** SB [40] **Diabetes** SB [41] **Huntington's disease** SB [42] **Mucopolysaccharidosis I & VII** SB [43,44] **α1-antitrypsin deficiency** PB [45]

**Table 2.** List of diseases corrected with *Sleeping Beauty* (SB) and *piggyBac* (PB)

Peripheral blood and umbilical cord T cells have been extensively modified with both viral and non-viral gene delivery systems for immunotherapeutic purposes [10]. This therapeutic avenue has been successfully used for the treatment of viral infections and Epstein Barr vi‐ rus (EBV) associated lymphoma post autologous bone marrow transplantation [46,47]. They also hold promise for treatment of other cancers [48-50]. But the use of of viral vectors for the generation of clinical grade T cells is expensive, time intensive and not free of risks. Nonviral gene delivery systems, including DNA transposons, are being increasingly explored as

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 275

A schematic of how primary human T lymphocytes can be gene modified with transposons is shown in Figure 3. The *sleeping beauty* system was used to successfully modify peripheral blood mononuclear cells with a CD19-specific chimeric antigen receptor (CAR)[9]. These modified PBMCs were then used to generate CAR+ T cells which preserved their CD4+, CD8+, central memory and effector-effector cell phenotypes. The *piggyBac* system has also

**5.1. Genetic modification of human T lymphocytes**

**Figure 3.** Schematic of transposon modificaiton of primary human T cells.

an alternative strategy.

#### **4. Challenges of transposon as gene delivery system**

Given the promise of transposons as gene delivery vehicle, it suffers from certain challenges e.g. reduced delivery, random integration profile and silencing of the integrated transgene.

#### **4.1. Low delivery efficiency**

Transposon systems are carried by naked DNA plasmids and there efficiency is limited to the efficiency of getting the plasmid into to the cell by chemical or physical means. Certain pri‐ mary cells and cell lines are easy to transfect (e.g. HEK293, HeLa, Hepatocytes) and transpo‐ sons have high transposition efficiency in these cells. But other clinically relevant cells (e.g. primary lymphocytes) are difficult to transfect. Often the method used for transfection (e.g. nucleofection and electroporation) is toxic to the cells and leads to excessive cell death thus re‐ ducing the efficiency of stable transfection. Efforts are on to circumvent these difficulties by de‐ veloping novel delivery methods e.g. cell-penetrating peptides (CPP) –*piggyBac* fusions [28] or using polyethylenimine [29]. Some investigators have encapsulated transposon systems with‐ in viruses to use the virus to deliver the DNA from which transposition occurs [30-34] This may improve efficiency, however, the issues with immunogenicity of viruses remain.

#### **4.2. Random integration profile**

Transposons as described above have uncontrolled or relatively random integration prefer‐ ence with regards to genomic elements. This leaves the transposed transgene open to influ‐ ence of the neighboring genomic region. Additional, uncontrolled or not site-directed integration increases the risk for possible genotoxicity.

#### **4.3. Silencing of the integrated transgene**

Gene silencing has been observed when using *sleeping beauty* in cultured cells [35]. Transgene silencing and epigenetic transgene modification has not been well studied with *piggyBac*.

#### **5. Applications**

Both *sleeping beauty* and *piggyBac* have demonstrated correct of disease phenotypes in ani‐ mal models or in human cells (Table 2).


**Table 2.** List of diseases corrected with *Sleeping Beauty* (SB) and *piggyBac* (PB)

#### **5.1. Genetic modification of human T lymphocytes**

grating into RefSeq genes and near TSS and CpG enriched motifs although this may be in‐ fluenced by the state of the cell or type of the cell. Both *sleeping beauty* and *piggyBac* are being engineered for site-directed gene delivery to improve the safety of gene transfer. True geno‐ toxic risk for viral vectors was not discovered until they were used in humans. Transposons

Given the promise of transposons as gene delivery vehicle, it suffers from certain challenges e.g. reduced delivery, random integration profile and silencing of the integrated transgene.

Transposon systems are carried by naked DNA plasmids and there efficiency is limited to the efficiency of getting the plasmid into to the cell by chemical or physical means. Certain pri‐ mary cells and cell lines are easy to transfect (e.g. HEK293, HeLa, Hepatocytes) and transpo‐ sons have high transposition efficiency in these cells. But other clinically relevant cells (e.g. primary lymphocytes) are difficult to transfect. Often the method used for transfection (e.g. nucleofection and electroporation) is toxic to the cells and leads to excessive cell death thus re‐ ducing the efficiency of stable transfection. Efforts are on to circumvent these difficulties by de‐ veloping novel delivery methods e.g. cell-penetrating peptides (CPP) –*piggyBac* fusions [28] or using polyethylenimine [29]. Some investigators have encapsulated transposon systems with‐ in viruses to use the virus to deliver the DNA from which transposition occurs [30-34] This may

Transposons as described above have uncontrolled or relatively random integration prefer‐ ence with regards to genomic elements. This leaves the transposed transgene open to influ‐ ence of the neighboring genomic region. Additional, uncontrolled or not site-directed

Gene silencing has been observed when using *sleeping beauty* in cultured cells [35]. Transgene silencing and epigenetic transgene modification has not been well studied with *piggyBac*.

Both *sleeping beauty* and *piggyBac* have demonstrated correct of disease phenotypes in ani‐

improve efficiency, however, the issues with immunogenicity of viruses remain.

have not yet been used in humans, though one clinical trial has be approved.

**4. Challenges of transposon as gene delivery system**

**4.1. Low delivery efficiency**

274 Gene Therapy - Tools and Potential Applications

**4.2. Random integration profile**

**5. Applications**

integration increases the risk for possible genotoxicity.

**4.3. Silencing of the integrated transgene**

mal models or in human cells (Table 2).

Peripheral blood and umbilical cord T cells have been extensively modified with both viral and non-viral gene delivery systems for immunotherapeutic purposes [10]. This therapeutic avenue has been successfully used for the treatment of viral infections and Epstein Barr vi‐ rus (EBV) associated lymphoma post autologous bone marrow transplantation [46,47]. They also hold promise for treatment of other cancers [48-50]. But the use of of viral vectors for the generation of clinical grade T cells is expensive, time intensive and not free of risks. Nonviral gene delivery systems, including DNA transposons, are being increasingly explored as an alternative strategy.

**Figure 3.** Schematic of transposon modificaiton of primary human T cells.

A schematic of how primary human T lymphocytes can be gene modified with transposons is shown in Figure 3. The *sleeping beauty* system was used to successfully modify peripheral blood mononuclear cells with a CD19-specific chimeric antigen receptor (CAR)[9]. These modified PBMCs were then used to generate CAR+ T cells which preserved their CD4+, CD8+, central memory and effector-effector cell phenotypes. The *piggyBac* system has also been optimized to achieve stable transgene expression in human T lymphocytes [51]. Fur‐ ther, primary lymphocytes have been modified with multiple transgenes to redirect their specificity for CD19 and make them resistant to off target effects of chemotherapeutic drugs like rapamycin [15]. Cytotoxic T lymphocytes specific for Epstein Barr Virus (EBV) have also been successfully modified with human epidermal growth factor receptor-2 specific CAR (HER2-CAR)[52]. The first clinical trial involving transposon modified autologous T cells with a second generation CD19-specific CAR has been approved by the Food and Drug Ad‐ ministration[10]. This trial will involve the infusion of *ex vivo* expanded autologous T cells in patients undergoing autologous hematopoietic stem cell (HSC) transplantation with high risk of relapsed B-cell malignancies.

The *piggyBac* system seems to be ideally suited for this as it can undergo precise excision and does not leave behind "foot print" mutations [5]. In contrast, the *sleeping beauty* system has been shown to excise imprecisely leaving behind altered insertion sites [3]. The *piggyBac* system has been successfully used to generate transgene free iPSCs from both mouse and human embryonic fibroblasts with efficiency comparable to retroviral vectors [59-60]. *Piggy‐ Bac* has also been used to successfully reprogram murine tail tip fibroblasts into fully differ‐ entiated melanocytes which are more compatible with cell therapy regimens [61]. The use of a *piggyBac* based inducible reprogramming system also proved to be more stable and quick‐

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 277

Transposons have been used for genetic modification of human embryonic stem cells [63]. More recently, transposons have been used to insert bacterial artificial chromosomes (BACs) in human ES cells [64]. Both *sleeping beauty* and *piggyBac* have been used to genetically modi‐ fy hematopoietic stem cells [65]. Transposons provide an effective mechanism for perma‐ nent (or reversible in the case of *piggyBac*) genetic modification of a variety of stem cell types

SB100X and native *piggyBac* both have similar activity levels in human cells which is 100 fold more than the native *sleeping beauty*. The hyperactive *piggyBac* transposase (hyPBase) has

been shown to have 2 to 3 fold more activity than SB100X or native PB [66] (Figure 5).

er than an inducible lentiviral system [62].

**6. Current hot topics and future directions**

**6.1. Generation of hyperactive transposon elements**

**Figure 5.** Comparison of transposase activity in human cells

**5.3. Genetic modification of stem cells**

for eventual use in therapy.

#### **5.2. Generation of induced pluripotent stem cells**

Induced pluripotent stem cells (iPSCs) generated from a patient's own differentiated somat‐ ic cells holds promise for regenerative medicine. Early successful attempts involved delivery of defined reprogramming factors using retroviral vectors [11,53]. Unfortunately 20% of the chimeric offspring obtained from germline transmission of retrovirally reprogrammed clones developed tumors due to reactivation of the c-myc oncogene [54]. In addition, ectopic expression of the reprogramming factor(s) has been linked to tumors and skin dysplasia [55-56]. One way to circumvent the use of viral delivery systems is to deliver the program‐ ming factors as recombinant proteins [57] or by repeated plasmid transfections [58], both of which have proven to be extremely slow and inefficient. The higher gene delivery efficiency of transposons together with their ability of being excised from the cells post reprogram‐ ming and differentiation make them an attractive choice for generating iPSCs.

Somatic cells have been transfected with *piggyBac* transposons carrying reprogramming fac‐ tors and transposase. Reprogrammed iPSCs are selected and propagated to obtain individu‐ al iPSC clones. To generate transgene-free iPSCs, the transposase is re-expressed to remove the reprogramming factors followed by negative selection to identify transgene-free iPSCs (Figure 4).

**Figure 4.** Generation of transgene-free iPSCs using the *piggyBac* system.

The *piggyBac* system seems to be ideally suited for this as it can undergo precise excision and does not leave behind "foot print" mutations [5]. In contrast, the *sleeping beauty* system has been shown to excise imprecisely leaving behind altered insertion sites [3]. The *piggyBac* system has been successfully used to generate transgene free iPSCs from both mouse and human embryonic fibroblasts with efficiency comparable to retroviral vectors [59-60]. *Piggy‐ Bac* has also been used to successfully reprogram murine tail tip fibroblasts into fully differ‐ entiated melanocytes which are more compatible with cell therapy regimens [61]. The use of a *piggyBac* based inducible reprogramming system also proved to be more stable and quick‐ er than an inducible lentiviral system [62].

#### **5.3. Genetic modification of stem cells**

been optimized to achieve stable transgene expression in human T lymphocytes [51]. Fur‐ ther, primary lymphocytes have been modified with multiple transgenes to redirect their specificity for CD19 and make them resistant to off target effects of chemotherapeutic drugs like rapamycin [15]. Cytotoxic T lymphocytes specific for Epstein Barr Virus (EBV) have also been successfully modified with human epidermal growth factor receptor-2 specific CAR (HER2-CAR)[52]. The first clinical trial involving transposon modified autologous T cells with a second generation CD19-specific CAR has been approved by the Food and Drug Ad‐ ministration[10]. This trial will involve the infusion of *ex vivo* expanded autologous T cells in patients undergoing autologous hematopoietic stem cell (HSC) transplantation with high

Induced pluripotent stem cells (iPSCs) generated from a patient's own differentiated somat‐ ic cells holds promise for regenerative medicine. Early successful attempts involved delivery of defined reprogramming factors using retroviral vectors [11,53]. Unfortunately 20% of the chimeric offspring obtained from germline transmission of retrovirally reprogrammed clones developed tumors due to reactivation of the c-myc oncogene [54]. In addition, ectopic expression of the reprogramming factor(s) has been linked to tumors and skin dysplasia [55-56]. One way to circumvent the use of viral delivery systems is to deliver the program‐ ming factors as recombinant proteins [57] or by repeated plasmid transfections [58], both of which have proven to be extremely slow and inefficient. The higher gene delivery efficiency of transposons together with their ability of being excised from the cells post reprogram‐

Somatic cells have been transfected with *piggyBac* transposons carrying reprogramming fac‐ tors and transposase. Reprogrammed iPSCs are selected and propagated to obtain individu‐ al iPSC clones. To generate transgene-free iPSCs, the transposase is re-expressed to remove the reprogramming factors followed by negative selection to identify transgene-free iPSCs

ming and differentiation make them an attractive choice for generating iPSCs.

risk of relapsed B-cell malignancies.

276 Gene Therapy - Tools and Potential Applications

(Figure 4).

**5.2. Generation of induced pluripotent stem cells**

**Figure 4.** Generation of transgene-free iPSCs using the *piggyBac* system.

Transposons have been used for genetic modification of human embryonic stem cells [63]. More recently, transposons have been used to insert bacterial artificial chromosomes (BACs) in human ES cells [64]. Both *sleeping beauty* and *piggyBac* have been used to genetically modi‐ fy hematopoietic stem cells [65]. Transposons provide an effective mechanism for perma‐ nent (or reversible in the case of *piggyBac*) genetic modification of a variety of stem cell types for eventual use in therapy.

#### **6. Current hot topics and future directions**

#### **6.1. Generation of hyperactive transposon elements**

SB100X and native *piggyBac* both have similar activity levels in human cells which is 100 fold more than the native *sleeping beauty*. The hyperactive *piggyBac* transposase (hyPBase) has been shown to have 2 to 3 fold more activity than SB100X or native PB [66] (Figure 5).

**Figure 5.** Comparison of transposase activity in human cells

Efficiency of transposition is perceived as a bottleneck to efficient gene delivery. Attempts to engineer hyperactive versions of transposase have resulted in versions with increasing transposition activity. Strategies employed include import of amino acids from related transposases [67], alanine scanning [68] and site directed mutagenesis [69]. The construction of the SB100X transposase with ~100 folds higher activity than the original *sleeping beauty* transposase employed a high throughput screen of mutant transposases obtained from DNA shuffling [70]. A hyperactive version of the *piggyBac* transposase (hyPBase) has also been engineered with 17-fold increase in excision and 9-fold increase in integration [71]. The hyPBase has 7 amino acid substitution identified from a screen of PBase mutants but none of the 7 substitutions are in the catalytic domain of the transposase. The hyPBase also has foot‐ print mutation frequency (<5%) comparable to the wild type transposase and no apparent effect on genomic integrity. Unlike SB100X which showed a 50% reduction, the addition of a 24 kDa ZFN tag did not significantly alter transposition efficiency [66]. In vivo, a mouse co‐ don optimized version of hyPBase showed 10-fold greater long term gene expression than both native *piggyBac* and SB100X.

widespread applicability than viral vectors, in combination with the potential for site-direct‐ ed gene delivery, make transposons a promising non-viral gene delivery system as an alter‐

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 279

SS is supported in part by the Howard Hughes Medical Institute Med-Into-Grad Scholar

1 Michael E. DeBakey VA Medical Center, Baylor College of Medicine, Houston TX, USA

2 Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor Col‐

3 Department of Medicine-Nephrology Division, Baylor College of Medicine, Houston TX,

[1] McClintock B. The Origin and Behavior of Mutable Loci in Maize. *ProcNatlAcadSci* U

[2] Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial se‐ quencing and analysis of the human genome. *Nature*. 2001 Feb 15;409(6822):860–921. [3] Ivics Z, Hackett PB, Plasterk RH, Izsvák Z. Molecular Reconstruction of Sleeping Beauty, a Tc1-like Transposon from Fish, and Its Transposition in Human Cells. *Cell*.

[4] Cary LC, Goebel M, Corsaro BG, Wang H-G, Rosen E, Fraser MJ. Transposon muta‐ genesis of baculoviruses: Analysis of Trichoplusiani transposon IFP2 insertions with‐ in the FP-locus of nuclear polyhedrosis viruses. *Virology*. 1989 Sep;172(1):156–69. [5] Fraser MJ, Clszczon T, Elick T, Bauser C. Precise excision of TTAA-specific lepidop‐ teran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome

4 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston TX, USA

grant to TBMM Program. MHW is supported in part by NIH R01 DK093660.

native to viral vectors.

**Acknowlegements**

**Author details**

USA

**References**

Sunandan Saha2,3 and Matthew H. Wilson1,2,3,4

lege of Medicine, Houston TX, USA

S A. 1950 Jun;36(6):344–55.

1997 Nov 14;91(4):501–10.

\*Address all correspondence to: mhwilson@bcm.edu

#### **6.2. Engineering transposon systems for site-directed integration**

Random integration of transgene during delivery have resulted in adverse events including leukemia [25,72]. Integration of transgenes at other genetic loci may also affect expression of critical genes. Engineering transposon systems for site-directed integration would allow transgene delivery to sites in the genome resulting in improved gene expression, reduced positional effects at the site of integration, and improved safety. Most studies have utilized fusion of DNA-binding domains to the transposase to achieve site directed integration, be‐ ginning with the engineering of the *sleeping beauty* system. *Sleeping beauty* has been engi‐ neered to bias integration into plasmids containing target sites [73-74] and near selected elements and repeat elements in the genome [75-76]. The *piggyBac* system seems to be more suited for transposase modifications as the addition of additional domains to the transpo‐ sase does not alter the systems efficiency [7,77-79]. A Gal4-*piggyBac* fusion transposase has been shown to bias integration near Gal4 sites in episomal plasmids [80] and the genome [81]. A chimeric transposase containing an engineered zinc finger protein (ZFP) fused to the native *piggyBac* transposase has also been successfully used to bias integration at the genom‐ ic level [79]. Researchers have also used transcription factor DNA binding domains fused to the *piggyBac* transposase to label nearby transcription factor binding sites in the genomes of cells [82]. Current approaches are hampered by the ability of the transposase to integrate on its own without the targeting machinery which can lead to off-target integration. Futher en‐ gineering modifications to both the transposase and transposon may overcome this limita‐ tion.

#### **7. Conclusion**

Transposon systems are well suited for *ex vivo* gene therapy and *in vivo* delivery to target organs may also become a reality in the future. The advantages of lower cost and more widespread applicability than viral vectors, in combination with the potential for site-direct‐ ed gene delivery, make transposons a promising non-viral gene delivery system as an alter‐ native to viral vectors.

#### **Acknowlegements**

Efficiency of transposition is perceived as a bottleneck to efficient gene delivery. Attempts to engineer hyperactive versions of transposase have resulted in versions with increasing transposition activity. Strategies employed include import of amino acids from related transposases [67], alanine scanning [68] and site directed mutagenesis [69]. The construction of the SB100X transposase with ~100 folds higher activity than the original *sleeping beauty* transposase employed a high throughput screen of mutant transposases obtained from DNA shuffling [70]. A hyperactive version of the *piggyBac* transposase (hyPBase) has also been engineered with 17-fold increase in excision and 9-fold increase in integration [71]. The hyPBase has 7 amino acid substitution identified from a screen of PBase mutants but none of the 7 substitutions are in the catalytic domain of the transposase. The hyPBase also has foot‐ print mutation frequency (<5%) comparable to the wild type transposase and no apparent effect on genomic integrity. Unlike SB100X which showed a 50% reduction, the addition of a 24 kDa ZFN tag did not significantly alter transposition efficiency [66]. In vivo, a mouse co‐ don optimized version of hyPBase showed 10-fold greater long term gene expression than

Random integration of transgene during delivery have resulted in adverse events including leukemia [25,72]. Integration of transgenes at other genetic loci may also affect expression of critical genes. Engineering transposon systems for site-directed integration would allow transgene delivery to sites in the genome resulting in improved gene expression, reduced positional effects at the site of integration, and improved safety. Most studies have utilized fusion of DNA-binding domains to the transposase to achieve site directed integration, be‐ ginning with the engineering of the *sleeping beauty* system. *Sleeping beauty* has been engi‐ neered to bias integration into plasmids containing target sites [73-74] and near selected elements and repeat elements in the genome [75-76]. The *piggyBac* system seems to be more suited for transposase modifications as the addition of additional domains to the transpo‐ sase does not alter the systems efficiency [7,77-79]. A Gal4-*piggyBac* fusion transposase has been shown to bias integration near Gal4 sites in episomal plasmids [80] and the genome [81]. A chimeric transposase containing an engineered zinc finger protein (ZFP) fused to the native *piggyBac* transposase has also been successfully used to bias integration at the genom‐ ic level [79]. Researchers have also used transcription factor DNA binding domains fused to the *piggyBac* transposase to label nearby transcription factor binding sites in the genomes of cells [82]. Current approaches are hampered by the ability of the transposase to integrate on its own without the targeting machinery which can lead to off-target integration. Futher en‐ gineering modifications to both the transposase and transposon may overcome this limita‐

Transposon systems are well suited for *ex vivo* gene therapy and *in vivo* delivery to target organs may also become a reality in the future. The advantages of lower cost and more

both native *piggyBac* and SB100X.

278 Gene Therapy - Tools and Potential Applications

tion.

**7. Conclusion**

**6.2. Engineering transposon systems for site-directed integration**

SS is supported in part by the Howard Hughes Medical Institute Med-Into-Grad Scholar grant to TBMM Program. MHW is supported in part by NIH R01 DK093660.

#### **Author details**

Sunandan Saha2,3 and Matthew H. Wilson1,2,3,4

\*Address all correspondence to: mhwilson@bcm.edu

1 Michael E. DeBakey VA Medical Center, Baylor College of Medicine, Houston TX, USA

2 Interdepartmental Program in Translational Biology & Molecular Medicine, Baylor Col‐ lege of Medicine, Houston TX, USA

3 Department of Medicine-Nephrology Division, Baylor College of Medicine, Houston TX, USA

4 Center for Cell and Gene Therapy, Baylor College of Medicine, Houston TX, USA

#### **References**


in cell lines from two species of Lepidoptera. *Insect Molecular Biology*. 1996;5(2):141– 51.

[18] Assessment of adenoviral vector safety and toxicity: report of the National Institutes of Health Recombinant DNA Advisory Committee. *Hum. Gene Ther*. 2002 Jan 1;13(1):

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 281

[19] Morsy MA, Caskey CT. Expanded-capacity adenoviral vectors--the helper-depend‐

[20] Thomas CE, Schiedner G, Kochanek S, Castro MG, Löwenstein PR. Peripheral infec‐ tion with adenovirus causes unexpected long-term brain inflammation in animals in‐ jected intracranially with first-generation, but not with high-capacity, adenovirus vectors: Toward realistic long-term neurological gene therapy for chronic diseases.

[21] Baccala R, Gonzalez-Quintial R, Lawson BR, Stern ME, Kono DH, Beutler B, et al. Sensors of the innate immune system: their mode of action. *Nature Reviews Rheuma‐*

[22] Sharma S, Fitzgerald KA. Innate immune sensing of DNA. *PLoSPathog*. 2011 Apr;

[23] Bushman F, Lewinski M, Ciuffi A, Barr S, Leipzig J, Hannenhalli S, et al. Genomewide analysis of retroviral DNA integration. *Nature Reviews Microbiology*. 2005 Nov

[24] Wu X, Li Y, Crise B, Burgess SM. Transcription Start Regions in the Human Genome Are Favored Targets for MLV Integration. *Science*. 2003 Jun 13;300(5626):1749–51.

[25] Hacein-Bey-Abina S, Kalle CV, Schmidt M, McCormack MP, Wulffraat N, Leboulch P, et al. LMO2-Associated Clonal T Cell Proliferation in Two Patients after Gene

[26] Yant SR, Wu X, Huang Y, Garrison B, Burgess SM, Kay MA. High-Resolution Ge‐ nome-Wide Mapping of Transposon Integration in Mammals. *Mol. Cell. Biol*. 2005

[27] Galvan DL, Nakazawa Y, Kaja A, Kettlun C, Cooper LJN, Rooney CM, et al. Ge‐ nome-wide Mapping of PiggyBac Transposon Integrations in Primary Human T

[28] Lee C-Y, Li J-F, Liou J-S, Charng Y-C, Huang Y-W, Lee H-J. A gene delivery system for human cells mediated by both a cell-penetrating peptide and a piggyBactranspo‐

[29] Kang Y, Zhang X, Jiang W, Wu C, Chen C, Zheng Y, et al. Tumor-directed gene ther‐ apy in mice using a composite nonviral gene delivery system consisting of the piggy‐

[30] Bak RO, Mikkelsen JG. Mobilization of DNA transposable elements from lentiviral

Bac transposon and polyethylenimine. *BMC Cancer*. 2009 Apr 27;9(1):126.

Therapy for SCID-X1. *Science*. 2003 Oct 17;302(5644):415–9.

Cells. *Journal of Immunotherapy*. 2009 Oct;32(8):837–44.

sase. *Biomaterials*. 2011 Sep;32(26):6264–76.

vectors. *Mob Genet Elements*. 2011;1(2):139–44.

ent vectors. *Mol Med Today*. 1999 Jan;5(1):18–24.

*PNAS*. 2000 Jun 20;97(13):7482–7.

*tology*. 2009 Jul 14;5(8):448–56.

7(4):e1001310.

1;3(11):848–58.

Mar 15;25(6):2085–94.

3–13.


[18] Assessment of adenoviral vector safety and toxicity: report of the National Institutes of Health Recombinant DNA Advisory Committee. *Hum. Gene Ther*. 2002 Jan 1;13(1): 3–13.

in cell lines from two species of Lepidoptera. *Insect Molecular Biology*. 1996;5(2):141–

[6] Mitra R, Fain-Thornton J, Craig NL. piggyBac can bypass DNA synthesis during cut

[7] Wilson MH, Coates CJ, George AL. PiggyBac Transposon-mediated Gene Transfer in

[8] Savoldo B, Ramos CA, Liu E, Mims MP, Keating MJ, Carrum G, et al. CD28 costimu‐ lation improves expansion and persistence of chimeric antigen receptor–modified T cells in lymphoma patients. *Journal of Clinical Investigation*. 2011 May 2;121(5):1822–6.

[9] Singh H, Manuri PR, Olivares S, Dara N, Dawson MJ, Huls H, et al. Redirecting Spe‐ cificity of T-Cell Populations For CD19 Using the Sleeping Beauty System. *Cancer*

[10] Hackett PB, Largaespada DA, Cooper LJ. A Transposon and Transposase System for

[11] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors. *Cell*.

[12] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. In‐ duced Pluripotent Stem Cell Lines Derived from Human Somatic Cells. *Science*. 2007

[13] Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vec‐

[14] Balciunas D, Wangensteen KJ, Wilber A, Bell J, Geurts A, Sivasubbu S, et al. Harness‐ ing a High Cargo-Capacity Transposon for Genetic Applications in Vertebrates. *PLoS*

[15] Huye LE, Nakazawa Y, Patel MP, Yvon E, Sun J, Savoldo B, et al. CombiningmTor Inhibitors With Rapamycin-resistant T Cells: A Two-pronged Approach to Tumor Elimination. *Molecular Therapy* [Internet]. 2011 [cited 2012 Jul 12]; Available from: http://www.nature.com.ezproxyhost.library.tmc.edu/mt/journal/vaop/ncurrent/full/

[16] Li MA, Turner DJ, Ning Z, Yusa K, Liang Q, Eckert S, et al. Mobilization of giant pig‐ gyBac transposons in the mouse genome. *Nucl. Acids Res*. 2011 Dec 1;39(22):e148–

[17] Kahlig KM, Saridey SK, Kaja A, Daniels MA, George AL, Wilson MH. Multiplexed transposon-mediated stable gene transfer in human cells. *ProcNatlAcadSci* U S A.

tors for gene therapy. *Nature Reviews Genetics*. 2003 May 1;4(5):346–58.

and paste transposition. *The EMBO Journal*. 2008 Apr 9;27(7):1097–109.

Human Cells. *Molecular Therapy*. 2007;15(1):139–45.

Human Application. *Molecular Therapy*. 2010;18(4):674–83.

*Res*. 2008 Apr 15;68(8):2961–71.

2007 Nov 30;131(5):861–72.

Dec 21;318(5858):1917–20.

*Genet*. 2006 Nov 10;2(11):e169.

mt2011179a.html

2010 Jan 26;107(4):1343–8.

e148.

51.

280 Gene Therapy - Tools and Potential Applications


[31] Mikkelsen JG, Yant SR, Meuse L, Huang Z, Xu H, Kay MA. Helper-Independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery and persistent gene expression in vivo. *Mol. Ther.* 2003 Oct;8(4):654–65.

poson-mediated gene delivery: implications for non-viral gene therapy of

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 283

[44] Aronovich EL, Bell JB, Khan SA, Belur LR, Gunther R, Koniar B, et al. Systemic Cor‐ rection of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty

[45] Yusa K, Rashid ST, Strick-Marchand H, Varela I, Liu P-Q, Paschon DE, et al. Targeted gene correction of α1-antitrypsin deficiency in induced pluripotent stem cells. *Nature*.

[46] Leen AM, Myers GD, Sili U, Huls MH, Weiss H, Leung KS, et al. Monoculture-de‐ rived T lymphocytes specific for multiple viruses expand and produce clinically rele‐ vant effects in immunocompromised individuals. *Nature Medicine*. 2006 Oct 1;12(10):

[47] Rooney C., Ng CY., Loftin S, Smith C., Li C, Krance R., et al. Use of gene-modified virus-specific T lymphocytes to control Epstein-Barr-virus-related lymphoprolifera‐

[48] Straathof KCM, Bollard CM, Popat U, Huls MH, Lopez T, Morriss MC, et al. Treat‐ ment of nasopharyngeal carcinoma with Epstein-Barr virus–specific T lymphocytes.

[49] Bollard CM, Gottschalk S, Leen AM, Weiss H, Straathof KC, Carrum G, et al. Com‐ plete responses of relapsed lymphoma following genetic modification of tumor-anti‐ gen presenting cells and T-lymphocyte transfer. *Blood*. 2007 Oct 15;110(8):2838–45. [50] Dudley ME, Wunderlich JR, Yang JC, Sherry RM, Topalian SL, Restifo NP, et al. Adoptive Cell Transfer Therapy Following Non-Myeloablative but Lymphodeplet‐ ing Chemotherapy for the Treatment of Patients With Refractory Metastatic Melano‐

[51] Nakazawa Y, Huye LE, Dotti G, Foster AE, Vera JF, Manuri PR, et al. Optimization of the PiggyBac Transposon System for the Sustained Genetic Modification of Human T

[52] Nakazawa Y, Huye LE, Salsman VS, Leen AM, Ahmed N, Rollins L, et al. PiggyBacmediated Cancer Immunotherapy Using EBV-specific Cytotoxic T-cells Expressing HER2-specific Chimeric Antigen Receptor. *Molecular Therapy* [Internet]. 2011 [cited 2012 Jul 12]; Available from: http:// www.nature.com.ezproxyhost.library .tmc.edu/mt/journal/vaop/ncurrent/full/

[53] Takahashi K, Yamanaka S. Induction of Pluripotent Stem Cells from Mouse Embry‐ onic and Adult Fibroblast Cultures by Defined Factors. *Cell*. 2006 Aug 25;126(4):663–

[54] Okita K, Ichisaka T, Yamanaka S. Generation of germline-competent induced pluri‐

Lymphocytes. *Journal of Immunotherapy*. 2009 Oct;32(8):826–36.

potent stem cells. *Nature*. 2007 Jun 6;448(7151):313–7.

mucopolysaccharidoses. *The Journal of Gene Medicine*. 2007;9(5):403–15.

Transposon System. *Molecular Therapy*. 2009;17(7):1136–44.

2011 Oct 12;478(7369):391–4.

tion. *The Lancet*. 1995 Jan 7;345(8941):9–13.

*Blood*. 2005 Mar 1;105(5):1898–904.

ma. *JCO*. 2005 Apr 1;23(10):2346–57.

mt2011131a.html

76.

1160–6.


poson-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses. *The Journal of Gene Medicine*. 2007;9(5):403–15.

[44] Aronovich EL, Bell JB, Khan SA, Belur LR, Gunther R, Koniar B, et al. Systemic Cor‐ rection of Storage Disease in MPS I NOD/SCID Mice Using the Sleeping Beauty Transposon System. *Molecular Therapy*. 2009;17(7):1136–44.

[31] Mikkelsen JG, Yant SR, Meuse L, Huang Z, Xu H, Kay MA. Helper-Independent Sleeping Beauty transposon-transposase vectors for efficient nonviral gene delivery

[32] Staunstrup NH, Moldt B, Mátés L, Villesen P, Jakobsen M, Ivics Z, et al. Hybrid lenti‐ virus-transposon vectors with a random integration profile in human cells. *Mol. Ther*.

[33] Hausl M, Zhang W, Voigtländer R, Müther N, Rauschhuber C, Ehrhardt A. Develop‐ ment of adenovirus hybrid vectors for Sleeping Beauty transposition in large mam‐

[34] Yant SR, Ehrhardt A, Mikkelsen JG, Meuse L, Pham T, Kay MA. Transposition from a gutless adeno-transposon vector stabilizes transgene expression in vivo. *Nature Bio‐*

[35] Garrison BS, Yant SR, Mikkelsen JG, Kay MA. Postintegrative gene silencing within the Sleeping Beauty transposition system. *Mol. Cell. Biol.* 2007 Dec;27(24):8824–33. [36] Yant SR, Meuse L, Chiu W, Ivics Z, Izsvak Z, Kay MA. Somatic integration and longterm transgene expression in normal and haemophilic mice using a DNA transposon

[37] Ohlfest JR, Frandsen JL, Fritz S, Lobitz PD, Perkinson SG, Clark KJ, et al. Phenotypic correction and long-term expression of factor VIII in hemophilic mice by immunoto‐ lerization and nonviral gene transfer using the Sleeping Beauty transposon system.

[38] Liu L, Mah C, Fletcher BS. Sustained FVIII Expression and Phenotypic Correction of Hemophilia A in Neonatal Mice Using an Endothelial-Targeted Sleeping Beauty

[39] Montini E, Held PK, Noll M, Morcinek N, Al-Dhalimy M, Finegold M, et al. In Vivo Correction of Murine Tyrosinemia Type I by DNA-Mediated Transposition. *Molecu‐*

[40] Ortiz-Urda S, Lin Q, Yant SR, Keene D, Kay MA, Khavari PA. Sustainable correction of junctionalepidermolysisbullosa via transposon-mediated nonviral gene transfer.

[41] He C-X, Shi D, Wu W-J, Ding Y-F. Insulin expression in livers of diabetic mice medi‐ ated by hydrodynamics-based administration. *World J Gastroenterol*. 2004 Feb

[42] Chen ZJ, Kren BT, Wong PY-P, Low WC, Steer CJ. Sleeping Beauty-mediated downregulation of huntingtin expression by RNA interference. *Biochemical and Biophysical*

[43] Aronovich EL, Bell JB, Belur LR, Gunther R, Koniar B, Erickson DCC, et al. Pro‐ longed expression of a lysosomal enzyme in mouse liver after Sleeping Beauty trans‐

and persistent gene expression in vivo. *Mol. Ther.* 2003 Oct;8(4):654–65.

2009 Jul;17(7):1205–14.

282 Gene Therapy - Tools and Potential Applications

mals. *Curr Gene Ther*. 2011 Oct;11(5):363–74.

system. *Nature Genetics*. 2000 May 1;25(1):35–41.

Transposon. *Molecular Therapy*. 2006;13(5):1006–15.

*Research Communications*. 2005 Apr 8;329(2):646–52.

*technology*. 2002;20(10):999–1005.

*Blood*. 2005 Apr 1;105(7):2691–8.

*lar Therapy*. 2002;6(6):759–69.

15;10(4):567–72.

*Gene Therapy*. 2003;10(13):1099–104.


[55] Hochedlinger K, Yamada Y, Beard C, Jaenisch R. Ectopic Expression of Oct-4 Blocks Progenitor-Cell Differentiation and Causes Dysplasia in Epithelial Tissues. *Cell*. 2005 May 6;121(3):465–77.

[69] Zayed H, Izsvák Z, Walisko O, Ivics Z. Development of Hyperactive Sleeping Beauty Transposon Vectors by Mutational Analysis. *Molecular Therapy*. 2004;9(2):292–304.

Transposons for Non-Viral Gene Transfer http://dx.doi.org/10.5772/52527 285

[70] Mátés L, Chuah MKL, Belay E, Jerchow B, Manoj N, Acosta-Sanchez A, et al. Molecu‐ lar evolution of a novel hyperactive Sleeping Beauty transposase enables robust sta‐

[71] Yusa K, Zhou L, Li MA, Bradley A, Craig NL. A hyperactive piggyBactransposase

[73] Ivics Z, Katzer A, Stüwe EE, Fiedler D, Knespel S, Izsvák Z. Targeted Sleeping Beau‐

[74] Yant SR, Huang Y, Akache B, Kay MA. Site-directed transposon integration in hu‐

[75] Voigt K, Gogol-Döring A, Miskey C, Chen W, Cathomen T, Izsvák Z, et al. Retarget‐ ing Sleeping Beauty Transposon Insertions by Engineered Zinc Finger DNA-binding Domains. *Molecular therapy*: the journal of the American Society of Gene Therapy [In‐ ternet]. 2012 Jul 10 [cited 2012 Aug 10]; Available from: http://

[76] Ammar I, Gogol-Döring A, Miskey C, Chen W, Cathomen T, Izsvák Z, et al. Retarget‐ ing transposon insertions by the adeno-associated virus Rep protein. *Nucleic Acids*

[77] Wu SC-Y, Meir Y-JJ, Coates CJ, Handler AM, Pelczar P, Moisyadi S, et al. piggyBac is a flexible and highly active transposon as compared to Sleeping Beauty, Tol2, and

[78] Cadiñanos J, Bradley A. Generation of an inducible and optimized piggyBac transpo‐

[79] Kettlun C, Galvan DL, Jr ALG, Kaja A, Wilson MH. Manipulating piggyBac Transpo‐ son Chromosomal Integration Site Selection in Human Cells. *Molecular Therapy*.

[80] Maragathavally KJ, Kaminski JM, Coates CJ. Chimeric Mos1 and piggyBactranspo‐

[81] Owens JB, Urschitz J, Stoytchev I, Dang NC, Stoytcheva Z, Belcaid M, et al. Chimeric piggyBactransposases for genomic targeting in human cells. *Nucl. Acids Res*. [Inter‐ net]. 2012 Apr 9 [cited 2012 Jul 5]; Available from: http://nar.oxfordjournals.org/

[82] Wang H, Mayhew D, Chen X, Johnston M, Mitra RD. "Calling cards" for DNA-bind‐

sases result in site-directed integration. *FASEB J*. 2006 Sep;20(11):1880–2.

ing proteins in mammalian cells. *Genetics*. 2012 Mar;190(3):941–9.

Mos1 in mammalian cells. *PNAS*. 2006 Oct 10;103(41):15008–13.

[72] Check E. Gene therapy: A tragic setback. *Nature*. 2002 Nov 14;420(6912):116–8.

ty Transposition in Human Cells. *Molecular Therapy*. 2007;15(6):1137–44.

ble gene transfer in vertebrates. *Nature Genetics*. 2009;41(6):753–61.

for mammalian applications. *PNAS*. 2011 Jan 25;108(4):1531–6.

man cells. *Nucleic Acids Res*. 2007;35(7):e50.

www.ncbi.nlm.nih.gov/pubmed/22776959

son system. *Nucleic Acids Res*. 2007;35(12):e87.

content/early/2012/04/08/nar.gks309

*Res*. 2012 Aug 1;40(14):6693–712.

2011;19(9):1636–44.


[69] Zayed H, Izsvák Z, Walisko O, Ivics Z. Development of Hyperactive Sleeping Beauty Transposon Vectors by Mutational Analysis. *Molecular Therapy*. 2004;9(2):292–304.

[55] Hochedlinger K, Yamada Y, Beard C, Jaenisch R. Ectopic Expression of Oct-4 Blocks Progenitor-Cell Differentiation and Causes Dysplasia in Epithelial Tissues. *Cell*. 2005

[56] Foster KW, Liu Z, Nail CD, Li X, Fitzgerald TJ, Bailey SK, et al. Induction of KLF4 in basal keratinocytes blocks the proliferation–differentiation switch and initiates squa‐

[57] Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, et al. Generation of Induced Pluripo‐ tent Stem Cells Using Recombinant Proteins. *Cell Stem Cell*. 2009 May 8;4(5):381–4. [58] Stadtfeld M, Nagaya M, Utikal J, Weir G, Hochedlinger K. Induced Pluripotent Stem Cells Generated Without Viral Integration. *Science*. 2008 Nov 7;322(5903):945–9. [59] Yusa K, Rad R, Takeda J, Bradley A. Generation of transgene-free induced pluripo‐ tent mouse stem cells by the piggyBac transposon. *Nature Methods*. 2009;6(5):363–9. [60] Woltjen K, Michael IP, Mohseni P, Desai R, Mileikovsky M, Hämäläinen R, et al. pig‐ gyBac transposition reprograms fibroblasts to induced pluripotent stem cells. *Nature*.

[61] Yang R, Jiang M, Kumar SM, Xu T, Wang F, Xiang L, et al. Generation of Melano‐ cytes from Induced Pluripotent Stem Cells. *Journal of Investigative Dermatology*.

[62] Wernig M, Lengner CJ, Hanna J, Lodato MA, Steine E, Foreman R, et al. A drug-in‐ ducible transgenic system for direct reprogramming of multiple somatic cell types.

[63] Wilber A, Linehan JL, Tian X, Woll PS, Morris JK, Belur LR, et al. Efficient and stable transgene expression in human embryonic stem cells using transposon-mediated

[64] Rostovskaya M, Fu J, Obst M, Baer I, Weidlich S, Wang H, et al. Transposon-mediat‐ ed BAC transgenesis in human ES cells. *Nucleic acids research* [Internet]. 2012 Jun 30 [cited 2012 Aug 9]; Available from: http://www.ncbi.nlm.nih.gov/pubmed/22753106

[65] Grabundzija I, Irgang M, Mátés L, Belay E, Matrai J, Gogol-Döring A, et al. Compara‐ tive Analysis of Transposable Element Vector Systems in Human Cells. *Molecular*

[66] Doherty JE, Huye LE, Yusa K, Zhou L, Craig NL, Wilson MH. Hyperactive piggyBac gene transfer in human cells and in vivo. *Hum. Gene Ther*. 2012 Mar;23(3):311–20. [67] Baus J, Liu L, Heggestad AD, Sanz S, Fletcher BS. Hyperactive Transposase Mutants of the Sleeping Beauty Transposon. *Molecular Therapy*. 2005;12(6):1148–56.

[68] Yant SR, Park J, Huang Y, Mikkelsen JG, Kay MA. Mutational Analysis of the N-Ter‐ minal DNA-Binding Domain of Sleeping Beauty Transposase: Critical Residues for DNA Binding and Hyperactivity in Mammalian Cells. *Mol. Cell. Biol.* 2004 Oct

mous epithelial dysplasia. *Oncogene*. 2005;24(9):1491–500.

May 6;121(3):465–77.

284 Gene Therapy - Tools and Potential Applications

2009 Mar 1;458(7239):766–70.

*Nat. Biotechnol*. 2008 Aug;26(8):916–24.

gene transfer. *Stem Cells.* 2007 Nov;25(11):2919–27.

2011;131(12):2458–66.

*Therapy*. 2010;18(6):1200–9.

15;24(20):9239–47.


**Chapter 12**

**Lentiviral Gene Therapy Vectors:**

**Challenges and Future Directions**

Hélio A. Tomás, Ana F. Rodrigues,

http://dx.doi.org/10.5772/52534

**1. Introduction**

**1.1. Lentiviruses**

Paula M. Alves and Ana S. Coroadinha

Additional information is available at the end of the chapter

Lentiviral vectors (LV) are efficient vehicles for gene transfer in mammalian cells due to their capacity to stably express a gene of interest in non-dividing and dividing cells. Their use has exponentially grown in the last years both in research and in gene therapy protocols, reaching 12% of the viral vector based clinical trials in 2011 [1]. This chapter reviews and

Lentiviruses are human and animal pathogens that are known to have long incubation peri‐ ods and persistent infection. The time between the initial infection and the appearance of the first symptoms can reach several months or years [2]. Nowadays lentiviruses are classified as one of the seven genus of *Retroviridae* family. *Lentivirus* genus is composed by nine virus

All Retroviruses share similarities in structure, genomic organization and replicative proper‐ ties. Retroviruses are spherical viruses of around 80-120 nm in diameter [4] and are character‐ ized by a genome comprising two positive-sense single stranded RNA. Also, they have a unique replicative strategy where the viral RNA is reverse transcribed into double stranded DNA that is integrated in the cellular genome [5]. Together with the RNA strands, the enzymes necessary for replication and the structural proteins form the nucleocapsid. The later is inside a proteic capsid that is surrounded by a double lipidic membrane [6]. Connecting the lipidic membrane and the capsid there are the matrix proteins. The lipidic membrane has its origin in

the host cells and presents at surface the envelope viral glycoproteins (Env) (Figure 2).

© 2013 Tomás et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

discusses the state of the art on the production of HIV-1- based lentiviral vectors.

species that include primate and non-primate retroviruses (Figure 1) [3].

**Chapter 12**

### **Lentiviral Gene Therapy Vectors: Challenges and Future Directions**

Hélio A. Tomás, Ana F. Rodrigues, Paula M. Alves and Ana S. Coroadinha

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52534

#### **1. Introduction**

Lentiviral vectors (LV) are efficient vehicles for gene transfer in mammalian cells due to their capacity to stably express a gene of interest in non-dividing and dividing cells. Their use has exponentially grown in the last years both in research and in gene therapy protocols, reaching 12% of the viral vector based clinical trials in 2011 [1]. This chapter reviews and discusses the state of the art on the production of HIV-1- based lentiviral vectors.

#### **1.1. Lentiviruses**

Lentiviruses are human and animal pathogens that are known to have long incubation peri‐ ods and persistent infection. The time between the initial infection and the appearance of the first symptoms can reach several months or years [2]. Nowadays lentiviruses are classified as one of the seven genus of *Retroviridae* family. *Lentivirus* genus is composed by nine virus species that include primate and non-primate retroviruses (Figure 1) [3].

All Retroviruses share similarities in structure, genomic organization and replicative proper‐ ties. Retroviruses are spherical viruses of around 80-120 nm in diameter [4] and are character‐ ized by a genome comprising two positive-sense single stranded RNA. Also, they have a unique replicative strategy where the viral RNA is reverse transcribed into double stranded DNA that is integrated in the cellular genome [5]. Together with the RNA strands, the enzymes necessary for replication and the structural proteins form the nucleocapsid. The later is inside a proteic capsid that is surrounded by a double lipidic membrane [6]. Connecting the lipidic membrane and the capsid there are the matrix proteins. The lipidic membrane has its origin in the host cells and presents at surface the envelope viral glycoproteins (Env) (Figure 2).

© 2013 Tomás et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**1.2. HIV-1 genome**

all retroviruses (Figure 3).

cell allowing for virus entrance into the cell [10].

HIV-1 genome has about 9-10 kb and is constituted by several non-coding sequences that control gene expression and protein synthesis, and genes that code for regulatory and acces‐ sory proteins in addition to the structural and enzymatic genes *gag*, *pol* and *env*, common to

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The *gag* gene codes for a polypeptide that is proteolytic cleaved by the viral protease (PR) originating three main structural proteins: matrix (MA), capsid (CA) and nucelocapsid (NC). The *pro* gene codes for a polypeptide that after cleavage by PR, during the virus maturation, originates PR, reverse transcriptase (RT) and integrase (IN). These enzymes play critical roles in the life cycle of retroviruses since their functions are the cleavage of viral polypepti‐ des (also involved in virus maturation), the reverse transcription of viral RNA to doublestranded DNA (provirus) and the integration of the provirus into the cellular genome [7]. Finally the *env* gene encodes a polypeptide that is cleaved by cellular proteases in two pro‐ teins, the SU (surface) and TM (transmembrane) subunits. Together, these two proteins are the structural units of the Env protein that will interact with cellular receptors of the host

Flanking the retroviral provirus there are the 5' and 3' Long Terminal Repeats (LTRs) com‐ posed by the 3' untranslated region (U3), repeat elements (R) and 5' untranslated region (U5). The LTRs contain the enhancer/promoter sequence that allows for gene expression, the att repeats important for provirus integration and the polyadenylation signal (polyA).

The HIV-1 genome also has other six genes that code for two regulatory proteins (Tat and Rev), and four accessory proteins: Vif, Nef, Vpr, and Vpu. Tat protein interacts with cellular proteins and the mRNA TAR sequence acting by increasing the viral transcription hundreds of times. Rev interacts with Rev Responsive Element (RRE), a cis-acting sequence located in the middle of the *env* gene allowing the efficient nuclear export of unspliced or singly spliced messenger RNA. The functions of accessory proteins are related with pathogenesis

The function of all HIV-1 proteins and their interactions with the host cells are not yet clear‐ ly understood but it is already reported that there are 2589 unique HIV-1–to–human protein

Additionally to the coding sequences, the lentivirus genome also has several non-coding cisacting sequences that play important roles in viral replication. The LTRs contains the Trans‐ activator Response element (TAR) for the interactions of the complex formed by the Tat protein and transcriptional factors. After the 5' LTR there are the primer binding site (PBS), where the reverse transcription starts, and the packaging signal (Ψ). Within the *pol* sequence there are also the central polypurine tract (cPPT) and the central termination sequence (CTS) contributing both for the efficient reverse transcription. Further there are the RRE in the middle of *env* gene and near the beginning of the 3´LTR the polypurine tract (PPT), a purine rich region where the synthesis of the plus strand DNA during the reverse transcription

of the virus and they are not crucial for the viral replication *in-vitro*.

interactions that are formed by 1448 human proteins [8,9].

starts [10].

**Figure 1.** Lentiviruses taxonomy by the International Committee on Taxonomy of Viruses (ICTV).

Within the *Retroviridae* family, retroviruses can be classified as simple or complex. The com‐ plex retroviruses include the lentiviruses and spumaviruses presenting a more complex ge‐ nome with additional regulation steps in their life cycle.

**Figure 2.** Schematic representation of a retrovirus particle. Abbreviations: NC – nucleopcapsid; MA – matrix; CA – cap‐ sid; SU – surface subunit of Env protein; TM – transmembrane subunit of Env protein; RT – reverse transcriptase; PR – protease; IN – integrase.

#### **1.2. HIV-1 genome**

**Figure 1.** Lentiviruses taxonomy by the International Committee on Taxonomy of Viruses (ICTV).

nome with additional regulation steps in their life cycle.

288 Gene Therapy - Tools and Potential Applications

protease; IN – integrase.

Within the *Retroviridae* family, retroviruses can be classified as simple or complex. The com‐ plex retroviruses include the lentiviruses and spumaviruses presenting a more complex ge‐

**Figure 2.** Schematic representation of a retrovirus particle. Abbreviations: NC – nucleopcapsid; MA – matrix; CA – cap‐ sid; SU – surface subunit of Env protein; TM – transmembrane subunit of Env protein; RT – reverse transcriptase; PR – HIV-1 genome has about 9-10 kb and is constituted by several non-coding sequences that control gene expression and protein synthesis, and genes that code for regulatory and acces‐ sory proteins in addition to the structural and enzymatic genes *gag*, *pol* and *env*, common to all retroviruses (Figure 3).

The *gag* gene codes for a polypeptide that is proteolytic cleaved by the viral protease (PR) originating three main structural proteins: matrix (MA), capsid (CA) and nucelocapsid (NC). The *pro* gene codes for a polypeptide that after cleavage by PR, during the virus maturation, originates PR, reverse transcriptase (RT) and integrase (IN). These enzymes play critical roles in the life cycle of retroviruses since their functions are the cleavage of viral polypepti‐ des (also involved in virus maturation), the reverse transcription of viral RNA to doublestranded DNA (provirus) and the integration of the provirus into the cellular genome [7]. Finally the *env* gene encodes a polypeptide that is cleaved by cellular proteases in two pro‐ teins, the SU (surface) and TM (transmembrane) subunits. Together, these two proteins are the structural units of the Env protein that will interact with cellular receptors of the host cell allowing for virus entrance into the cell [10].

Flanking the retroviral provirus there are the 5' and 3' Long Terminal Repeats (LTRs) com‐ posed by the 3' untranslated region (U3), repeat elements (R) and 5' untranslated region (U5). The LTRs contain the enhancer/promoter sequence that allows for gene expression, the att repeats important for provirus integration and the polyadenylation signal (polyA).

The HIV-1 genome also has other six genes that code for two regulatory proteins (Tat and Rev), and four accessory proteins: Vif, Nef, Vpr, and Vpu. Tat protein interacts with cellular proteins and the mRNA TAR sequence acting by increasing the viral transcription hundreds of times. Rev interacts with Rev Responsive Element (RRE), a cis-acting sequence located in the middle of the *env* gene allowing the efficient nuclear export of unspliced or singly spliced messenger RNA. The functions of accessory proteins are related with pathogenesis of the virus and they are not crucial for the viral replication *in-vitro*.

The function of all HIV-1 proteins and their interactions with the host cells are not yet clear‐ ly understood but it is already reported that there are 2589 unique HIV-1–to–human protein interactions that are formed by 1448 human proteins [8,9].

Additionally to the coding sequences, the lentivirus genome also has several non-coding cisacting sequences that play important roles in viral replication. The LTRs contains the Trans‐ activator Response element (TAR) for the interactions of the complex formed by the Tat protein and transcriptional factors. After the 5' LTR there are the primer binding site (PBS), where the reverse transcription starts, and the packaging signal (Ψ). Within the *pol* sequence there are also the central polypurine tract (cPPT) and the central termination sequence (CTS) contributing both for the efficient reverse transcription. Further there are the RRE in the middle of *env* gene and near the beginning of the 3´LTR the polypurine tract (PPT), a purine rich region where the synthesis of the plus strand DNA during the reverse transcription starts [10].

as a tool for study HIV infection [11]. Few months after, the same group presented the first rep‐ lication-defective HIV-1 vector. In a trans-complementation assay for measuring the replica‐ tive potential of HIV-1 envelope glycoprotein mutants they used an identical HIV-1 provirus construction but with a deletion in the *env* gene. The Env glycoproteins were supplied by an in‐ dependent expression plasmid. The co-transfection of these two plasmids allowed for the pro‐ duction of replication-defective viruses [12]. These vectors were structural identical to the wild-type virus, but lacked in their genome the *env* gene. They could only perform a single cy‐ cle of replication because their host cells, after infection, did not have the *env* gene to produce infectious virus. Although the principal aim of these studies was not the creation of viral vec‐ tors, they were the basis of lentiviral vector development, suggesting that lentiviruses could be adapted as a tool for genetic material transfer and permanent modification of animal cells.

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291

Other preliminary studies were being conducted and several important discoveries or inno‐ vations had also contributed for the development of LVs. The introduction of the resistance marker gene *hypoxanthine–guanine phosphoribosyl-transferase* (*gpt*) under the expression con‐ trol of SV40 promoter in the place of *env* gene deletion allowed the first quantification of in‐ fectious LVs produced [13]. Like it had been observed for other γ-retroviral vectors (γ-RVs) it was possible to produce infectious lentiviral particles with Env glycoproteins from other viruses (pseudotyping); for example the Moloney Murine Leukemia Virus amphotrophic en‐ velope 4070A (A-MoMLV) [13], and Human T-cell Leukemia Virus Type I (HTLV-I) enve‐ lope [14] were successfully used suggesting that *env* gene was not necessary for virion particle formation. The localization and sequence of packaging signal was identified as the main responsible for the packaging of viral RNA [15] suggesting that modified RNAs with Ψ could also be packaged into virions. The discovery of the great stability conferred to LV pseudotyped with Vesicular Stomatitis Virus-G protein protein (VSV-G) allowed to concen‐

fectivity [16,17]. It was shown that LVs can transduce efficiently non-dividing cells, their

All these steps showed the potential of using modified lentiviruses as vectors, stimulating the iterative studies and the evolution of LVs in the next years. Their further development was based in safety principles (most of them already used in the development of oncoretro‐ viral vectors) such as the splitting of the genome into several independent expression cas‐ settes: the packaging cassette with the structural and enzymatic elements, the transfer cassettes with the gene of interest and the envelope expression cassette. In addition, the elimination of non-essential viral elements and the homology reduction among the expres‐ sion cassettes also contributed to avoid the possibility of recombination, vector mobilization

Four generations of LVs are currently considered; these differ from each other in terms of the number of genetic constructs used to drive the expression of the viral components, the number of wild-type genes retained as well as the number and type of heterologous *cis*-ele‐

principal advantage over the oncoretroviral vectors [16,18,19].

and the generation of replicative competent lentiviral vectors (RCLVs).

ments used to increase vector titers and promote vector safety.

**2.1. Four generations of packaging constructs**

by ultracentrifugation or ultrafiltration without significant loss of in‐

trate the LV up to 109

**Figure 3.** Schematic representation of HIV-1 provirus genome. Abbreviations: LTR - long terminal repeat; attL and attR - left and right attachment sites; U3 - 3' untranslated region; R - repeat element; U5 - 5' untranslated; TAR - transacti‐ vation response element; PBS - primer binding site; DIS - dimerization signal; SD - splice donor site; SA - splice acceptor site; ψ - packaging signal; cPPT - central polypurine tract; CTS - central termination sequence; RRE - Rev response ele‐ ment; PPT - 3' polypurine tract; polyA - polyadenylation signal.

#### **1.3. HIV-1 life cycle**

The HIV-1 Life cycle starts when the Env glycoproteins GP120 located at surface of the viral envelope bind the CD4 cellular receptor and co-receptor CCR5, CSCR4 or both. This binding induces conformational changes of Env glycoproteins that allows for the fusion of the viral envelope with the cell membrane and the consequent entry of the viral core into the cell. Once inside the cell the capsid starts to disintegrate and the RT enzyme begins the reverse transcription where a double-stranded proviral DNA is synthesized using one of the posi‐ tive single-strand viral RNA molecules as template. When reverse transcription is complet‐ ed the double-strand DNA now called provirus forms a complex with viral proteins (RT, IN, NC, Vpr and MA) and cellular proteins termed pre-integration complex (PIC) that is import‐ ed to the cell nucleus by an ATP-dependent manner. It is this energy-dependent mechanism that allows the transduction of non-dividing cells by lentiviruses, unlike γ-retrovirus.

In the nucleus the linear provirus is integrated into the cellular genome by the integrase. Now all the requisites to produce new viruses are filled and the proviral DNA is transcribed into mRNA by the cellular RNA polymerase II. Still inside the nucleus some transcripts suf‐ fer a splicing event. The mRNA transcripts are exported from the nucleus to cytoplasm to be transcribed and to start to form the viral particles; two full-length RNA transcripts will be packaged in the viral particles.

The assembly of the viral proteins and the viral RNA occurs near the cellular membrane, in specific regions called lipid-rafts that are rich in cholesterol and sphingolipds. The immature viral particles are released from cells by budding. After leaving the cells, the viral protease cleaves the Gag and Pol proteins precursors to finally generate a mature infectious virion (reviewed by [5,10]).

#### **2. Lentiviral vector development**

The development of lentiviral vectors (LVs) started in 1989 when an HIV-1 provirus with a*chloramphenicol acetyltransferase* (*cat*) reporter gene in place of the non-essential *nef* gene was constructed. The transfection of Jurkat cells with this modified provirus plasmid produced in‐ fectious replicative competent viruses, very similar with wild-type HIV-1, that could be used as a tool for study HIV infection [11]. Few months after, the same group presented the first rep‐ lication-defective HIV-1 vector. In a trans-complementation assay for measuring the replica‐ tive potential of HIV-1 envelope glycoprotein mutants they used an identical HIV-1 provirus construction but with a deletion in the *env* gene. The Env glycoproteins were supplied by an in‐ dependent expression plasmid. The co-transfection of these two plasmids allowed for the pro‐ duction of replication-defective viruses [12]. These vectors were structural identical to the wild-type virus, but lacked in their genome the *env* gene. They could only perform a single cy‐ cle of replication because their host cells, after infection, did not have the *env* gene to produce infectious virus. Although the principal aim of these studies was not the creation of viral vec‐ tors, they were the basis of lentiviral vector development, suggesting that lentiviruses could be adapted as a tool for genetic material transfer and permanent modification of animal cells.

Other preliminary studies were being conducted and several important discoveries or inno‐ vations had also contributed for the development of LVs. The introduction of the resistance marker gene *hypoxanthine–guanine phosphoribosyl-transferase* (*gpt*) under the expression con‐ trol of SV40 promoter in the place of *env* gene deletion allowed the first quantification of in‐ fectious LVs produced [13]. Like it had been observed for other γ-retroviral vectors (γ-RVs) it was possible to produce infectious lentiviral particles with Env glycoproteins from other viruses (pseudotyping); for example the Moloney Murine Leukemia Virus amphotrophic en‐ velope 4070A (A-MoMLV) [13], and Human T-cell Leukemia Virus Type I (HTLV-I) enve‐ lope [14] were successfully used suggesting that *env* gene was not necessary for virion particle formation. The localization and sequence of packaging signal was identified as the main responsible for the packaging of viral RNA [15] suggesting that modified RNAs with Ψ could also be packaged into virions. The discovery of the great stability conferred to LV pseudotyped with Vesicular Stomatitis Virus-G protein protein (VSV-G) allowed to concen‐ trate the LV up to 109 by ultracentrifugation or ultrafiltration without significant loss of in‐ fectivity [16,17]. It was shown that LVs can transduce efficiently non-dividing cells, their principal advantage over the oncoretroviral vectors [16,18,19].

All these steps showed the potential of using modified lentiviruses as vectors, stimulating the iterative studies and the evolution of LVs in the next years. Their further development was based in safety principles (most of them already used in the development of oncoretro‐ viral vectors) such as the splitting of the genome into several independent expression cas‐ settes: the packaging cassette with the structural and enzymatic elements, the transfer cassettes with the gene of interest and the envelope expression cassette. In addition, the elimination of non-essential viral elements and the homology reduction among the expres‐ sion cassettes also contributed to avoid the possibility of recombination, vector mobilization and the generation of replicative competent lentiviral vectors (RCLVs).

#### **2.1. Four generations of packaging constructs**

**Figure 3.** Schematic representation of HIV-1 provirus genome. Abbreviations: LTR - long terminal repeat; attL and attR - left and right attachment sites; U3 - 3' untranslated region; R - repeat element; U5 - 5' untranslated; TAR - transacti‐ vation response element; PBS - primer binding site; DIS - dimerization signal; SD - splice donor site; SA - splice acceptor site; ψ - packaging signal; cPPT - central polypurine tract; CTS - central termination sequence; RRE - Rev response ele‐

The HIV-1 Life cycle starts when the Env glycoproteins GP120 located at surface of the viral envelope bind the CD4 cellular receptor and co-receptor CCR5, CSCR4 or both. This binding induces conformational changes of Env glycoproteins that allows for the fusion of the viral envelope with the cell membrane and the consequent entry of the viral core into the cell. Once inside the cell the capsid starts to disintegrate and the RT enzyme begins the reverse transcription where a double-stranded proviral DNA is synthesized using one of the posi‐ tive single-strand viral RNA molecules as template. When reverse transcription is complet‐ ed the double-strand DNA now called provirus forms a complex with viral proteins (RT, IN, NC, Vpr and MA) and cellular proteins termed pre-integration complex (PIC) that is import‐ ed to the cell nucleus by an ATP-dependent manner. It is this energy-dependent mechanism

that allows the transduction of non-dividing cells by lentiviruses, unlike γ-retrovirus.

In the nucleus the linear provirus is integrated into the cellular genome by the integrase. Now all the requisites to produce new viruses are filled and the proviral DNA is transcribed into mRNA by the cellular RNA polymerase II. Still inside the nucleus some transcripts suf‐ fer a splicing event. The mRNA transcripts are exported from the nucleus to cytoplasm to be transcribed and to start to form the viral particles; two full-length RNA transcripts will be

The assembly of the viral proteins and the viral RNA occurs near the cellular membrane, in specific regions called lipid-rafts that are rich in cholesterol and sphingolipds. The immature viral particles are released from cells by budding. After leaving the cells, the viral protease cleaves the Gag and Pol proteins precursors to finally generate a mature infectious virion

The development of lentiviral vectors (LVs) started in 1989 when an HIV-1 provirus with a*chloramphenicol acetyltransferase* (*cat*) reporter gene in place of the non-essential *nef* gene was constructed. The transfection of Jurkat cells with this modified provirus plasmid produced in‐ fectious replicative competent viruses, very similar with wild-type HIV-1, that could be used

ment; PPT - 3' polypurine tract; polyA - polyadenylation signal.

**1.3. HIV-1 life cycle**

290 Gene Therapy - Tools and Potential Applications

packaged in the viral particles.

**2. Lentiviral vector development**

(reviewed by [5,10]).

Four generations of LVs are currently considered; these differ from each other in terms of the number of genetic constructs used to drive the expression of the viral components, the number of wild-type genes retained as well as the number and type of heterologous *cis*-ele‐ ments used to increase vector titers and promote vector safety.

The system of three expression cassettes developed in 1996 by Naldini *et al.* [16] is consid‐ ered the first generation of LVs. In this system the packaging cassette has all structural pro‐ teins, with exception of Env glycoproteins, and all accessory and regulatory proteins. Later the 5' LTR was substituted by a strong promoter (CMV or RSV) and the 3' LTR by an SV40 or insulin poly(A) site to reduce the homology between the cassettes. To prevent the packag‐ ing of viral elements the Ψ and PBS were deleted. In the *env* expression cassette the gp120 from HIV-1 was replaced by other *env* genes as VSV-G or amphotrophic MLV envelope (Figure 4). Finally the transgene cassette was composed by the 5' LTR, the ψ with a truncat‐ ed *gag* gene, the RRE cis-acting region and the gene of interest under the control of a heterol‐ ogous promoter (usually CMV) and the 3'LTR [16,20].This system allowed in an easy way to achieve good titers but its level of safety was not very high. RCL could be generated just with three recombination events by homologous recombination between the viral sequences in all cassettes or endogenous retroviral sequences in cells. In order to improve the safety and decrease the cytotoxicity of LVs, the three plasmid system was maintained, but all ac‐ cessory genes not required for viral replication *in vitro* (vif, vpr, vpu, and nef) [21] were re‐ moved without negative effects on vector yield or infectivity. And in this way the second generation of LVs was developed (Figure 4) [22–25]. In the second generation, if by chance some RCL was generated, it would be unlikely to be pathogenic [26]. However the number of homologous events to generate RCL was the same as in the previous generation.

structs the homology between them was eliminated. These changes also allowed an inde‐ pendent expression of Rev since the sequences with suboptimal codon usage in HIV-1 genome, that conferred RNA instability and consequently lower expression, disappeared [32]. In the fourth generation (Figure 4) the homology between constructs were severely re‐ duced but the titers had also been affected comparing with systems with the Rev/RRE [32]. Also, with the independence of Rev/RRE system, the level of biosafety decreased since the number of homologous recombination events for RCL formation was again three. Maybe due to these drawbacks the fourth generation has not been extensively used. However the codon optimization technology had been used to decrease the homology between sequen‐

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

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293

Regarding the biosafety concerns about RNA mobilization and the possibility of generating RCLs, other improvements in packaging constructs were used and tested in transient LVs productions. These improvements relied on the concept of *split-genome* used for retroviral and lentiviral vector development but this time applied to the packaging construct. The *gagpol* sequences were divided by two or three independent expression cassettes, disarming the functional *gag-pol* structure that is essential for vector mobilization [34]. In these systems ad‐ ditional recombination events between the several expression cassettes are necessary to gen‐ erate RCLs which seems to contribute to a significant decrease of recombinant vectors formation with a functional *gag-pol* structure [35,36]. Although this increased LV safety the transduction efficiency and the LV production are challenged by the higher number of plas‐

**Figure 4.** Schematic representation of the four generations of lentiviral packaging constructs: A) First generation packaging vector. B) Second generation packaging vector. C) Third generation packaging vector. D) Fourth generation

ces, improve the expression of viral components and viral titers [33].

mids required [37].

packaging vector.

Reducing the lentiviral sequences by eliminating the *tat* and place the *rev* in an independent plasmid was the further step that originated the third generation of LV [27]. The *tar* sequence was replaced by a strong heterologous promoter. Therefore Tat protein was no longer neces‐ sary to increase the transgene transcription and *tat* gene was eliminated. This contributed for the reduction of lentiviral elements in the constructs. Rev was placed in an independent nonoverlapping plasmid increasing the safety since now four events of homologous recombina‐ tion were required for RCL formation [27]. With these new features, the vectors of third generation (Figure 4) presented a higher level of biosafety and, as the titers did not decreased, their use was widespread. Today they are the most commonly used LVs.

Although the formation of RCL was improbable, homologous recombination between the constructs was still possible since RRE sequence and part of packaging sequence in *gag* gene was in both transfer and structural packaging constructs. To solve these problems other sol‐ utions were developed originating the fourth generation of LV. The first approach used con‐ sisted in the replacement of the RRE sequences by heterologous sequences with similar functions that do not need the Rev protein. Some of these sequences were the Mason-Pfizer monkey virus constitutive transport element (CTE), the posttranscriptional control element (PCE) of the spleen necrosis virus and the human nuclear protein Sam68 [28–31]. The heter‐ ologous sequences increased the stability of the transcripts allowing their nuclear export. However the titers have decreased.

In 2000 a different approach based on codon optimization was implemented in lentiviral vector design [33]. This approach consists in perform silent mutations, changing the codon that codes for a certain aminoacid for other that codes for the same aminoacid, in principle, with a higher intracellular availability [32]. Applying this to the packaging and transfer con‐ structs the homology between them was eliminated. These changes also allowed an inde‐ pendent expression of Rev since the sequences with suboptimal codon usage in HIV-1 genome, that conferred RNA instability and consequently lower expression, disappeared [32]. In the fourth generation (Figure 4) the homology between constructs were severely re‐ duced but the titers had also been affected comparing with systems with the Rev/RRE [32]. Also, with the independence of Rev/RRE system, the level of biosafety decreased since the number of homologous recombination events for RCL formation was again three. Maybe due to these drawbacks the fourth generation has not been extensively used. However the codon optimization technology had been used to decrease the homology between sequen‐ ces, improve the expression of viral components and viral titers [33].

The system of three expression cassettes developed in 1996 by Naldini *et al.* [16] is consid‐ ered the first generation of LVs. In this system the packaging cassette has all structural pro‐ teins, with exception of Env glycoproteins, and all accessory and regulatory proteins. Later the 5' LTR was substituted by a strong promoter (CMV or RSV) and the 3' LTR by an SV40 or insulin poly(A) site to reduce the homology between the cassettes. To prevent the packag‐ ing of viral elements the Ψ and PBS were deleted. In the *env* expression cassette the gp120 from HIV-1 was replaced by other *env* genes as VSV-G or amphotrophic MLV envelope (Figure 4). Finally the transgene cassette was composed by the 5' LTR, the ψ with a truncat‐ ed *gag* gene, the RRE cis-acting region and the gene of interest under the control of a heterol‐ ogous promoter (usually CMV) and the 3'LTR [16,20].This system allowed in an easy way to achieve good titers but its level of safety was not very high. RCL could be generated just with three recombination events by homologous recombination between the viral sequences in all cassettes or endogenous retroviral sequences in cells. In order to improve the safety and decrease the cytotoxicity of LVs, the three plasmid system was maintained, but all ac‐ cessory genes not required for viral replication *in vitro* (vif, vpr, vpu, and nef) [21] were re‐ moved without negative effects on vector yield or infectivity. And in this way the second generation of LVs was developed (Figure 4) [22–25]. In the second generation, if by chance some RCL was generated, it would be unlikely to be pathogenic [26]. However the number

of homologous events to generate RCL was the same as in the previous generation.

their use was widespread. Today they are the most commonly used LVs.

However the titers have decreased.

292 Gene Therapy - Tools and Potential Applications

Reducing the lentiviral sequences by eliminating the *tat* and place the *rev* in an independent plasmid was the further step that originated the third generation of LV [27]. The *tar* sequence was replaced by a strong heterologous promoter. Therefore Tat protein was no longer neces‐ sary to increase the transgene transcription and *tat* gene was eliminated. This contributed for the reduction of lentiviral elements in the constructs. Rev was placed in an independent nonoverlapping plasmid increasing the safety since now four events of homologous recombina‐ tion were required for RCL formation [27]. With these new features, the vectors of third generation (Figure 4) presented a higher level of biosafety and, as the titers did not decreased,

Although the formation of RCL was improbable, homologous recombination between the constructs was still possible since RRE sequence and part of packaging sequence in *gag* gene was in both transfer and structural packaging constructs. To solve these problems other sol‐ utions were developed originating the fourth generation of LV. The first approach used con‐ sisted in the replacement of the RRE sequences by heterologous sequences with similar functions that do not need the Rev protein. Some of these sequences were the Mason-Pfizer monkey virus constitutive transport element (CTE), the posttranscriptional control element (PCE) of the spleen necrosis virus and the human nuclear protein Sam68 [28–31]. The heter‐ ologous sequences increased the stability of the transcripts allowing their nuclear export.

In 2000 a different approach based on codon optimization was implemented in lentiviral vector design [33]. This approach consists in perform silent mutations, changing the codon that codes for a certain aminoacid for other that codes for the same aminoacid, in principle, with a higher intracellular availability [32]. Applying this to the packaging and transfer con‐

Regarding the biosafety concerns about RNA mobilization and the possibility of generating RCLs, other improvements in packaging constructs were used and tested in transient LVs productions. These improvements relied on the concept of *split-genome* used for retroviral and lentiviral vector development but this time applied to the packaging construct. The *gagpol* sequences were divided by two or three independent expression cassettes, disarming the functional *gag-pol* structure that is essential for vector mobilization [34]. In these systems ad‐ ditional recombination events between the several expression cassettes are necessary to gen‐ erate RCLs which seems to contribute to a significant decrease of recombinant vectors formation with a functional *gag-pol* structure [35,36]. Although this increased LV safety the transduction efficiency and the LV production are challenged by the higher number of plas‐ mids required [37].

**Figure 4.** Schematic representation of the four generations of lentiviral packaging constructs: A) First generation packaging vector. B) Second generation packaging vector. C) Third generation packaging vector. D) Fourth generation packaging vector.

#### **2.2. Transfer vector**

The transfer vector is the expression cassette of the transgene that will be packaged into the viral vector and integrated in the cellular genome of the target cells. Besides the gene of in‐ terest and the commonly heterologous promoter for transgene expression, the transfer ex‐ pression cassette must have: the sequences responsible for the expression of the full-length transcript and its packaging into the newly formed virions in the producer cells; the sequen‐ ces that will interact with viral and cellular proteins to allow an efficient reverse transcrip‐ tion, transport into the cellular nucleus and proviral integration into target cells genome. Despite the simple design and the lack of sequences that code for viral proteins, the transfer vector also evolved over the time. This evolution was primarily focused on safety by reduc‐ ing and replacing the viral sequences by heterologous elements and in optimizing both safe‐ ty and efficiency by the addition of several *cis*-acting elements to the transfer cassette [10].

er/promoter sequences deleted can have a role in an efficient transcription termination [45]. In this context several improvements were done by the addition of heterologous elements to increase safety, expression and efficacy of LVs: heterologous polyadenylation signals in the 3´LTR could increase the efficiency of LVs and are particularly beneficial in the case of SIN LVs avoiding the read-through of cellular genes [40,46]; the chromatin insulators as the chicken hypersensitive site 4 (cHS4) sequence core from the β-globin locus control region (LCR) can reduce the interference from the neighboring regions in the vector and transgene expression [48]. Also these can improve the LV safety avoiding the full-length vector tran‐ scription or reducing long-distance effects of the integrated transgene promoter on neigh‐ boring cellular genes in the target cells. Additionally to the increased safety, insulators can help to maintain the gene expression over time preventing transcriptional silencing events in both producer and target cells [47–49]; incorporation of certain post-transcriptional regu‐ latory elements (PRE) like the woodchuck hepatitis virus posttranscriptional regulatory ele‐ ment (WPRE) near the 3' untranslated region can also decrease the read-through in SIN vectors increasing the transgene expression and viral titers, [50–53]. The firsts WPRE se‐ quences used contained part of a sequence that codes for a protein (WHV X) that has been reported a few times as related with carcinoma formation, posing safety concerns. However a further improved WPRE was created without this potential harmful sequence [54]; The cPPT sequence contributes for efficient reverse transcription and the proviral nuclear import processes. Although this non-essential sequence was not used in the firsts transfer vectors,

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

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295

its re-insertion increased the gene transfer efficiency [55–57].

**Figure 5.** Schematic representation of a non-SIN transfer vector (A) and a SIN transfer vector (B).

LVs, as other retroviral vectors, can incorporate in their viral particles Env glycoproteins from other enveloped viruses, a feature denominated pseudotyping. This was firstly demon‐ strated for the HIV-1-based lentiviral vector using a Moloney Murine Leukemia Virus am‐ photropic envelope 4070A (A-MoMLV) [13] and an Human T-cell Leukemia Virus Type I

**3. Pseudotyping**

(HTLV-I) envelope [14].

The transfer vectors usually used in the first and second generation of packaging constructs LVs were composed by the 5' LTR which include the TAR sequence, the PBS, the SD, the Ψ, the 5' part of *gag* gene, the RRE sequence, the SA, an heterologous promoter followed by the gene of interest, the PPT and polyA within the 3' LTR. The first hundreds of base pairs of *gag* are included after the packaging signal to increase the packaging efficiency (Figure 5). To avoid *gag* translation the initiation codon is usually mutated or cloned out of frame [16,20]. However, like it was previously found for γ-RVs, this transfer vector design with both wild-type LTRs can lead to integration genotoxicity and facilitates the mobilization of the transgene in the case of posterior infection of transduced cells [38]. To overcome these biosafety problems the LTRs of the transfer vector suffered additional changes. One of the first modifications was the replacement of the enhancer/promoter and Tar sequence of the 5' LTR by a strong heterologous promoter allowing the transcription of the full-length viral RNA in a Tat-independent manner [25]. In addition the wild-type enhancer/promoter se‐ quences in the U3 region of the 3´LTR were deleted originating the self-inactivating (SIN) LVs [27,39,40], as it had already been done for γ-RVs [41].

The SIN design (Figure 5) generates in the target cells a proviral vector without enhancer/ promoter sequences in both LTRs. In producer cells the packaged RNA transcript does have the heterologous promoter in the 5' end. Afterwards, in the target cells, during the reverse transcription, the U3 region of 3' LTR is copied and transferred to the 5'LTR. This transcrip‐ tional inactivation offered by the SIN design presents several safety advantages: prevents the formation of potentially packageable viral transcripts from the 5´LTR and consequently prevents vector mobilization by prior infection with a replicative retrovirus [39,42]; reduces the risk of insertional mutagenesis induced by the transcriptional interference of the LTRs in the neighboring sequences that can lead to the activation or up-regulation of oncogenes [43]; and lowers the risk of RCL formation by the reduction of the sequences with homology with wild-type virus.

The adoption of SIN design did not affected LV production as it happened with γ-RVs [27,39,40,44]. However both LVs and γ-RVs displayed high frequencies of read-through of the 3' polyadenylation signal which can lead to the transcription of cellular sequences as on‐ cogenes. This inefficient termination of transcription could suggest that some of the enhanc‐ er/promoter sequences deleted can have a role in an efficient transcription termination [45]. In this context several improvements were done by the addition of heterologous elements to increase safety, expression and efficacy of LVs: heterologous polyadenylation signals in the 3´LTR could increase the efficiency of LVs and are particularly beneficial in the case of SIN LVs avoiding the read-through of cellular genes [40,46]; the chromatin insulators as the chicken hypersensitive site 4 (cHS4) sequence core from the β-globin locus control region (LCR) can reduce the interference from the neighboring regions in the vector and transgene expression [48]. Also these can improve the LV safety avoiding the full-length vector tran‐ scription or reducing long-distance effects of the integrated transgene promoter on neigh‐ boring cellular genes in the target cells. Additionally to the increased safety, insulators can help to maintain the gene expression over time preventing transcriptional silencing events in both producer and target cells [47–49]; incorporation of certain post-transcriptional regu‐ latory elements (PRE) like the woodchuck hepatitis virus posttranscriptional regulatory ele‐ ment (WPRE) near the 3' untranslated region can also decrease the read-through in SIN vectors increasing the transgene expression and viral titers, [50–53]. The firsts WPRE se‐ quences used contained part of a sequence that codes for a protein (WHV X) that has been reported a few times as related with carcinoma formation, posing safety concerns. However a further improved WPRE was created without this potential harmful sequence [54]; The cPPT sequence contributes for efficient reverse transcription and the proviral nuclear import processes. Although this non-essential sequence was not used in the firsts transfer vectors, its re-insertion increased the gene transfer efficiency [55–57].

**Figure 5.** Schematic representation of a non-SIN transfer vector (A) and a SIN transfer vector (B).

#### **3. Pseudotyping**

**2.2. Transfer vector**

294 Gene Therapy - Tools and Potential Applications

wild-type virus.

The transfer vector is the expression cassette of the transgene that will be packaged into the viral vector and integrated in the cellular genome of the target cells. Besides the gene of in‐ terest and the commonly heterologous promoter for transgene expression, the transfer ex‐ pression cassette must have: the sequences responsible for the expression of the full-length transcript and its packaging into the newly formed virions in the producer cells; the sequen‐ ces that will interact with viral and cellular proteins to allow an efficient reverse transcrip‐ tion, transport into the cellular nucleus and proviral integration into target cells genome. Despite the simple design and the lack of sequences that code for viral proteins, the transfer vector also evolved over the time. This evolution was primarily focused on safety by reduc‐ ing and replacing the viral sequences by heterologous elements and in optimizing both safe‐ ty and efficiency by the addition of several *cis*-acting elements to the transfer cassette [10]. The transfer vectors usually used in the first and second generation of packaging constructs LVs were composed by the 5' LTR which include the TAR sequence, the PBS, the SD, the Ψ, the 5' part of *gag* gene, the RRE sequence, the SA, an heterologous promoter followed by the gene of interest, the PPT and polyA within the 3' LTR. The first hundreds of base pairs of *gag* are included after the packaging signal to increase the packaging efficiency (Figure 5). To avoid *gag* translation the initiation codon is usually mutated or cloned out of frame [16,20]. However, like it was previously found for γ-RVs, this transfer vector design with both wild-type LTRs can lead to integration genotoxicity and facilitates the mobilization of the transgene in the case of posterior infection of transduced cells [38]. To overcome these biosafety problems the LTRs of the transfer vector suffered additional changes. One of the first modifications was the replacement of the enhancer/promoter and Tar sequence of the 5' LTR by a strong heterologous promoter allowing the transcription of the full-length viral RNA in a Tat-independent manner [25]. In addition the wild-type enhancer/promoter se‐ quences in the U3 region of the 3´LTR were deleted originating the self-inactivating (SIN)

The SIN design (Figure 5) generates in the target cells a proviral vector without enhancer/ promoter sequences in both LTRs. In producer cells the packaged RNA transcript does have the heterologous promoter in the 5' end. Afterwards, in the target cells, during the reverse transcription, the U3 region of 3' LTR is copied and transferred to the 5'LTR. This transcrip‐ tional inactivation offered by the SIN design presents several safety advantages: prevents the formation of potentially packageable viral transcripts from the 5´LTR and consequently prevents vector mobilization by prior infection with a replicative retrovirus [39,42]; reduces the risk of insertional mutagenesis induced by the transcriptional interference of the LTRs in the neighboring sequences that can lead to the activation or up-regulation of oncogenes [43]; and lowers the risk of RCL formation by the reduction of the sequences with homology with

The adoption of SIN design did not affected LV production as it happened with γ-RVs [27,39,40,44]. However both LVs and γ-RVs displayed high frequencies of read-through of the 3' polyadenylation signal which can lead to the transcription of cellular sequences as on‐ cogenes. This inefficient termination of transcription could suggest that some of the enhanc‐

LVs [27,39,40], as it had already been done for γ-RVs [41].

LVs, as other retroviral vectors, can incorporate in their viral particles Env glycoproteins from other enveloped viruses, a feature denominated pseudotyping. This was firstly demon‐ strated for the HIV-1-based lentiviral vector using a Moloney Murine Leukemia Virus am‐ photropic envelope 4070A (A-MoMLV) [13] and an Human T-cell Leukemia Virus Type I (HTLV-I) envelope [14].

In general the pseudotyped LVs have the tropism of the virus where glycoproteins are de‐ rived from, but there are some exceptions such as the glycoprotein of the Mokola virus, where the pseudotyped vectors did not presented the specific neurotropism of the paren‐ tal virus [58]. This ability of LVs to be pseudotyped showed to be advantageous since sev‐ eral glycoproteins could be tested to improve the transduction of cells with different receptors. As an example, HIV-based LVs pseudotyped with glycoprotein derived from the Rabies virus PV strain exhibited a great efficiency and neuronal tropism among the tested envelopes [59].

**Species/Envelope Vectors Comments References**

Very wide tropism. Presents resistance to high-speed centrifugation. Cytotoxic for producer cells if expressed constitutively. Susceptible to complement-mediated degradation which can be minimized by PEGylation

More efficient and less toxic than VSV-G in

Transduces hepatocytes, glia cells and

vector targeting

Ebola HIV-1 Efficiently transduces airway epithelium [72]

cells of the hematopoietic system [70][71]

Low toxicity [73]

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

neuronal axons [24]

subretinal injection [24][74]

Able to transduce most cells [18][16]

neurons [75][76]

[16][64][65][66][66][66][67 - 67][67 – 69]

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297

[77]

HIV-1 HIV-2 FIV EIAV SIV BIV JDV CAEV

HIV-1 SIV

SIV HIV-1 FIV

HIV-1 FIV

HIV-1 SIV

ic vesicles formed after antibody binding [86].

improve infection specificity of LVs [87–91].

Rabies HIV-1 Rabies confers retrograde transport in

Mokola EIAV Mokola selectively transduces RPE upon

Sindbis virus HIV-1 pH-dependentendosomal entry. Useful for

Influenza virus hemagglutinin HIV-1 Transduces airway epithelium [72]

**Table 1.** Lentiviral Vectors pseudotyped with various heterologous viral glycoproteins. Adapted from [78,79].

The envelope proteins engineered by fusion of natural ligands were in general able to bind to target cells. However the fusion domain of Env resulted generally in low vector titers since the ligand inhibits the fusogenic properties of the Env protein that allows for viral en‐ try [85]. This approach seems to be more challenging but there are already improvements. One example is the LV pseudotyped with a chimeric glycoprotein of Sindbis virus covalent‐ ly linked with mouse/human chimerical CD20-specific antibody which resulted in specific and stable transduction of CD20+ human lymphoid B cells. In this case the membrane fusion is triggered by the glycoprotein, in a pH-dependent manner, and it happens inside endocyt‐

Other glycoproteins and ligands are being tested and used as well as alternative strategies to

Vesicular stomatitis virus (VSV-G)

Feline endogenous retrovirus (RD114)

Lymphocytic choriomeningitis virus (LCMV)

Ross River virus

Moloney murine leukemia virus 4070 envelope

In addition to the tropism of LVs, the Env glycoproteins also affect vector structure and sta‐ bility, the interactions with the target cells and the LV behavior during the infection. One example is LVs pseudotyped with rabies virus glycoprotein which allow for the retrograde axonal transport and access to the nervous system after peripheral infection [60]. Another example is the stability conferred to LVs by the VSV-G glycoproteins. The VSV-G glycopro‐ teins are one of the most used Env proteins due to their wide tropism, with high titers ach‐ ieved, great stability and resistance conferred to the LVs that allows for their concentration by ultracentrifugation. In addition they resist to freeze-thaw cycles, an important factor for storage of the vectors [16,18,19,61]. Despite these positive characteristics, the VSV-G protein is toxic for producer cells if expressed constitutively [17] and is inactivated by human serum complement [62], although this inactivation can be minimized using VSV-G conjugated with poly(ethylene glycol) [63].

Up to the present, several glycoproteins were used to pseudotype LVs (Table 1) each one presenting specific advantages and disadvantages that also depend on the LV application.

Although LVs pseudotyped with different Env glycoproteins present different tropisms, be‐ ing some tropisms more selective than others, in general these are not specific for a particu‐ lar cell type as happens with HIV-1 glycoproteins [80,81]. For instance, the Ebola Zaire (EboZ) glycoprotein seems to be superior to other glycoproteins in the transduction of apical airway epithelia [72]. However also has been shown to transduce liver, heart, and muscle tissues [82].

This lack of specificity is not ideal from a clinical point of view, especially for *in vivo* gene therapy applications since it can lead to the infection of cells that do not need to be trans‐ duced [83].

Several strategies have been used to increase the specificity of infection in order to retarget the LVs to a cell of interest. These strategies consisted in genetic engineering of virus envel‐ ops by deletion of some domains or fusing molecule-ligands (growth factors, hormones, peptides or single-chain antibodies) in several locations of the viral glycoproteins. The pur‐ pose is to choose cellular receptors specifically expressed on the target cells that will interact with the chimeric glycoproteins, restricting this way the vector tropism. A successful exam‐ ple was the removal of the heparan sulfate binding domain from the Sindbis virus envelope protein which effectively restricted the tropism of pseudotyped LVs to dendritic cells. This genetic modified Env protein interacts solely with the C-type lectin-like receptor almost ex‐ clusively on primary dendritic cells unlike its natural counterpart [84].


In general the pseudotyped LVs have the tropism of the virus where glycoproteins are de‐ rived from, but there are some exceptions such as the glycoprotein of the Mokola virus, where the pseudotyped vectors did not presented the specific neurotropism of the paren‐ tal virus [58]. This ability of LVs to be pseudotyped showed to be advantageous since sev‐ eral glycoproteins could be tested to improve the transduction of cells with different receptors. As an example, HIV-based LVs pseudotyped with glycoprotein derived from the Rabies virus PV strain exhibited a great efficiency and neuronal tropism among the

In addition to the tropism of LVs, the Env glycoproteins also affect vector structure and sta‐ bility, the interactions with the target cells and the LV behavior during the infection. One example is LVs pseudotyped with rabies virus glycoprotein which allow for the retrograde axonal transport and access to the nervous system after peripheral infection [60]. Another example is the stability conferred to LVs by the VSV-G glycoproteins. The VSV-G glycopro‐ teins are one of the most used Env proteins due to their wide tropism, with high titers ach‐ ieved, great stability and resistance conferred to the LVs that allows for their concentration by ultracentrifugation. In addition they resist to freeze-thaw cycles, an important factor for storage of the vectors [16,18,19,61]. Despite these positive characteristics, the VSV-G protein is toxic for producer cells if expressed constitutively [17] and is inactivated by human serum complement [62], although this inactivation can be minimized using VSV-G conjugated with

Up to the present, several glycoproteins were used to pseudotype LVs (Table 1) each one presenting specific advantages and disadvantages that also depend on the LV application.

Although LVs pseudotyped with different Env glycoproteins present different tropisms, be‐ ing some tropisms more selective than others, in general these are not specific for a particu‐ lar cell type as happens with HIV-1 glycoproteins [80,81]. For instance, the Ebola Zaire (EboZ) glycoprotein seems to be superior to other glycoproteins in the transduction of apical airway epithelia [72]. However also has been shown to transduce liver, heart, and muscle

This lack of specificity is not ideal from a clinical point of view, especially for *in vivo* gene therapy applications since it can lead to the infection of cells that do not need to be trans‐

Several strategies have been used to increase the specificity of infection in order to retarget the LVs to a cell of interest. These strategies consisted in genetic engineering of virus envel‐ ops by deletion of some domains or fusing molecule-ligands (growth factors, hormones, peptides or single-chain antibodies) in several locations of the viral glycoproteins. The pur‐ pose is to choose cellular receptors specifically expressed on the target cells that will interact with the chimeric glycoproteins, restricting this way the vector tropism. A successful exam‐ ple was the removal of the heparan sulfate binding domain from the Sindbis virus envelope protein which effectively restricted the tropism of pseudotyped LVs to dendritic cells. This genetic modified Env protein interacts solely with the C-type lectin-like receptor almost ex‐

clusively on primary dendritic cells unlike its natural counterpart [84].

tested envelopes [59].

296 Gene Therapy - Tools and Potential Applications

poly(ethylene glycol) [63].

tissues [82].

duced [83].

**Table 1.** Lentiviral Vectors pseudotyped with various heterologous viral glycoproteins. Adapted from [78,79].

The envelope proteins engineered by fusion of natural ligands were in general able to bind to target cells. However the fusion domain of Env resulted generally in low vector titers since the ligand inhibits the fusogenic properties of the Env protein that allows for viral en‐ try [85]. This approach seems to be more challenging but there are already improvements. One example is the LV pseudotyped with a chimeric glycoprotein of Sindbis virus covalent‐ ly linked with mouse/human chimerical CD20-specific antibody which resulted in specific and stable transduction of CD20+ human lymphoid B cells. In this case the membrane fusion is triggered by the glycoprotein, in a pH-dependent manner, and it happens inside endocyt‐ ic vesicles formed after antibody binding [86].

Other glycoproteins and ligands are being tested and used as well as alternative strategies to improve infection specificity of LVs [87–91].

#### **4. Lentiviral vector production**

The continuous research in LV development in the last twenty years and the acquired knowledge from the previous development of γ-RVs allowed the production of LVs with a significant biosafety level. However to apply LVs to clinical use they need to be easily and inexpensively produced and purified at a large-scale since, high concentrations of lentiviral particles are usually needed for efficient gene transfer in primary cells and the treatment of a single patient may require several liters of viral supernatant [92,93]

phate or polyethylenimine) and after 24 to 72 hours the LV are harvested [93]. This produc‐ tion system is fast and can be easily adapted to produce LVs with new genes of interest or with other Env glycoproteins. It is a simple process to apply at small scales commonly used in research, unlike the laborious development of a packaging cell line. However transient production is not the ideal choice for large and clinical LV productions since it is difficult to scale-up and requires large amounts of Good Manufacturing Practices (GMP) grade plasmid expressions cassettes turning the production more expensive [93,103]. In addition, transient LV production brings some biosafety problems like recombination between expression cas‐ settes that could originate or facilitate the RCL formation. The recombination can occur in the mixture of transfection, inside the producer cells or during reverse transcription in the target cells since, generally after transfection cells have several copies of the plasmids which can contribute for the co-packaging of RNA transcripts [33,104]. Also batch to batch variabil‐ ity is common in transient productions since a population of transfected cells that expresses viral elements from episomal cassettes is generated. This can further complicate biosafety

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299

Nevertheless transient LV production is commonly used and recently it was shown that high titers of HIV-based LVs for clinical applications can be obtained by transient calcium

> **Maximal titers (I.P./ml**)

3rd VSV-G 1x106 PEI-mediated

3rd VSV-G 1x108 PEI-mediated

3rd VSV-G 2x109 Transfection with

3rd VSV-G 1x108 Transfection by Flow

**Table 2.** Transient LV productions. In this table they are presented several features of recent lentiviral productions in a

There are several transfection agents that can be used to transfect mammalian cells as cal‐ cium phosphate, polyethylenimine (PEI) and cationic molecules (such as LipofectAMINE® and FuGENE®). For large scale only Ca-phosphate and PEI are used since the others are much more expensive. Both reagents are efficient but PEI is usually preferred since Caphosphate efficiency is highly sensitive to pH variations and can require serum or albu‐ min to reduce Ca-phosphate cytotoxicity, unlike PEI [93]. However their use can raise some purity problems and can be cost-ineffective. Recently a method that does not use chemicals for transfection, flow electroporation, was used for transiently LV production at

**Observations Reference**

transfection [107]

transfection [101]

calcium phosphate [99]

Electroporation [105]

phosphate transfection at large-scale under GMP conditions (Table 2) [99].

**generation Envelope**

validation steps.

293 E

HEK293

293T

293T

transient manner.

**Cell origin Vector Packaging**

SIN HIV-1 based

> HIV-1 based

> HIV-1 based

> HIV-1 based

For large-scale and clinical-grade LV productions, a stable LV producer cell line seems to be the best approach for increased safety and well characterized production process. However, unlike γ-RVs, the development of LV packaging cell lines has been more challenging be‐ cause of the cytotoxicity of some viral proteins like Tat, Nef, Vpr, Rev and PR [94]. Also VSV-G envelope, the typically envelope of choice for LV production because of its wide tropism and stability conferred to viral particles, is toxic for the producer cells. The VSV-G envelope can however be replaced by other non-toxic envelopes as the feline endogenous virus RD114 or the amphotropic MLV 4070A Env glycoproteins [33,95] and thus among the cytotoxic lentiviral proteins just the protease is still necessary for lentiviral vector produc‐ tion with the current packaging systems [93].

HIV protease mediates its toxicity *in vitro* and *in vivo* by cleaving procaspase 8, originating the casp8p41 fragment. This fragment induces mitochondrial depolarization leading to mi‐ tochondrial release of cytochrome C, activation of the downstream caspases 9 and 3 and nu‐ clear fragmentation [96–98]. This cytotoxicity has hampered the development of stable cell lines.

The most used cells for LV production are the human embryonic kidney HEK-293 cell line and its genetic derivates the 293T (expressing the SV40 large-T antigen) and 293E (express‐ ing the Epstein-Barr virus nuclear antigen-1, EBNA-1) cell lines. For clinical application hu‐ man 293 and 293T cells have been the exclusive cell substrates [93]. Both cell lines can be used to produce LV in adherent systems and both can be easily adapted to serum-free sus‐ pension cultures. The 293T cells are most widely used because presents superior LV pro‐ ductivities compared with HEK-293 cells. However the HEK-293 cell line may have an advantage in terms of safety as it lacks the SV40 large T antigen encoding gene (expressed in 293T cells) which is oncogenic [93,99,100]. In some research works other human or mon‐ key derived cells have been used (other 293 derived clones, HeLa, HT1080, TE671, COS-1, COS-7, CV-1), although most of them showed lower LV titers [101]. However, COS-1 cells have shown to be capable of producing 3-4 times improved vector quality (expressed in in‐ fectious vector titer per ng of CA protein, p24), comparing with 293T cells, under serumcontaining conditions [102].

#### **4.1. Transient lentiviral vector production**

Commonly LVs are produced by co-transfecting cells with the several expression cassettes harboring the transgene and the viral elements using chemical agents (e.g. calcium phos‐ phate or polyethylenimine) and after 24 to 72 hours the LV are harvested [93]. This produc‐ tion system is fast and can be easily adapted to produce LVs with new genes of interest or with other Env glycoproteins. It is a simple process to apply at small scales commonly used in research, unlike the laborious development of a packaging cell line. However transient production is not the ideal choice for large and clinical LV productions since it is difficult to scale-up and requires large amounts of Good Manufacturing Practices (GMP) grade plasmid expressions cassettes turning the production more expensive [93,103]. In addition, transient LV production brings some biosafety problems like recombination between expression cas‐ settes that could originate or facilitate the RCL formation. The recombination can occur in the mixture of transfection, inside the producer cells or during reverse transcription in the target cells since, generally after transfection cells have several copies of the plasmids which can contribute for the co-packaging of RNA transcripts [33,104]. Also batch to batch variabil‐ ity is common in transient productions since a population of transfected cells that expresses viral elements from episomal cassettes is generated. This can further complicate biosafety validation steps.

**4. Lentiviral vector production**

298 Gene Therapy - Tools and Potential Applications

tion with the current packaging systems [93].

lines.

containing conditions [102].

**4.1. Transient lentiviral vector production**

The continuous research in LV development in the last twenty years and the acquired knowledge from the previous development of γ-RVs allowed the production of LVs with a significant biosafety level. However to apply LVs to clinical use they need to be easily and inexpensively produced and purified at a large-scale since, high concentrations of lentiviral particles are usually needed for efficient gene transfer in primary cells and the treatment of a

For large-scale and clinical-grade LV productions, a stable LV producer cell line seems to be the best approach for increased safety and well characterized production process. However, unlike γ-RVs, the development of LV packaging cell lines has been more challenging be‐ cause of the cytotoxicity of some viral proteins like Tat, Nef, Vpr, Rev and PR [94]. Also VSV-G envelope, the typically envelope of choice for LV production because of its wide tropism and stability conferred to viral particles, is toxic for the producer cells. The VSV-G envelope can however be replaced by other non-toxic envelopes as the feline endogenous virus RD114 or the amphotropic MLV 4070A Env glycoproteins [33,95] and thus among the cytotoxic lentiviral proteins just the protease is still necessary for lentiviral vector produc‐

HIV protease mediates its toxicity *in vitro* and *in vivo* by cleaving procaspase 8, originating the casp8p41 fragment. This fragment induces mitochondrial depolarization leading to mi‐ tochondrial release of cytochrome C, activation of the downstream caspases 9 and 3 and nu‐ clear fragmentation [96–98]. This cytotoxicity has hampered the development of stable cell

The most used cells for LV production are the human embryonic kidney HEK-293 cell line and its genetic derivates the 293T (expressing the SV40 large-T antigen) and 293E (express‐ ing the Epstein-Barr virus nuclear antigen-1, EBNA-1) cell lines. For clinical application hu‐ man 293 and 293T cells have been the exclusive cell substrates [93]. Both cell lines can be used to produce LV in adherent systems and both can be easily adapted to serum-free sus‐ pension cultures. The 293T cells are most widely used because presents superior LV pro‐ ductivities compared with HEK-293 cells. However the HEK-293 cell line may have an advantage in terms of safety as it lacks the SV40 large T antigen encoding gene (expressed in 293T cells) which is oncogenic [93,99,100]. In some research works other human or mon‐ key derived cells have been used (other 293 derived clones, HeLa, HT1080, TE671, COS-1, COS-7, CV-1), although most of them showed lower LV titers [101]. However, COS-1 cells have shown to be capable of producing 3-4 times improved vector quality (expressed in in‐ fectious vector titer per ng of CA protein, p24), comparing with 293T cells, under serum-

Commonly LVs are produced by co-transfecting cells with the several expression cassettes harboring the transgene and the viral elements using chemical agents (e.g. calcium phos‐

single patient may require several liters of viral supernatant [92,93]

Nevertheless transient LV production is commonly used and recently it was shown that high titers of HIV-based LVs for clinical applications can be obtained by transient calcium phosphate transfection at large-scale under GMP conditions (Table 2) [99].


**Table 2.** Transient LV productions. In this table they are presented several features of recent lentiviral productions in a transient manner.

There are several transfection agents that can be used to transfect mammalian cells as cal‐ cium phosphate, polyethylenimine (PEI) and cationic molecules (such as LipofectAMINE® and FuGENE®). For large scale only Ca-phosphate and PEI are used since the others are much more expensive. Both reagents are efficient but PEI is usually preferred since Caphosphate efficiency is highly sensitive to pH variations and can require serum or albu‐ min to reduce Ca-phosphate cytotoxicity, unlike PEI [93]. However their use can raise some purity problems and can be cost-ineffective. Recently a method that does not use chemicals for transfection, flow electroporation, was used for transiently LV production at large-scale [105]. The electroporation systems are normally used to transfect small vol‐ umes but flow electroporation addresses this limitation by continuously passing the de‐ sired volume of a cell and DNA suspension between two electrodes [106]. The procedure can be effectively scaled up for large bioprocessing avoiding additional costs and purifica‐ tion problems (Table 2) [105].

In conditional packaging cell lines the expression of cytotoxic proteins is under control of inducible promoters and the number ofcells and growth conditions can be controlled, start‐ ing the LV production at a defined moment by adding an inductor or removing the suppres‐ sor from the culture medium. Originally the titers were low but further improvements in the expression cassettes design and optimization of the induction parameters led to similar lev‐ els of transient productions. However, such systems can only produce LV for a few days be‐ cause of the activity of the cytotoxic viral proteins. In addition these packaging cells have often shown to be instable due to leaky expression of the cytotoxic viral elements that are under control of the inducible promoters and the need to add an inductor to the medium in

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301

In 2003 Ikeda and co-workers have reported the development of a non-inducible packaging cell line that continuously produces LV for three months in culture (Table 3). However, sig‐ nificant titers could only be obtained after MLV-based vector transduction. This procedure raises serious problems from the biosafety point of view, since it increases the chances of RCL by homologous recombination, posing further concerns of co-packaging [37]. Never‐ theless it was shown that it is possible to establish a cell line that can continuously produce

Lentiviral vectors have emerged as powerful and versatile vectors for *ex vivo* and *in vivo* gene transfer into dividing and non-dividing cells. The particular characteristics of LVs al‐ lied to their marked development during the last years have triggered the attention of differ‐ ent fields, consequently a vast range of applications for these vectors, from fundamental biological research to human gene therapy have appeared. One of the applications of LVs is in genome-wide functional studies. The combination of synthetic siRNAs (small interfering RNA) or shRNAs (short hairpin RNAs) that can suppress the expression of genes of interest in mammalian cells [114], with engineered LVs allowed the formation of libraries like the Netherlands Cancer Institute (NKI) libraries, the RNAi consortium (TRC) libraries, the Han‐ non–Elledge libraries, and the System Biosciences (SystemBio) libraries for high-throughput loss-of-function screens in a wide range of mammalian cells [115]. For example, the TRC shRNA library has nearly 300,000 shRNAs targeting for 60,000 human and mouse genes [116]. The ability of LVs to achieve stable high-efficiency gene silencing in a wide variety of cells including primary cells, that are difficult to transduce, or non-dividing cells such as

Other application for LVs is in animal transgenesis. Genetic-modified animals can be created by infection of fertilized or unfertilized oocytes, single-cell embryos, early blastocysts, em‐ bryonic stem cells or by transduction of cells that are used as donors of nucleus for somatic cell nuclear transfer (SCNT) [10]. These animals (transgenic mices, rats, pigs, cows, chicken, monkeys) are used to understand gene function or biological processes, for validation of drug targets, for production of human therapeutic proteins and as preclinical models for hu‐

some systems can add further difficulties to the purification process [93].

LV although, until now no additional reports for this system appeared.

neurons thus greatly expanded the possibility of the RNAi screens [117].

**5. Lentiviral vector applications**

man diseases [118].

#### **4.2. Stable lentiviral vector production**

To overcome the biosafey problems in LV transient productions, inducible packaging cells lines have been developed (Table 3). The development of these systems is more time-con‐ suming since after insertion of each expression cassette the population of stably transfected cells is usually screened for the best producer clone, like for γ-RVs, to maximize the LV pro‐ duction. However, these packaging cell lines are derived from one clone, therefore all the cells have the same growth and LV production behavior being the LV productions reprodu‐ cible. This allows the generation of GMP cell banks, increasing safety conditions.


**Table 3.** Lentiviral vector packaging cell lines. In this table they are presented several features of available packaging cell lines for LV production.

In conditional packaging cell lines the expression of cytotoxic proteins is under control of inducible promoters and the number ofcells and growth conditions can be controlled, start‐ ing the LV production at a defined moment by adding an inductor or removing the suppres‐ sor from the culture medium. Originally the titers were low but further improvements in the expression cassettes design and optimization of the induction parameters led to similar lev‐ els of transient productions. However, such systems can only produce LV for a few days be‐ cause of the activity of the cytotoxic viral proteins. In addition these packaging cells have often shown to be instable due to leaky expression of the cytotoxic viral elements that are under control of the inducible promoters and the need to add an inductor to the medium in some systems can add further difficulties to the purification process [93].

In 2003 Ikeda and co-workers have reported the development of a non-inducible packaging cell line that continuously produces LV for three months in culture (Table 3). However, sig‐ nificant titers could only be obtained after MLV-based vector transduction. This procedure raises serious problems from the biosafety point of view, since it increases the chances of RCL by homologous recombination, posing further concerns of co-packaging [37]. Never‐ theless it was shown that it is possible to establish a cell line that can continuously produce LV although, until now no additional reports for this system appeared.

#### **5. Lentiviral vector applications**

large-scale [105]. The electroporation systems are normally used to transfect small vol‐ umes but flow electroporation addresses this limitation by continuously passing the de‐ sired volume of a cell and DNA suspension between two electrodes [106]. The procedure can be effectively scaled up for large bioprocessing avoiding additional costs and purifica‐

To overcome the biosafey problems in LV transient productions, inducible packaging cells lines have been developed (Table 3). The development of these systems is more time-con‐ suming since after insertion of each expression cassette the population of stably transfected cells is usually screened for the best producer clone, like for γ-RVs, to maximize the LV pro‐ duction. However, these packaging cell lines are derived from one clone, therefore all the cells have the same growth and LV production behavior being the LV productions reprodu‐

> **Maximal titers (I.P./ml)**

2nd VSV-G 1x107 Tet-off [108]

3rd VSV-G 3.4x107 Tet-on [111]

3rd VSV-G 7.4x105 Tet-on [112]

1.2x107 1.6x106 8.5x106

**Table 3.** Lentiviral vector packaging cell lines. In this table they are presented several features of available packaging

**Observations Reference**

[109]

[110]

[103]

[113]

Ecdysone inducible system. Codonoptimized gag-pol

Ponasterone inducible system. Codonoptimized gag-pol

Tet-off. Codonoptimized gag-pol

Introduction of vector by concatemeric array transfection. Tet-off

Continuous system.

Codon-optimized gag-pol [33]

cible. This allows the generation of GMP cell banks, increasing safety conditions.

3rd VSV-G 1.8x105

3rd VSV-G 1x105

2nd VSV-G 3x105

3rd VSV-G 5x107

Ampho GaLV RDpro

**generation Envelope**

tion problems (Table 2) [105].

300 Gene Therapy - Tools and Potential Applications

**Cell origin Vector Packaging**

HIV-1 based

HIV-1 based

SIVbased

HIV-1 based

HIV-1 based

EIAV based

SIVbased

HIV-1 based

2nd

293T

293T

293T

293T

293T

293T

293T

293T

cell lines for LV production.

**4.2. Stable lentiviral vector production**

Lentiviral vectors have emerged as powerful and versatile vectors for *ex vivo* and *in vivo* gene transfer into dividing and non-dividing cells. The particular characteristics of LVs al‐ lied to their marked development during the last years have triggered the attention of differ‐ ent fields, consequently a vast range of applications for these vectors, from fundamental biological research to human gene therapy have appeared. One of the applications of LVs is in genome-wide functional studies. The combination of synthetic siRNAs (small interfering RNA) or shRNAs (short hairpin RNAs) that can suppress the expression of genes of interest in mammalian cells [114], with engineered LVs allowed the formation of libraries like the Netherlands Cancer Institute (NKI) libraries, the RNAi consortium (TRC) libraries, the Han‐ non–Elledge libraries, and the System Biosciences (SystemBio) libraries for high-throughput loss-of-function screens in a wide range of mammalian cells [115]. For example, the TRC shRNA library has nearly 300,000 shRNAs targeting for 60,000 human and mouse genes [116]. The ability of LVs to achieve stable high-efficiency gene silencing in a wide variety of cells including primary cells, that are difficult to transduce, or non-dividing cells such as neurons thus greatly expanded the possibility of the RNAi screens [117].

Other application for LVs is in animal transgenesis. Genetic-modified animals can be created by infection of fertilized or unfertilized oocytes, single-cell embryos, early blastocysts, em‐ bryonic stem cells or by transduction of cells that are used as donors of nucleus for somatic cell nuclear transfer (SCNT) [10]. These animals (transgenic mices, rats, pigs, cows, chicken, monkeys) are used to understand gene function or biological processes, for validation of drug targets, for production of human therapeutic proteins and as preclinical models for hu‐ man diseases [118].

Lentiviral vectors are being increasingly used for the cell genetic modification leading to cell-engineering applications. Stable gene transduction can be used for *in vivo* imaging of vector infected cells. *In vivo* imaging studies of cells, including stem cells, have become in‐ creasingly important to understand cell distribution, differentiation, migration, function, and transgene expression in animal models. As an example, LVs expressing the firefly luci‐ ferase gene were used to monitor human embryonic stem cell (hESC) engraftment and proliferation in live mice after transplantation [119]. LVs can also be used to cellular re‐ programming of somatic cells. More specifically, the promising induced pluripotent stem cells (iPS) can be generated from a somatic cell by transduction of four key transcription factors, Oct4, Sox2, Klf4, and c-Myc, using LVs[120,121]. iPS can be used to study stem cell biology, as a cellular platform for pharmacological and toxicological [122] and are considered a possible source of autologous stem cells for use in regenerative medicine. LVs also have been used in biotechnology to engineer cell lines for the production of pro‐ teins of interest [123].

**6. Conclusions and outlook**

reducing homology between them.

to increase their productivity, quality and safety.

grants (SFRH/BD/79022/2011 and SFRH/BD/48393/2008).

higher biosafety.

**Acknowledgments**

**Author details**

Portugal

The major concerns associated with the use of all retroviral vectors are the formation of rep‐ lication competent retroviral vectors (RCR), the mutational integration of the provirus into the host cellular genome and mobilization of structural viral genes to target cells. In addi‐ tion, the majority of developed LVs are HIV-derived raising further safety concerns since this is a well known human pathogen. Significant efforts have been made to develop LVs with improved biosafety and increased transduction efficiency. Some of those biosafety fea‐ tures include the splitting of viral elements by several expression cassettes, the use of selfinactivating vectors (SIN), decreasing to a minimum the number of viral elements and

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

303

Lentiviral vectors have already won its place as valuable and flexible tool for gene delivery, being used in several applications but further research is still ongoing towards the develop‐ ment of a lentiviral vector providing higher titers, higher robustness, lower toxicity and

Lentiviral vector gene therapy is becoming a real alternative vector for therapy with dozens of clinical trials either been already performed or ongoing. These, together with the future incoming clinical trials, will enable to assess overall the pros and cons of the newcomer len‐ tiviral vectors and will provide insights to further vector innovations that will be important

The authors acknowledge the financial support received from the Fundaçãopara a Ciência e a Tecnologia-Portugal (FCT) (PTDC/EBB-BIO/100491/2008 and PTDC/EBB-BIO/ 118621/2010). Hélio Antunes Tomás and Ana F. Rodrigues acknowledge FCT for their PhD

Hélio A. Tomás1,2, Ana F. Rodrigues1,2, Paula M. Alves1,2 and Ana S. Coroadinha1,2

2 Instituto de Biologia Experimental Tecnológica, Oeiras, Portugal

1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras,

The main goal of LV technology is their use in clinical gene therapy applications. Within this purpose considerable efforts have been made to increase the safety and efficacy of LVs. Proof-of-concept has been established in preclinical animal models since several re‐ search groups have reported that LVs could treat or cure a disease including β-thalassae‐ mia[124], sickle cell anemia [125], hemophilia B [126] and ζ-chain-associated protein kinase of 70 kDa immunodeficiency [127]. Moreover, improvements in other genetic disor‐ ders like Parkinson's disease [128], cystic fibrosis [129] and spinal muscular atrophy [130] have been reported.

LVs have more recently moved beyond the preclinical stage into the clinical arena. The first human clinical trial using LVs was initiated in 2003. In this, a VSV-G-pseudotyped HIVbased vector was engineered to conditionally express an antisense RNA against envelope glycoprotein in the presence of regulatory proteins provided by wild-type virus. Five sub‐ jects with chronic HIV infection received a single dose of gene-modified autologous CD4+ T cells which resulted in an increase of CD4+ T cells (in four out of the five subjects) and de‐ crease in the viral load (in all five participants) after 1 year. Further studies over 2 years have not detected any adverse clinical events [131].

Since this first gene therapy clinical trial until June 2012, about 54 gene therapy clinical tri‐ al using LVs are ongoing or have been approved. Among them there are 12 trials for the treatment of HIV infection, 22 for the treatment of monogenic diseases (X linked cerebral adrenoleukodystrophy, Sickle cell anemia, Wiskott-Aldrich Syndrome, Metachromatic Leukodystrophy, X-Linked Chronic Granulomatous Disease, Inherited Skin Disease Neth‐ erton Syndrome, mucopolysaccharidosis type VII, β-thalassemia, Fanconi Anemia Comple‐ mentation Group A, X-Linked Severe Combined Deficiency, Adenosine Deaminase Deficient Severe Combined Immunodeficiency, Hemophilia A), 15 against various cancers, 2 for Parkinson's disease, 3 for ocular diseases and 1 for patients with Stargardt Macular Degeneration [1].

#### **6. Conclusions and outlook**

Lentiviral vectors are being increasingly used for the cell genetic modification leading to cell-engineering applications. Stable gene transduction can be used for *in vivo* imaging of vector infected cells. *In vivo* imaging studies of cells, including stem cells, have become in‐ creasingly important to understand cell distribution, differentiation, migration, function, and transgene expression in animal models. As an example, LVs expressing the firefly luci‐ ferase gene were used to monitor human embryonic stem cell (hESC) engraftment and proliferation in live mice after transplantation [119]. LVs can also be used to cellular re‐ programming of somatic cells. More specifically, the promising induced pluripotent stem cells (iPS) can be generated from a somatic cell by transduction of four key transcription factors, Oct4, Sox2, Klf4, and c-Myc, using LVs[120,121]. iPS can be used to study stem cell biology, as a cellular platform for pharmacological and toxicological [122] and are considered a possible source of autologous stem cells for use in regenerative medicine. LVs also have been used in biotechnology to engineer cell lines for the production of pro‐

The main goal of LV technology is their use in clinical gene therapy applications. Within this purpose considerable efforts have been made to increase the safety and efficacy of LVs. Proof-of-concept has been established in preclinical animal models since several re‐ search groups have reported that LVs could treat or cure a disease including β-thalassae‐ mia[124], sickle cell anemia [125], hemophilia B [126] and ζ-chain-associated protein kinase of 70 kDa immunodeficiency [127]. Moreover, improvements in other genetic disor‐ ders like Parkinson's disease [128], cystic fibrosis [129] and spinal muscular atrophy [130]

LVs have more recently moved beyond the preclinical stage into the clinical arena. The first human clinical trial using LVs was initiated in 2003. In this, a VSV-G-pseudotyped HIVbased vector was engineered to conditionally express an antisense RNA against envelope glycoprotein in the presence of regulatory proteins provided by wild-type virus. Five sub‐ jects with chronic HIV infection received a single dose of gene-modified autologous CD4+ T cells which resulted in an increase of CD4+ T cells (in four out of the five subjects) and de‐ crease in the viral load (in all five participants) after 1 year. Further studies over 2 years have

Since this first gene therapy clinical trial until June 2012, about 54 gene therapy clinical tri‐ al using LVs are ongoing or have been approved. Among them there are 12 trials for the treatment of HIV infection, 22 for the treatment of monogenic diseases (X linked cerebral adrenoleukodystrophy, Sickle cell anemia, Wiskott-Aldrich Syndrome, Metachromatic Leukodystrophy, X-Linked Chronic Granulomatous Disease, Inherited Skin Disease Neth‐ erton Syndrome, mucopolysaccharidosis type VII, β-thalassemia, Fanconi Anemia Comple‐ mentation Group A, X-Linked Severe Combined Deficiency, Adenosine Deaminase Deficient Severe Combined Immunodeficiency, Hemophilia A), 15 against various cancers, 2 for Parkinson's disease, 3 for ocular diseases and 1 for patients with Stargardt Macular

teins of interest [123].

302 Gene Therapy - Tools and Potential Applications

have been reported.

Degeneration [1].

not detected any adverse clinical events [131].

The major concerns associated with the use of all retroviral vectors are the formation of rep‐ lication competent retroviral vectors (RCR), the mutational integration of the provirus into the host cellular genome and mobilization of structural viral genes to target cells. In addi‐ tion, the majority of developed LVs are HIV-derived raising further safety concerns since this is a well known human pathogen. Significant efforts have been made to develop LVs with improved biosafety and increased transduction efficiency. Some of those biosafety fea‐ tures include the splitting of viral elements by several expression cassettes, the use of selfinactivating vectors (SIN), decreasing to a minimum the number of viral elements and reducing homology between them.

Lentiviral vectors have already won its place as valuable and flexible tool for gene delivery, being used in several applications but further research is still ongoing towards the develop‐ ment of a lentiviral vector providing higher titers, higher robustness, lower toxicity and higher biosafety.

Lentiviral vector gene therapy is becoming a real alternative vector for therapy with dozens of clinical trials either been already performed or ongoing. These, together with the future incoming clinical trials, will enable to assess overall the pros and cons of the newcomer len‐ tiviral vectors and will provide insights to further vector innovations that will be important to increase their productivity, quality and safety.

#### **Acknowledgments**

The authors acknowledge the financial support received from the Fundaçãopara a Ciência e a Tecnologia-Portugal (FCT) (PTDC/EBB-BIO/100491/2008 and PTDC/EBB-BIO/ 118621/2010). Hélio Antunes Tomás and Ana F. Rodrigues acknowledge FCT for their PhD grants (SFRH/BD/79022/2011 and SFRH/BD/48393/2008).

#### **Author details**

Hélio A. Tomás1,2, Ana F. Rodrigues1,2, Paula M. Alves1,2 and Ana S. Coroadinha1,2

1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Oeiras, Portugal

2 Instituto de Biologia Experimental Tecnológica, Oeiras, Portugal

#### **References**

[1] The Journal of Gene Medicine: Gene Therapy Clinical Trials Worldwide www.wi‐ ley.com/legacy/wileychi/genmed/clinical/ (accessed 1 August 2012)

lope Glycoprotein M. Journal of Virology. 1990;64(5):2416–20. jvi.asm.org/content/

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

305

[13] Page K a, Landau NR, Littman DR. Construction and use of a human immunodefi‐ ciency virus vector for analysis of virus infectivity. Journal of virology. 1990;64(11):

5270–6. jvi.asm.org/content/64/11/5270.full.pdf+html (accessed 1 August 2012) [14] Landau NR, Page K a, Littman DR. Pseudotyping with human T-cell leukemia virus type I broadens the human immunodeficiency virus host range. Journal of virology.

[15] Clever J, Sassetti C, Parslow TG. RNA secondary structure and binding sites for gag gene products in the 5 ' packaging signal of human immunodeficiency virus type 1. Journal of Virology. 1995;69(4):2101–9. jvi.asm.org/content/69/4/2101.full.pdf (ac‐

[16] Naldini L, Blömer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene deliv‐ ery and stable transduction of nondividing cells by a lentiviral vector. Science (New York, N.Y.). 1996;272(5259):263–7. www.sciencemag.org/content/272/5259/263.long

[17] Burns JC, Friedmann T, Driever W, Burrascano M, Yee JK. Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proceedings of the National Academy of Sciences of the United States of America. 1993;90(17):8033–7.

[18] Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M. Transduc‐ tion of nondividing cells using pseudotyped defective high-titer HIV type 1 particles. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(26):15266–71. www.pnas.org/content/93/26/15266.long (accessed 1 August

[19] Akkina RK, Walton RM, Chen ML, Li QX, Planelles V, Chen IS. High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retro‐ viral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G. Journal of virology. 1996;70(4):2581–5. jvi.asm.org/content/70/4/2581.long (accessed 1

[20] Naldini L, Blömer U, Gage FH, Trono D, Verma IM. Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. Proceedings of the National Academy of Sciences of the United States of America. 1996;93(21):11382–8. www.pnas.org/content/93/21/11382.full.pdf

[21] Gibbs JS, Regier DA, Desrosiers RC. Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes. AIDS research and human retrovi‐ ruses. 1994;10(4):343–50.http://www.ncbi.nlm.nih.gov/pubmed/8068414(accessed 1

www.pnas.org/content/90/17/8033.full.pdf (accessed 1 August 2012)

1991;65(1):162–9. jvi.asm.org/content/65/1/162 (accessed 1 August 2012)

64/5/2416.full.pdf+html (accessed 1 August 2012)

cessed 1 August 2012)

(accessed 1 August 2012)

2012)

August 2012)

August 2012)

(accessed 1 August 2012)


lope Glycoprotein M. Journal of Virology. 1990;64(5):2416–20. jvi.asm.org/content/ 64/5/2416.full.pdf+html (accessed 1 August 2012)

[13] Page K a, Landau NR, Littman DR. Construction and use of a human immunodefi‐ ciency virus vector for analysis of virus infectivity. Journal of virology. 1990;64(11): 5270–6. jvi.asm.org/content/64/11/5270.full.pdf+html (accessed 1 August 2012)

**References**

304 Gene Therapy - Tools and Potential Applications

[1] The Journal of Gene Medicine: Gene Therapy Clinical Trials Worldwide www.wi‐

[2] Campbell RS, Robinson WF. The comparative pathology of the lentiviruses. Journal‐

[3] International Committee on Taxonomy of Viruses(ICTV) http://ictvonline.org/viru‐

[4] Vogt VM, Simon MN. Mass determination of rous sarcoma virus virions by scanning transmission electron microscopy. Journal of virology. 1999;73(8):7050–5. jvi.asm.org/

[5] Coffin JM, Hughes SH, Varmus H. 1997. Retroviruses: Cold Spring Harbor.

[6] Adamson CS, Jones IM. The molecular basis of HIV capsid assembly--five years of progress. Reviews in medical virology. ;14(2):107–21. onlinelibrary.wiley.com/doi/

[7] Katz RA, Skalka AM. The retroviral enzymes. Annual review of biochemistry. 1994;63:133–73. www.annualreviews.org/doi/abs/10.1146/annurev.bi.

[8] Ptak RG, Fu W, Sanders-Beer BE, Dickerson JE, Pinney JW, Robertson DL, et al. Cata‐ loguing the HIV type 1 human protein interaction network. AIDS research and hu‐ man retroviruses. 2008;24(12):1497–502. online.liebertpub.com/doi/abs/10.1089/aid.

[9] Fu W, Sanders-Beer BE, Katz KS, Maglott DR, Pruitt KD, Ptak RG. Human immuno‐ deficiency virus type 1, human protein interaction database at NCBI. Nucleic acids research. 2009;37 (Database issue) :D417–22. nar.oxfordjournals.org/content/37/

[10] Pluta K, Kacprzak MM. Use of HIV as a gene transfer vector. Acta biochimica Poloni‐ ca. 2009;56(4):531–95. www.actabp.pl/pdf/4\_2009/531.pdf (accessed 1August 2012) [11] Terwilliger EF, Godin B, Sodroski JG, Haseltine W a. Construction and use of a repli‐ cation-competent human immunodeficiency virus (HIV-1) that expresses the chlor‐ amphenicol acetyltransferase enzyme. Proceedings of the National Academy of Sciences of the United States of America. 1989 May;86(10):3857–61. http://

www.pnas.org/content/86/10/3857.full.pdf+html (accessed 1 August 2012)

[12] Helseth E, Kowalski M, Gabuzda D, Olshevsky UDY, Haseltine W, Sodroskil J. Rapid complementation assays measuring replicative potential of human immunodeficien‐ cy virus type 1 envelope glycoprotein mutants. Rapid Complementation Assays Measuring Replicative Potential of Human Immunodeficiency Virus Type 1 Enve‐

ley.com/legacy/wileychi/genmed/clinical/ (accessed 1 August 2012)

of comparative pathology. 1998; 119(4):333–95.

sTaxonomy.asp?version=2011(accessed 1 August 2012)

content/73/8/7050.full.pdf+html (accessed 1 August 2012)

10.1002/rmv.418/citedby (accessed 1 August 2012)

63.070194.001025 (accessed 1 August 2012)

2008.0113(accessed 1 August 2012)

suppl\_1/D417 (accessed 1 August 2012)

www.ncbi.nlm.nih.gov/books/NBK19376/(accessed 1 August 2012)


[22] Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature biotechnology. 1997;15(9):871– 5. www.nature.com/nbt/journal/v15/n9/full/nbt0997-871.html (accessed 1 August 2012)

a codon-optimized HIV-1 gag-pol gene. Journal of virology. 2000;74(10):4839–52.

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

307

[33] Ikeda Y, Takeuchi Y, Martin F, Cosset F-L, Mitrophanous K, Collins M. Continuous high-titer HIV-1 vector production. 2003. www.nature.com/nbt/journal/v21/n5/full/

[34] Kappes JC, Wu X. Safety considerations in vector development. Somatic cell and mo‐ lecular genetics. 2001;26(1-6):147–58. www.springerlink.com/content/

[35] Wu X, Wakefield JK, Liu H, Xiao H, Kralovics R, Prchal JT, et al. Development of a novel trans-lentiviral vector that affords predictable safety. Molecular therapy: the journal of the American Society of Gene Therapy. 2000;2(1):47–55 www.nature.com/mt/journal/v2/n1/pdf/mt2000140a.pdf (accessed 1 August 2012)

[36] Westerman KA, Ao Z, Cohen EA, Leboulch P. Design of a trans protease lentiviral packaging system that produces high titer virus. Retrovirology. 2007;4(96):1–14. www.retrovirology.com/content/pdf/1742-4690-4-96.pdf (accessed 1 August 2012)

[37] Pauwels K, Gijsbers R, Toelen J, Schambach A, Willard-Gallo K, Verheust C, et al. State-of-the-art Lentiviral Vectors for Research Use: Risk Assessment and Biosafety Recommendations. Current gene therapy. 2009;9(6):459–74. www.benthamdirect.org/ pages/content.php?CGT/2009/00000009/00000006/0002Q.SGM (accessed 1 August

[38] Montini E, Cesana D, Schmidt M, Sanvito F, Bartholomae CC, Ranzani M, et al. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. The Journal of clini‐ cal investigation. 2009;119(4):964–75. www.jci.org/articles/view/37630 (accessed 1

[39] Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, et al. Self-Inactivat‐ ing Lentivirus Vector for Safe and Efficient In Vivo Gene Delivery. J. Virol.. 1998;72(12):9873–80. jvi.asm.org/cgi/content/abstract/72/12/9873(accessed 1 August

[40] Iwakuma T, Cui Y, Chang LJ. Self-inactivating lentiviral vectors with U3 and U5 modifications. Virology. 1999;261(1):120–32. www.sciencedirect.com/science/

[41] Yu S-F. Self-Inactivating Retroviral Vectors Designed for Transfer of Whole Genes in‐ to Mammalian Cells. Proceedings of the National Academy of Sciences. 1986;83(10): 3194–8. www.pnas.org/cgi/content/abstract/83/10/3194(accessed 1 August2012)

[42] Bukovsky AA, Song JP, Naldini L. Interaction of human immunodeficiency virus-de‐ rived vectors with wild-type virus in transduced cells. Journal of virology. 1999;73(8):

7087–92. jvi.asm.org/content/73/8/7087.long (accessed 1 August 2012)

article/pii/S0042682299998501(accessed 1 August 2012)

jvi.asm.org/content/74/10/4839.long (accessed 1 August 2012)

w93t23118464r17k/?MUD=MP (accessed 1 August 2012)

nbt815.html (accessed 1 August 2012)

2012)

2012)

August 2012)


a codon-optimized HIV-1 gag-pol gene. Journal of virology. 2000;74(10):4839–52. jvi.asm.org/content/74/10/4839.long (accessed 1 August 2012)

[33] Ikeda Y, Takeuchi Y, Martin F, Cosset F-L, Mitrophanous K, Collins M. Continuous high-titer HIV-1 vector production. 2003. www.nature.com/nbt/journal/v21/n5/full/ nbt815.html (accessed 1 August 2012)

[22] Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature biotechnology. 1997;15(9):871– 5. www.nature.com/nbt/journal/v15/n9/full/nbt0997-871.html (accessed 1 August

[23] Kafri T, Blömer U, Peterson DA, Gage FH, Verma IM. Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nature genetics. 1997;17(3):314–7. www.nature.com/ng/journal/v17/n3/abs/ng1197-314.html (accessed

[24] Mochizuki H, Schwartz JP, Tanaka K, Brady RO, Reiser J. High-titer human immune deficiency virus type 1-based vector systems for gene delivery into nondividing cells. Journal of virology. 1998;72(11):8873–83. jvi.asm.org/content/72/11/8873.long (ac‐

[25] Kim VN, Mitrophanous K, Kingsman SM, Kingsman a J. Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1. Journal of virolo‐

gy. 1998 Jan;72(1):811–6. jvi.asm.org/content/72/1/811(accessed 1 August 2012) [26] Naldini L, Verma IM. Lentiviral vectors. Advances in virus research. 2000;55:599– 609. http://www.ncbi.nlm.nih.gov/pubmed/11050959(accessed 1 August 2012) [27] Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, et al. A third-genera‐ tion lentivirus vector with a conditional packaging system. Journal of virology. 1998;72(11):8463–71. jvi.asm.org/content/72/11/8463.long (accessed 1 August 2012) [28] Bray M, Prasad S, Dubay JW, Hunter E, Jeang KT, Rekosh D, et al. A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent. Proceedings of the National Academy of Sciences of the United States of America. 1994;91(4):1256–60.

www.pnas.org/content/91/4/1256.full.pdf+html?sid=f828b8ae-496c-476c-bace-

[29] Reddy TR, Xu W, Mau JK, Goodwin CD, Suhasini M, Tang H, et al. Inhibition of HIV replication by dominant negative mutants of Sam68, a functional homolog of HIV-1 Rev. Nature medicine. 1999;5(6):635–42. www.nature.com/nm/journal/v5/n7/full/

[30] Roberts TM, Boris-Lawrie K. The 5' RNA terminus of spleen necrosis virus stimulates translation of nonviral mRNA. Journal of virology. 2000;74(17):8111–8. jvi.asm.org/

[31] Pandya S, Boris-Lawrie K, Leung NJ, Akkina R, Planelles V. Development of an Re‐ vindependent, minimal simian immunodeficiency virus-derived vector system. Hu‐ man gene therapy. 2001 May 1;12(7):847–57. online.liebertpub.com/doi/pdfplus/

[32] Kotsopoulou E, Kim VN, Kingsman AJ, Kingsman SM, Mitrophanous KA. A Rev-in‐ dependent human immunodeficiency virus type 1 (HIV-1)-based vector that exploits

2012)

1 August 2012)

306 Gene Therapy - Tools and Potential Applications

cessed 1 August 2012)

e85aeee47e57(accessed 1 August 2012)

nm0799\_849c.html (accessed 1 August 2012)

content/74/17/8111.long (accessed 1 August 2012)

10.1089/104303401750148847 (accessed 1 August 2012)


[43] Bokhoven M, Stephen SL, Knight S, Gevers EF, Robinson IC, Takeuchi Y, et al. Inser‐ tional gene activation by lentiviral and gammaretroviral vectors. Journal of virology. 2009;83(1):283–94. jvi.asm.org/content/83/1/283 (accessed 1 August 2012)

bloodjournal.hematologylibrary.org/content/96/10/3392.long (accessed 1 August

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

309

[54] Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D, et al. Wood chuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene thera‐ py. 2006;13(7):641–5. www.nature.com/gt/journal/v13/n7/full/3302698a.html (ac‐

[55] Manganini M, Serafini M, Bambacioni F, Casati C, Erba E, Follenzi A, et al. A human immunodeficiency virus type 1 pol gene-derived sequence (cPPT/CTS) increases the efficiency of transduction of human nondividing monocytes and T lymphocytes by lentiviral vectors. Human gene therapy. 2002;13(15):1793–807. online.liebertpub.com/doi/abs/10.1089/104303402760372909 (accessed 1 August 2012)

[56] Sirven A, Pflumio F, Zennou V, Titeux M, Vainchenker W, Coulombel L, et al. The human immunodeficiency virus type-1 central DNA flap is a crucial determinant for lentiviral vector nuclear import and gene transduction of human hematopoietic stem cells. Blood. 2000;96(13):4103–10. bloodjournal.hematologylibrary.org/content/

[57] Logan AC, Nightingale SJ, Haas DL, Cho GJ, Pepper K a, Kohn DB. Factors influenc‐ ing the titer and infectivity of lentiviral vectors. Human gene therapy. 2004;15(10): 976–88. online.liebertpub.com/doi/abs/10.1089/hum.2004.15.976 (accessed 1 August

[58] Desmaris N, Bosch A, Salaün C, Petit C, Prévost MC, Tordo N, et al. Production and neurotropism of lentivirus vectors pseudotyped with lyssavirus envelope glycopro‐ teins. Molecular therapy: the journal of the American Society of Gene Therapy.;4(2): 149–56. www.nature.com/mt/journal/v4/n2/abs/mt2001210a.html (accessed 1 August

[59] Federici T, Kutner R, Zhang X-Y, Kuroda H, Tordo N, Boulis NM, et al. Comparative analysis of HIV-1-based lentiviral vectors bearing lyssavirus glycoproteins for neuro‐ nal gene transfer. Genetic vaccines and therapy. 2009;7(1):1. www.gvt-journal.com/

[60] Mazarakis ND. Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral deliv‐ ery. Human Molecular Genetics. 2001;10(19):2109–21. hmg.oxfordjournals.org/cgi/

[61] Bartz SR, Rogel ME, Emerman M. Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which dif‐ fers from DNA damage checkpoint control. Journal of virology. 1996;70(4):2324–31.

jvi.asm.org/content/70/4/2324.full.pdf+html?sid=9792589f-078b-4868-a8a5-

2012)

2012)

2012)

cessed 1 August 2012)

96/13/4103.long (accessed 1 August 2012)

content/7/1/1(accessed 1 August 2012)

f0926b6b2dee (accessed 1 August 2012)

content/abstract/10/19/2109(accessed 1 August 2012)


bloodjournal.hematologylibrary.org/content/96/10/3392.long (accessed 1 August 2012)

[54] Schambach A, Bohne J, Baum C, Hermann FG, Egerer L, von Laer D, et al. Wood chuck hepatitis virus post-transcriptional regulatory element deleted from X protein and promoter sequences enhances retroviral vector titer and expression. Gene thera‐ py. 2006;13(7):641–5. www.nature.com/gt/journal/v13/n7/full/3302698a.html (ac‐ cessed 1 August 2012)

[43] Bokhoven M, Stephen SL, Knight S, Gevers EF, Robinson IC, Takeuchi Y, et al. Inser‐ tional gene activation by lentiviral and gammaretroviral vectors. Journal of virology.

[44] Miyoshi H, Blomer U, Takahashi M, Gage FH, Verma IM. Development of a Self-In‐ activating Lentivirus Vector. J. Virol.. 1998;72(10):8150–7. jvi.asm.org/cgi/content/

[45] Yang Q, Lucas A, Son S, Chang L-J. Overlapping enhancer/promoter and transcrip‐ tional termination signals in the lentiviral long terminal repeat. Retrovirology.

[46] Schambach A, Galla M, Maetzig T, Loew R, Baum C. Improving transcriptional ter‐ mination of self-inactivating gamma-retroviral and lentiviral vectors. Molecular ther‐ apy: the journal of the American Society of Gene Therapy. 2007;15(6):1167–73. www.nature.com/mt/journal/v15/n6/full/6300152a.html (accessed 1 August 2012) [47] Hanawa H, Persons DA, Nienhuis AW. Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors. Journal of virology. 2005;79(13):

[48] Hino S, Fan J, Taguwa S, Akasaka K, Matsuoka M. Sea urchin insulator protects lenti‐ viral vector from silencing by maintaining active chromatin structure. Gene therapy. 2004;11(10):819–28. www.nature.com/gt/journal/v11/n10/full/3302227a.html (ac‐

[49] Arumugam PI, Scholes J, Perelman N, Xia P, Yee J-K, Malik P. Improved human be‐ ta-globin expression from self-inactivating lentiviral vectors carrying the chicken hy‐ persensitive site-4 (cHS4) insulator element. Molecular therapy: the journal of the American Society of Gene Therapy. 2007;15(10):1863–71. www.nature.com/mt/jour‐

[50] Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Hepatitis Virus Posttranscrip‐ tional Regulatory Element Enhances Expression of Transgenes Delivered by Retrovi‐ ral Vectors. J. Virol. 1999;73(4):2886–92. jvi.asm.org/cgi/content/abstract/

[51] Oh T, Bajwa A, Jia G, Park F. Lentiviral vector design using alternative RNA export elements. Retrovirology. 2007;4(1):38. www.retrovirology.com/content/4/1/38 (ac‐

[52] Pistello M, Vannucci L, Ravani A, Bonci F, Chiuppesi F, del Santo B, et al. Stream‐ lined design of a self-inactivating feline immunodeficiency virus vector for transduc‐ ing ex vivo dendritic cells and T lymphocytes. Genetic vaccines and therapy.

[53] Salmon P, Kindler V, Ducrey O, Chapuis B, Zubler RH, Trono D. High-level trans‐ gene expression in human hematopoietic progenitors and differentiated blood line‐ ages after transduction with improved lentiviral vectors. Blood. 2000;96(10):3392–8.

2007;5(1):8. www.gvt-journal.com/content/5/1/8(accessed 1 August 2012)

2009;83(1):283–94. jvi.asm.org/content/83/1/283 (accessed 1 August 2012)

2007;4:4. www.retrovirology.com/content/4/1/4 (accessed 1 August 2012)

8410–21. jvi.asm.org/content/79/13/8410.long (accessed 1 August 2012)

nal/v15/n10/full/6300259a.html (accessed 1 August 2012)

abstract/72/10/8150(accessed 1 August 2012)

cessed 1 August 2012)

308 Gene Therapy - Tools and Potential Applications

73/4/2886(accessed 1 August 2012)

cessed 1 August 2012)


[62] DePolo NJ, Reed JD, Sheridan PL, Townsend K, Sauter SL, Jolly DJ, et al. VSV-G pseudotypedlentiviral vector particles produced in human cells are inactivated by human serum. Molecular therapy: the journal of the American Society of Gene Ther‐ apy. 2000;2(3):218–22. www.nature.com/mt/journal/v2/n3/abs/mt2000164a.html (ac‐ cessed 1 August 2012)

[71] Zhang X-Y, La Russa VF, Reiser J. Transduction of bone-marrow-derived mesenchy‐ mal stem cells by using lentivirus vectors pseudotyped with modified RD114 enve‐ lope glycoproteins. Journal of virology. 2004;78(3):1219–29 jvi.asm.org/content/

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

311

[72] Kobinger GP, Weiner DJ, Yu QC, Wilson JM. Filovirus-pseudotypedlentiviral vector can efficiently and stably transduce airway epithelia in vivo. Nature biotechnology. 2001;19(3):225–30. www.nature.com/nbt/journal/v19/n3/full/nbt0301\_225.html (ac‐

[73] Beyer WR, Westphal M, Ostertag W, von Laer D. Oncoretrovirus and lentivirus vec‐ tors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range. Journal of virology. 2002;76(3):1488–95.

[74] Wong L-F, Azzouz M, Walmsley LE, Askham Z, Wilkes FJ, Mitrophanous KA, et al. Transduction patterns of pseudotyped lentiviral vectors in the nervous system. Mo‐ lecular therapy: the journal of the American Society of Gene Therapy.;9(1):101–11. www.nature.com/mt/journal/v9/n1/full/mt200415a.html (accessed 1 August 2012) [75] Kahl CA, Marsh J, Fyffe J, Sanders DA, Cornetta K. Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus. Journal of virology. 2004;78(3):1421–

[76] Kang Y, Stein CS, Heth JA, Sinn PL, Penisten AK, Staber PD, et al. In vivo gene trans‐ fer using a nonprimatelentiviral vector pseudotyped with Ross River Virus glycopro‐ teins. Journal of virology. 2002;76(18):9378–88. jvi.asm.org/content/76/18/9378.long

[77] Morizono K, Bristol G, Xie YM, Kung SK, Chen IS. Antibody-directed targeting of retroviral vectors via cell surface antigens. Journal of virology. 2001;75(17):8016–20.

[78] Cronin J, Zhang X-Y, Reiser J. Altering the Tropism of Lentiviral Vectors through Pseudotyping. Current gene therapy. 2005;5(4):387–98. www.benthamdirect.org/ pages/content.php?CGT/2005/00000005/00000004/0003Q.SGM (accessed 1 August

[79] Felder JM, Sutton RE. Lentiviral Vectors. In: Gene and Cell Therapy: Therapeutic

[80] Lodge R, Subbramanian R a, Forget J, Lemay G, Cohen E a. MuLV-based vectors pseudotyped with truncated HIV glycoproteins mediate specific gene transfer in CD4+ peripheral blood lymphocytes. Gene therapy. 1998;5(5):655–64. www.na‐

[81] Thaler S, Schnierle BS. A packaging cell line generating CD4-specific retroviral vec‐ tors for efficient gene transfer into primary human T-helper lymphocytes. Molecular

ture.com/gt/journal/v5/n5/abs/3300646a.html (accessed 1 August 2012)

jvi.asm.org/content/76/3/1488.long (accessed 1 August 2012)

30. jvi.asm.org/content/78/3/1421.long (accessed 1 August 2012)

jvi.asm.org/content/75/17/8016 (accessed 1 August 2012)

78/3/1219.long (accessed 1 August 2012)

cessed 1 August 2012)

(accessed 1 August 2012)

Mechanisms and Strategies. 2009.

2012)


[71] Zhang X-Y, La Russa VF, Reiser J. Transduction of bone-marrow-derived mesenchy‐ mal stem cells by using lentivirus vectors pseudotyped with modified RD114 enve‐ lope glycoproteins. Journal of virology. 2004;78(3):1219–29 jvi.asm.org/content/ 78/3/1219.long (accessed 1 August 2012)

[62] DePolo NJ, Reed JD, Sheridan PL, Townsend K, Sauter SL, Jolly DJ, et al. VSV-G pseudotypedlentiviral vector particles produced in human cells are inactivated by human serum. Molecular therapy: the journal of the American Society of Gene Ther‐ apy. 2000;2(3):218–22. www.nature.com/mt/journal/v2/n3/abs/mt2000164a.html (ac‐

[63] Croyle MA, Callahan SM, Auricchio A, Schumer G, Linse KD, Wilson JM, et al. PE‐ Gylation of a vesicular stomatitis virus G pseudotypedlentivirus vector prevents in‐ activation in serum. Journal of virology. 2004;78(2):912–21. jvi.asm.org/content/

[64] Berkowitz R, Ilves H, Lin WY, Eckert K, Coward A, Tamaki S, et al. Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus. Journal of virology. 2001;75(7):3371–82. jvi.asm.org/content/75/7/3371. long (ac‐

[65] Metharom P, Takyar S, Xia HQ, Ellem KA, Wilcox GE, Wei MQ. Development of dis‐ abled, replication-defective gene transfer vectors from the Jembrana disease virus, a new infectious agent of cattle. Veterinary microbiology. 2001;80(1):9–22. www.scien‐

cedirect.com/science/article/pii/S037811350000376X (accessed 1 August 2012)

[66] Mselli-Lakhal L, Guiguen F, Greenland T, Mornex J-F, Chebloune Y. Gene transfer system derived from the caprine arthritis-encephalitis lentivirus. Journal of virologi‐ cal methods. 2006;136(1-2):177–84. http://www.sciencedirect.com/science/article/pii/

[67] Duisit G, Conrath H, Saleun S, Folliot S, Provost N, Cosset F-L, et al. Five recombi‐ nant simian immunodeficiency virus pseudotypes lead to exclusive transduction of retinal pigmented epithelium in rat. Molecular therapy: the journal of the American Society of Gene Therapy. 2002;6(4):446–54. www.nature.com/mt/journal/v6/n4/pdf/

[68] Kobinger GP, Deng S, Louboutin J-P, Vatamaniuk M, Matschinsky F, Markmann JF, et al. Transduction of human islets with pseudotypedlentiviral vectors. Human gene therapy. 2004;15(2):211–9. online.liebertpub.com/doi/abs/

[69] Mochizuki H, Schwartz JP, Tanaka K, Brady RO, Reiser J. High-Titer Human Immu‐ nodeficiency Virus Type 1-Based Vector Systems for Gene Delivery into Nondividing Cells. J. Virol.. 1998;72(11):8873–83. jvi.asm.org/cgi/content/abstract/

[70] Hanawa H, Kelly PF, Nathwani AC, Persons DA, Vandergriff JA, Hargrove P, et al. Comparison of various envelope proteins for their ability to pseudotypelentiviral vectors and transduce primitive hematopoietic cells from human blood. Molecular therapy: the journal of the American Society of Gene Therapy. 2002;5(3):242–51. www.nature.com/mt/journal/v5/n3/full/mt200238a.html (accessed 1 August 2012)

cessed 1 August 2012)

310 Gene Therapy - Tools and Potential Applications

cessed 1 August 2012)

78/2/912 (accessed 1 August 2012)

S0166093406001571(accessed 1 August 2012)

mt2002197a.pdf (accessed 1 August 2012)

72/11/8873(accessed 1 August 2012)

10.1089/104303404772680010 (accessed 1August 2012)


therapy: the journal of the American Society of Gene Therapy. 2001;4(3):273–9. www.nature.com/mt/journal/v4/n3/abs/mt2001227a.html (accessed 1 August 2012)

[92] Haas DL, Case SS, Crooks GM, Kohn DB. Critical factors influencing stable transduc‐ tion of human CD34(+) cells with HIV-1-derived lentiviral vectors. Molecular thera‐ py : the journal of the American Society of Gene Therapy. 2000;2(1):71–80. www.nature.com/mt/journal/v2/n1/abs/mt2000143a.html (accessed 1 August 2012)

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

313

[93] Schweizer M, Merten O-W. Large-scale production means for the manufacturing of lentiviral vectors. Current gene therapy. 2010;10(6):474–86. www.benthamdirect.org/ pages/content.php?CGT/2010/00000010/00000006/0006Q.SGM (accessed 1 August

[94] Gougeon M-L. Apoptosis as an HIV strategy to escape immune attack. Nature re‐ views. Immunology. 2003;3(5):392–404. www.nature.com/nri/journal/v3/n5/full/

[95] Bell AJ, Fegen D, Ward M, Bank A. RD114 envelope proteins provide an effective and versatile approach to pseudotypelentiviral vectors. Experimental biology and medicine (Maywood, N.J.). 2010;235(10):1269–76. ebm.rsmjournals.com/content/

[96] Sainski AM, Natesampillai S, Cummins NW, Bren GD, Taylor J, Saenz DT, et al. The HIV-1-specific protein Casp8p41 induces death of infected cells through Bax/Bak. Journal of virology. 2011;85(16):7965–75. jvi.asm.org/content/85/16/7965.long (ac‐

[97] Algeciras-Schimnich A, Belzacq-Casagrande A-S, Bren GD, Nie Z, Taylor J a, Rizza S a, et al. Analysis of HIV Protease Killing Through Caspase 8 Reveals a Novel Interac‐ tion Between Caspase 8 and Mitochondria. The open virology journal. 2007;1:39–46. benthamscience.com/open/openaccess.php?tovj/articles/V001/39TOVJ.htm (accessed

[98] Nie Z, Phenix BN, Lum JJ, Alam a, Lynch DH, Beckett B, et al. HIV-1 protease proc‐ esses procaspase 8 to cause mitochondrial release of cytochrome c, caspase cleavage and nuclear fragmentation. Cell death and differentiation. 2002;9(11):1172–84. www.nature.com/cdd/journal/v9/n11/full/4401094a.html (accessed 1 August 2012)

[99] Merten O-W, Charrier S, Laroudie N, Fauchille S, Dugué C, Jenny C, et al. Largescale manufacture and characterization of a lentiviral vector produced for clinical ex vivo gene therapy application. Human gene therapy. 2011;22(3):343–56 online.liebert‐

[100] Gama-Norton L, Botezatu L, Herrmann S, Schweizer M, Alves PM, Hauser H, et al. Lentivirus production is influenced by SV40 large T-antigen and chromosomal inte‐ gration of the vector in HEK293 cells. Human gene therapy. 2011;22(10):1269–79. on‐

line.liebertpub.com/doi/abs/10.1089/hum.2010.143 (accessed 1 August 2012)

[101] Ansorge S, Lanthier S, Transfiguracion J, Durocher Y, Henry O, Kamen A. Develop‐ ment of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures. The journal of gene medicine.

pub.com/doi/abs/10.1089/hum.2010.060 (accessed 1 August 2012)

2012)

nri1087.html (accessed 1 August 2012)

235/10/1269.long (accessed 1 August 2012)

cessed 1 August 2012)

1 August 2012)


[92] Haas DL, Case SS, Crooks GM, Kohn DB. Critical factors influencing stable transduc‐ tion of human CD34(+) cells with HIV-1-derived lentiviral vectors. Molecular thera‐ py : the journal of the American Society of Gene Therapy. 2000;2(1):71–80. www.nature.com/mt/journal/v2/n1/abs/mt2000143a.html (accessed 1 August 2012)

therapy: the journal of the American Society of Gene Therapy. 2001;4(3):273–9. www.nature.com/mt/journal/v4/n3/abs/mt2001227a.html (accessed 1 August 2012)

[82] MacKenzie TC, Kobinger GP, Kootstra NA, Radu A, Sena-Esteves M, Bouchard S, et al. Efficient transduction of liver and muscle after in utero injection of lentiviral vec‐ tors with different pseudotypes. Molecular therapy: the journal of the American Soci‐ ety of Gene Therapy. 2002;6(3):349–58. www.nature.com/mt/journal/v6/n3/pdf/

[83] Peng KW, Pham L, Ye H, Zufferey R, Trono D, Cosset FL, et al. Organ distribution of gene expression after intravenous infusion of targeted and untargeted lentiviral vec‐ tors. Gene therapy. 2001;8(19):1456–63. www.nature.com/gt/journal/v8/n19/full/

[84] Yang L, Yang H, Rideout K, Cho T, Joo KI, Ziegler L, et al. Engineered lentivector targeting of dendritic cells for in vivo immunization. Nature biotechnology. 2008;26(3):326–34. www.nature.com/nbt/journal/v26/n3/full/nbt1390.html (accessed 1

[85] Escors D, Breckpot K. Lentiviral vectors in gene therapy: their current status and fu‐ ture potential. Archivum immunologiaeettherapiaeexperimentalis. 2010;58(2):107–19.

[86] Ziegler L, Yang L, Joo K il, Yang H, Baltimore D, Wang P. Targeting lentiviral vectors to antigen-specific immunoglobulins. Human gene therapy. 2008;19(9):861–72. on‐

[87] Frecha C, Szécsi J, Cosset F-L, Verhoeyen E. Strategies for targeting lentiviral vectors. Current gene therapy. 2008;8(6):449–60. www.benthamdirect.org/pages/content.php?

[88] Trimby C. STRATEGIES FOR TARGETING LENTIVIRAL VECTORS. http://uknowl‐

[89] Zhang X-Y, Kutner RH, Bialkowska A, Marino MP, Klimstra WB, Reiser J. Cell-spe‐ cific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or avian sarcoma/leukosis virus. Retrovirology. 2010;7(1):3. http://www.retrovirology.com/content/7/1/3(accessed 1 August 2012)

[90] Yang L, Bailey L, Baltimore D, Wang P. Targeting lentiviral vectors to specific cell types in vivo. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(31):11479–84. www.pnas.org/cgi/content/abstract/

[91] Lei Y, Joo K-I, Zarzar J, Wong C, Wang P. Targeting lentiviral vector to specific cell types through surface displayed single chain antibody and fusogenic molecule. Vi‐ rology journal. 2010;7:35. www.virologyj.com/content/7/1/35 (accessed 1 August

www.springerlink.com/content/725574uvq7552u2g/ (accessed 1 August 2012)

line.liebertpub.com/doi/abs/10.1089/hgt.2007.149 (accessed 1 August 2012)

CGT/2008/00000008/00000006/0005Q.SGM (accessed 1 August 2012)

edge.uky.edu/gradschool\_diss/157(accessed 1 August 2012)

103/31/11479(accessed 1 August 2012)

2012)

mt2002180a.pdf (accessed 1 August 2012)

3301552a.html (accessed 1 August 2012)

August 2012)

312 Gene Therapy - Tools and Potential Applications


2009;11(10):868–76. onlinelibrary.wiley.com/doi/10.1002/jgm.1370/abstract (accessed 1 August 2012)

Therapy. 2008;16(3):500–7. www.nature.com/mt/journal/v16/n3/full/6300383a.html

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

315

[112] Stewart HJ, Leroux-Carlucci MA, Sion CJM, Mitrophanous KA, Radcliffe PA. Devel‐ opment of inducible EIAV-based lentiviral vector packaging and producer cell lines. Gene therapy. 2009;16(6):805–14. www.nature.com/gt/journal/v16/n6/full/

[113] Throm RE, Ouma A a, Zhou S, Chandrasekaran A, Lockey T, Greene M, et al. Effi‐ cient construction of producer cell lines for a SIN lentiviral vector for SCID-X1 gene therapy by concatemeric array transfection. Blood. 2009;113(21):5104–10 bloodjour‐

nal.hematologylibrary.org/content/113/21/5104.long (accessed 1 August 2012) [114] Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, Conklin DS. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & develop‐ ment. 2002;16(8):948–58. genesdev.cshlp.org/content/16/8/948.long (accessed 1 Au‐

[115] Hu G, Luo J. A primer on using pooled shRNA libraries for functional genomic screens. Acta biochimic et biophysica Sinica. 2012 ;44(2):103–12. abbs.oxford‐jour‐

[116] Moffat J, Grueneberg DA, Yang X, Kim SY, Kloepfer AM, Hinkle G, et al. A lentivir‐ alRNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell. 2006;124(6):1283–98. www.cell.com/retrieve/pii/S0092867406002388 (ac‐

[117] Singer O, Verma IM. Applications of lentiviral vectors for shRNA delivery and trans‐ genesis. Current gene therapy. 2008;8(6):483–8. www.benthamdirect.org/pages/ content.php?CGT/2008/00000008/00000006/0008Q.SGM (accessed 1 August 2012) [118] Pfeifer A. Lentiviral transgenesis--a versatile tool for basic research and gene thera‐ py. Current gene therapy. 2006;6(4):535–42. www.benthamdirect.org/pages/ content.php?CGT/2006/00000006/00000004/0006Q.SGM (accessed 1 August 2012) [119] Pomper MG, Hammond H, Yu X, Ye Z, Foss CA, Lin DD, et al. Serial imaging of hu‐ man embryonic stem-cell engraftment and teratoma formation in live mouse models. Cell research. 2009;19(3):370–9. www.nature.com/cr/journal/v19/n3/full/

[120] Welstead GG, Brambrink T, Jaenisch R. Generating iPS cells from MEFS through forced expression of Sox-2, Oct-4, c-Myc, and Klf4. Journal of visualized experi‐ ments : JoVE.2008;(14). www.jove.com/video/734/generating-ips-cells-from-mefs-

[121] Brambrink T, Foreman R, Welstead GG, Lengner CJ, Wernig M, Suh H, et al. Sequen‐ tial expression of pluripotency markers during direct reprogramming of mouse so‐ matic cells. Cell stem cell. 2008;2(2):151–9. www.cell.com/cell-stem-cell/abstract/

through-forced-expression-sox-2-oct-4 (accessed 1 August 2012)

nals.org/cgi/content/abstract/44/2/103(accessed 1 August 2012)

(accessed 1 August 2012)

gust 2012)

cessed 1 August 2012)

cr2008329a.html (accessed 1 August 2012)

S1934-5909(08)00005-2 (accessed 1 August 2012)

gt200920a.html (accessed 1 August 2012)


Therapy. 2008;16(3):500–7. www.nature.com/mt/journal/v16/n3/full/6300383a.html (accessed 1 August 2012)

[112] Stewart HJ, Leroux-Carlucci MA, Sion CJM, Mitrophanous KA, Radcliffe PA. Devel‐ opment of inducible EIAV-based lentiviral vector packaging and producer cell lines. Gene therapy. 2009;16(6):805–14. www.nature.com/gt/journal/v16/n6/full/ gt200920a.html (accessed 1 August 2012)

2009;11(10):868–76. onlinelibrary.wiley.com/doi/10.1002/jgm.1370/abstract (accessed

[102] Smith SL, Shioda T. Advantages of COS-1 monkey kidney epithelial cells as packag‐ ing host for small-volume production of high-quality recombinant lentiviruses. Jour‐ nal of virological methods. 2009;157(1):47–54. www.sciencedirect.com/science/

[103] Ni Y, Sun S, Oparaocha I, Humeau L, Davis B, Cohen R, et al. Generation of a pack‐ aging cell line for prolonged large-scale production of high-titer HIV-1-based lentivi‐ ral vector. The journal of gene medicine. 2005;7(6):818–34. onlinelibrary.wiley.com/doi/10.1002/jgm.726/abstract (accessed 1 August 2012) [104] Mann R, Mulligan RC, Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983;33(1):153–9. online‐

library.wiley.com/doi/10.1002/jgm.726/abstract (accessed 1 August 2012)

[105] Witting SR, Li L-H, Jasti A, Allen C, Cornetta K, Brady J, et al. Efficient large volume lentiviral vector production using flow electroporation. Human gene therapy. 2012;23(2):243–9. online.liebertpub.com/doi/abs/10.1089/hum.2011.088 (accessed 1

[106] Rols MP, Coulet D, Teissié J. Highly efficient transfection of mammalian cells by elec‐ tric field pulses. Application to large volumes of cell culture by using a flow system. European journal of biochemistry / FEBS. 1992;206(1):115–21. onlinelibrary.wi‐ ley.com/doi/10.1111/j.1432-1033.1992.tb16908.x/abstract (accessed 1 August 2012) [107] Segura M, Garnier A, Durocher Y, Coelho H. Production of Lentiviral Vectors by Large-Scale Transient Transfection of Suspension Cultures and Affinity Chromatog‐ raphy Purification. 2007;98(4):789–99. onlinelibrary.wiley.com/doi/10.1002/bit.

[108] Kafri T, van Praag H, Ouyang L, Gage FH, Verma IM. A Packaging Cell Line for Len‐ tivirus Vectors. Journal of Virology. 1999;73(1):576–84. jvi.asm.org/cgi/content/

[109] Pacchiaa L, Adelson ME, Kaul M, Ron Y, Dougherty JP. An inducible packaging cell system for safe, efficient lentiviral vector production in the absence of HIV-1 accesso‐ ry proteins. Virology. 2001 Mar 30;282(1):77–86. www.sciencedirect.com/science/arti‐

[110] Kuate S, Wagner R, Uberla K. Development and characterization of a minimal indu‐ cible packaging cell line for simian immunodeficiency virus-based lentiviral vectors. The journal of gene medicine;4(4):347–55. onlinelibrary.wiley.com/doi/10.1002/jgm.

[111] Broussau S, Jabbour N, Lachapelle G, Durocher Y, Tom R, Transfiguracion J, et al. In‐ ducible packaging cells for large-scale production of lentiviral vectors in serum-free suspension culture. Molecular therapy : the journal of the American Society of Gene

article/pii/S0166093408004412 (accessed 1 August 2012)

1 August 2012)

314 Gene Therapy - Tools and Potential Applications

August 2012)

21467/pdf (accessed 1 August 2012)

abstract/73/1/576 (accessed 1 August 2012)

290/abstract (accessed 1 August 2012)

cle/pii/S0042682200907876 (accessed 1 August 2012)


[122] Lian Q, Chow Y, Esteban MA, Pei D, Tse H-F. Future perspective of induced pluripo‐ tent stem cells for diagnosis, drug screening and treatment of human diseases. Thrombosis and haemostasis. 2010;104(1):39–44. www.schattauer.de/en/magazine/ subject-areas/journals-a-z/thrombosis-and-haemostasis/contents/archive/issue/1093/ manuscript/13188.html (accessed 1 August 2012)

[131] Levine BL, Humeau LM, Boyer J, MacGregor R-R, Rebello T, Lu X, et al. Gene trans‐

www.pnas.org/cgi/content/abstract/103/46/17372 (accessed 1 August 2012)

fer in humans using a conditionally replicating lentiviral vector. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(46):17372–7.

Lentiviral Gene Therapy Vectors: Challenges and Future Directions

http://dx.doi.org/10.5772/52534

317


[131] Levine BL, Humeau LM, Boyer J, MacGregor R-R, Rebello T, Lu X, et al. Gene trans‐ fer in humans using a conditionally replicating lentiviral vector. Proceedings of the National Academy of Sciences of the United States of America. 2006;103(46):17372–7. www.pnas.org/cgi/content/abstract/103/46/17372 (accessed 1 August 2012)

[122] Lian Q, Chow Y, Esteban MA, Pei D, Tse H-F. Future perspective of induced pluripo‐ tent stem cells for diagnosis, drug screening and treatment of human diseases. Thrombosis and haemostasis. 2010;104(1):39–44. www.schattauer.de/en/magazine/ subject-areas/journals-a-z/thrombosis-and-haemostasis/contents/archive/issue/1093/

[123] Spencer HT, Denning G, Gautney RE, Dropulic B, Roy AJ, Baranyi L, et al. Lentiviral vector platform for production of bioengineered recombinant coagulation factor VIII. Molecular therapy: the journal of the American Society of Gene Therapy. 2011;19(2): 302–9. www.nature.com/mt/journal/v19/n2/full/mt2010239a.html (accessed 1 August

[124] Malik P, Arumugam PI, Yee J-K, Puthenveetil G. Successful correction of the human Cooley's anemia beta-thalassemia major phenotype using a lentiviral vector flanked by the chicken hypersensitive site 4 chromatin insulator. Annals of the New York Academy of Sciences. 2005;1054:238–49. onlinelibrary.wiley.com/doi/10.1196/annals.

[125] Pawliuk R, Westerman KA, Fabry ME, Payen E, Tighe R, Bouhassira EE, et al. Cor‐ rection of sickle cell disease in transgenic mouse models by gene therapy. Science (New York, N.Y.). 2001;294(5550):2368–71. www.sciencemag.org/content/

[126] Brown BD, Cantore A, Annoni A, Sergi LS, Lombardo A, Della Valle P, et al. A mi‐ croRNA-regulated lentiviral vector mediates stable correction of hemophilia B mice. Blood. 2007;110(13):4144–52. bloodjournal.hematologylibrary.org/content/

[127] Adjali O, Marodon G, Steinberg M, Mongellaz C, Thomas-Vaslin V, Jacquet C, et al. In vivo correction of ZAP-70 immunodeficiency by intrathymic gene transfer. The Journal of clinical investigation. 2005;115(8):2287–95. www.jci.org/articles/view/23966

[128] Lo Bianco C, Schneider BL, Bauer M, Sajadi A, Brice A, Iwatsubo T, et al. Lentiviral vector delivery of parkin prevents dopaminergic degeneration in an alpha-synuclein rat model of Parkinson's disease. Proceedings of the National Academy of Sciences of the United States of America. 2004;101(50):17510–5. www.pnas.org/cgi/content/

[129] Wang G, Slepushkin V, Zabner J, Keshavjee S, Johnston JC, Sauter SL, et al. Feline immunodeficiency virus vectors persistently transduce nondividing airway epithelia and correct the cystic fibrosis defect. The Journal of clinical investigation.

1999;104(11):R55–62. www.jci.org/articles/view/8390 (accessed 1 August 2012) [130] Azzouz M, Le T, Ralph GS, Walmsley L, Monani UR, Lee DCP, et al. Lentivectormediated SMN replacement in a mouse model of spinal muscular atrophy. The Jour‐ nal of clinical investigation. 2004;114(12):1726–31. www.jci.org/articles/view/

manuscript/13188.html (accessed 1 August 2012)

1345.030/abstract (accessed 1 August 2012)

294/5550/2368.abstract (accessed 1 August 2012)

110/13/4144.long (accessed 1 August 2012)

abstract/101/50/17510(accessed 1 August 2012)

(accessed 1 August 2012)

22922(accessed 1 August 2012)

2012)

316 Gene Therapy - Tools and Potential Applications

**Chapter 13**

**Lentiviral Vectors in Immunotherapy**

Ines Dufait, Therese Liechtenstein, Alessio Lanna,

Grazyna Kochan, Karine Breckpot and David Escors

Genetic immunotherapy can be defined as a therapeutic approach in which therapeutic genes are introduced into defined target cell types to modulate immune responses. A major challenge for this therapeutic strategy is the delivery of these genes into target cells in an efficient, stable manner. Possibly one of the best systems to achieve this is the use of lentivi‐ ral vectors (lentivectors) as gene carriers, as they are capable of transducing both dividing

Lentivectors are mainly derived from the human immunodeficiency virus (HIV-1) genome, a member of the *Retroviridae* family. The defining characteristic of retroviruses is their ca‐ pacity to stably integrate their RNA genome into the host cell chromosomes, in the form of a cDNA copy (Figure 1). Therefore, retrovirus and lentivirus vectors have been used exten‐ sively in research since they are ideal gene carriers into target cells. Moreover, both retrovi‐ rus and lentivirus vectors have been successfully applied in human gene therapy for the treatment of several genetic/metabolic inherited diseases (Cartier et al, 2009; Cavazzana-Cal‐

vo et al, 2010; Gaspar et al, 2004; Grez et al, 2010; Ott et al, 2006; Thrasher et al, 2006).

Lentiviruses are spherical enveloped viruses with a diameter around 80 to 120 nm and con‐ tain two copies of a single-stranded RNA genome (Figure 2) [2]. The genome is enclosed within a core composed of the structural and enzymatic proteins nucleocapsid (NC), capsid (CA), reverse transcriptase (RT), integrase (IN) and protease (PR). The core is surrounded by a protein layer of matrix (MA) protein. The envelope protein (ENV) is embedded in the viri‐ on lipid envelope, and it binds to the target cellular receptor and mediates virion entry.

> © 2013 Dufait et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Additional information is available at the end of the chapter

Roberta Laranga, Antonella Padella, Christopher Bricogne, Frederick Arce,

http://dx.doi.org/10.5772/50717

**1. Introduction**

and resting cells [1].

### **Lentiviral Vectors in Immunotherapy**

Ines Dufait, Therese Liechtenstein, Alessio Lanna, Roberta Laranga, Antonella Padella, Christopher Bricogne, Frederick Arce, Grazyna Kochan, Karine Breckpot and David Escors

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/50717

#### **1. Introduction**

Genetic immunotherapy can be defined as a therapeutic approach in which therapeutic genes are introduced into defined target cell types to modulate immune responses. A major challenge for this therapeutic strategy is the delivery of these genes into target cells in an efficient, stable manner. Possibly one of the best systems to achieve this is the use of lentivi‐ ral vectors (lentivectors) as gene carriers, as they are capable of transducing both dividing and resting cells [1].

Lentivectors are mainly derived from the human immunodeficiency virus (HIV-1) genome, a member of the *Retroviridae* family. The defining characteristic of retroviruses is their ca‐ pacity to stably integrate their RNA genome into the host cell chromosomes, in the form of a cDNA copy (Figure 1). Therefore, retrovirus and lentivirus vectors have been used exten‐ sively in research since they are ideal gene carriers into target cells. Moreover, both retrovi‐ rus and lentivirus vectors have been successfully applied in human gene therapy for the treatment of several genetic/metabolic inherited diseases (Cartier et al, 2009; Cavazzana-Cal‐ vo et al, 2010; Gaspar et al, 2004; Grez et al, 2010; Ott et al, 2006; Thrasher et al, 2006).

Lentiviruses are spherical enveloped viruses with a diameter around 80 to 120 nm and con‐ tain two copies of a single-stranded RNA genome (Figure 2) [2]. The genome is enclosed within a core composed of the structural and enzymatic proteins nucleocapsid (NC), capsid (CA), reverse transcriptase (RT), integrase (IN) and protease (PR). The core is surrounded by a protein layer of matrix (MA) protein. The envelope protein (ENV) is embedded in the viri‐ on lipid envelope, and it binds to the target cellular receptor and mediates virion entry.

© 2013 Dufait et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Lentivectors are classified as complex retroviruses according to their genome organisation, as it contains accessory and regulatory genes absent in other retroviruses. Nevertheless, the retrovirus genome shares a common 5' to the 3' gene organisation, with Gag, Pol and Env genes [1, 3]. Gag encodes MA, CA and NC as a polyprotein. Pol encodes enzymatic proteins associated to reverse transcription, that is, the reverse transcriptase (RT), integrase (IN) and protease (PR). RT synthesizes a single cDNA copy from the retrovirus genomic RNA [4]. IN mediates cDNA integration in the host cell chromosome, while PR cleaves Gag and Gag-Pol

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 321

The integrated cDNA genome is flanked by two long terminal repeats (LTRs) subdivided in U3, R and U5 regions. U3 is the HIV-1 promoter. The R region marks the starting point of transcription, and U5 region is critical for reverse transcription. The other key elements are the packaging signal (Ψ) and the polypurine tract (PPT). The packaging signal, as in many other virus species, allows RNA genome encapsidation during virion assembly in the cyto‐

Lentivectors are usually obtained following a three-plasmid co-transfection in 293T cells (Figure 3) [6, 7]. The first one, the packaging plasmid, provides the structural and RT pro‐ teins (Gag-Pol). The second one, the envelope plasmid, encodes a glycoprotein to pseudo‐ type the lentivector particles. This process consists on the incorporation of an heterologous Env in the viral lipid envelope. This will allow the lentivector to exhibit the specific cell trop‐ ism given by the Env used in pseudotyping. One of the most used Env is the Vesicular Sto‐ matitis Virus (VSV) Glycoprotein (G). The VSV-G confers stability to the lentivector particles and a very broad tropism for human and non-human cells [8]. Lastly, the third one, the transfer plasmid, contains the cis-acting sequences for replication/transcription and packag‐ ing (Figure 3) [9]. By including promoters within the transfer plasmid, any gene of interest can be expressed either constitutively or inducibly, in a cell type-specific of unspecific man‐ ner (Figure 3) [1]. Therefore, lentiviral vectors can also incorporate genes with immunoregu‐

Two main cell types of the immune system have been preferential targets for genetic immuno‐ therapy: antigen presenting cells (APCs) and effector T lymphocytes. These two cell types are key controllers of immune responses. By expressing transgenes of interest in APCs, such as DCs, they can be processed and presented to antigen-specific T cells in the immunological syn‐ apse. This antigen presentation is the first step in either starting of suppressing immune re‐ sponses. Therefore, if genes with modulating properties of APC functions can be co-expressed with antigens, the strength and type of immune response can be controlled. In fact, genetic modification of cells from the immune system can circumvent the limitations of current immu‐ notherapeutic protocols. Using targeted lentiviral vectors, specificity and effectiveness can be

Although more challenging than DCs, T lymphocytes can also be genetically modified using lentiviral vectors. Vectors expressing T cell receptors (TCRs) specific for antigens of interest can modify the specificity of T cell populations, or expand their antigen profile. Therefore, these genetically modified T cells can be adoptively transferred in the human patients. This

achieved by targeting key cells that modulate and polarize immune responses.

plasm. The PPT element is a key element for reverse transcription [5].

latory properties in cells from the immune system.

polyproteins during virion maturation.

**Figure 1.** The retrovirus life cycle.The life cycle of retroviruses, including lentiviruses is shown in this figure as a multi‐ step mechanism, starting with virion binding to the cellular receptor (R), leading to direct fusion or endocytosis. Then, the internal core is released and the two RNA molecules undergo reverse transcription as indicated, ending up with a single cDNA molecule. The core is then transported to the nucleus (in the case of lentiviruses) and the cDNA is inte‐ grated into the cell chromosome. The integrated genome (provirus) undergoes transcription, producing more RNA genome copies (and also spliced mRNAs, not shown here), which are also translated into structural and enzymatic proteins. These are then assembled into virions that bud out of the infected cells.

**Figure 2.** In this scheme, the lentivirus virion is represented as a sphere containing a genome made of two RNA mole‐ cules associated to the nucleocapsid (NC) protein. The nucleocapsid is enclosed by a core made of capsid (CA) protein, which is surrounded by a shell of matrix (MA) protein that associates to the virion envelope. The two subunits of the HIV-1 envelope are also indicated (SU and TM). In addition, other enzymatic (IN, RT, PR), accessory (Vpr, p6) and cellu‐ lar (Cyclophilin A, Cy-A) proteins are shown, which are incorporated into lentivirus particles.

Lentivectors are classified as complex retroviruses according to their genome organisation, as it contains accessory and regulatory genes absent in other retroviruses. Nevertheless, the retrovirus genome shares a common 5' to the 3' gene organisation, with Gag, Pol and Env genes [1, 3]. Gag encodes MA, CA and NC as a polyprotein. Pol encodes enzymatic proteins associated to reverse transcription, that is, the reverse transcriptase (RT), integrase (IN) and protease (PR). RT synthesizes a single cDNA copy from the retrovirus genomic RNA [4]. IN mediates cDNA integration in the host cell chromosome, while PR cleaves Gag and Gag-Pol polyproteins during virion maturation.

The integrated cDNA genome is flanked by two long terminal repeats (LTRs) subdivided in U3, R and U5 regions. U3 is the HIV-1 promoter. The R region marks the starting point of transcription, and U5 region is critical for reverse transcription. The other key elements are the packaging signal (Ψ) and the polypurine tract (PPT). The packaging signal, as in many other virus species, allows RNA genome encapsidation during virion assembly in the cyto‐ plasm. The PPT element is a key element for reverse transcription [5].

Lentivectors are usually obtained following a three-plasmid co-transfection in 293T cells (Figure 3) [6, 7]. The first one, the packaging plasmid, provides the structural and RT pro‐ teins (Gag-Pol). The second one, the envelope plasmid, encodes a glycoprotein to pseudo‐ type the lentivector particles. This process consists on the incorporation of an heterologous Env in the viral lipid envelope. This will allow the lentivector to exhibit the specific cell trop‐ ism given by the Env used in pseudotyping. One of the most used Env is the Vesicular Sto‐ matitis Virus (VSV) Glycoprotein (G). The VSV-G confers stability to the lentivector particles and a very broad tropism for human and non-human cells [8]. Lastly, the third one, the transfer plasmid, contains the cis-acting sequences for replication/transcription and packag‐ ing (Figure 3) [9]. By including promoters within the transfer plasmid, any gene of interest can be expressed either constitutively or inducibly, in a cell type-specific of unspecific man‐ ner (Figure 3) [1]. Therefore, lentiviral vectors can also incorporate genes with immunoregu‐ latory properties in cells from the immune system.

**Figure 1.** The retrovirus life cycle.The life cycle of retroviruses, including lentiviruses is shown in this figure as a multi‐ step mechanism, starting with virion binding to the cellular receptor (R), leading to direct fusion or endocytosis. Then, the internal core is released and the two RNA molecules undergo reverse transcription as indicated, ending up with a single cDNA molecule. The core is then transported to the nucleus (in the case of lentiviruses) and the cDNA is inte‐ grated into the cell chromosome. The integrated genome (provirus) undergoes transcription, producing more RNA genome copies (and also spliced mRNAs, not shown here), which are also translated into structural and enzymatic

**Figure 2.** In this scheme, the lentivirus virion is represented as a sphere containing a genome made of two RNA mole‐ cules associated to the nucleocapsid (NC) protein. The nucleocapsid is enclosed by a core made of capsid (CA) protein, which is surrounded by a shell of matrix (MA) protein that associates to the virion envelope. The two subunits of the HIV-1 envelope are also indicated (SU and TM). In addition, other enzymatic (IN, RT, PR), accessory (Vpr, p6) and cellu‐

lar (Cyclophilin A, Cy-A) proteins are shown, which are incorporated into lentivirus particles.

proteins. These are then assembled into virions that bud out of the infected cells.

320 Gene Therapy - Tools and Potential Applications

Two main cell types of the immune system have been preferential targets for genetic immuno‐ therapy: antigen presenting cells (APCs) and effector T lymphocytes. These two cell types are key controllers of immune responses. By expressing transgenes of interest in APCs, such as DCs, they can be processed and presented to antigen-specific T cells in the immunological syn‐ apse. This antigen presentation is the first step in either starting of suppressing immune re‐ sponses. Therefore, if genes with modulating properties of APC functions can be co-expressed with antigens, the strength and type of immune response can be controlled. In fact, genetic modification of cells from the immune system can circumvent the limitations of current immu‐ notherapeutic protocols. Using targeted lentiviral vectors, specificity and effectiveness can be achieved by targeting key cells that modulate and polarize immune responses.

Although more challenging than DCs, T lymphocytes can also be genetically modified using lentiviral vectors. Vectors expressing T cell receptors (TCRs) specific for antigens of interest can modify the specificity of T cell populations, or expand their antigen profile. Therefore, these genetically modified T cells can be adoptively transferred in the human patients. This strategy is particularly important to generate T cells with high affinity TCRs towards tu‐ mour-associated antigens.

PD-L1 with PD-1, are strongly inhibitory[16, 17]. Apart from these two signals, T cells re‐ quire a third signal which drives their differentiation into distinct subtypes that will regulate different types of immune responses [13, 18]. This third signal is usually provided by differ‐ ent combination of cytokines present within the immunological synapse (Figure 4). For ex‐ ample, the presence of high levels of IL12 will polarise T cell differentation towards a Th1 type (cellular cytotoxic immunity). On the other hand, high levels of IL10 will drive polari‐

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 323

**Figure 4.** In this scheme, DCs (left) present antigens to specific CD8 and CD4 T cells (as indicated) in the context of MHC class I (I) or class II (II) molecules. These T cells receive further stimuli by co-stimulation through ligand-receptor interactions between the DC and T cells (as indicated in the figure). Simultaneously, activated DCs secrete cytokines and chemokines (indicated in the figure as spheres) that will drive T cell activation, proliferation and differentation

An ideal immunotherapeutic approach would be to use lentiviral vectors to deliver the antigen of interest together with the three signals required for the desired T cell polarisation. Lentivec‐ tors have been extensively used for this purpose, because they are particularly effective in transducing DCs without affecting their functionality, unlike other vectors such as those based on adenoviruses [8, 19-26]. In fact, the stable integration of the lentivector genome allows longterm, sustained transgene expression[1, 9]. In addition, the expressed transgene is processed and its antigen peptides loaded in MHC I and MHC II molecules [27]. This is of the outmost im‐ portance for immunotherapy, since expressed proteins can be processed and loaded onto MHC-II molecules through several pathways. While secreted proteins can enter the endocytic pathway and membrane proteins can be recycled towards the endosomal pathway, cytoplas‐ mic proteins can still enter the MHC II pathway by autophagy[28]. Nevertheless, to improve MHC II loading of peptides from cytoplasmic proteins, endocytic localisation sequences can be fused to the transgene, with the lysosomal-associated membrane protein 1 or with the amino-

The use of lentivectors to express whole trangenes rather than antigen petides circumvents the necessity of designing specific peptide/protein vaccines for loading into specific MHC genotypes [27]. Thus, lentivectors expressing model antigens have been extensively used as a proof of principle. Amongst others, the antigens chicken ovalbumin (OVA), tumour-tu‐ mour associated antigens such as MELAN-A, tyrosinase related protein (Trp), NY-ESO or antigens from infectious agents have been expressed in DCs. These modified DCs induced

strong activation and proliferation of antigen-specific T cells. [17, 33-38].

sation towards Th2 (antibody responses).

into either cytotoxic CD8 T cells or T helper cells, as shown on the right.

terminal portion of the MHC II invariant chain [29-32].

**Figure 3.** The HIV-1 genome is shown at the top of the figure. All structural, accessory and enzymatic genes are indicat‐ ed throughout the genome. The two LTRs are shown as present in an integrated provirus. The three functional regions of the HIV LTR are shown on top of the 5- LTR. Numbers indicate nucleotide positions. The HIV genome is splitted in three different plasmids to engineer a gene vector. The transfer (vector) plasmid is indicated, with only the HIV-1 LTRs con‐ taining an internal promoter driving the expression of a gene of interest. In the packaging plasmid, only the Gag-Pro-Pol and Rev, Tat genes have been retained. This increases biosafety. Transcription of these genes takes place under the control of the cytomegalovirus promoter (CMV), as indicated in the figure. Lastly, a third plasmid encoding an enve‐ lope glycoprotein is shown on the bottom of the figure. This envelope pseudotypes the lentivector particle.

#### **2. Genetic modification of DCs with lentivectors**

For the elimination of cancer cells and chronic infections such as HIV, hepatitits B and ma‐ laria, a strong, effective T cell response is required. To initiate these strong T cell responses, the interaction between antigen-specific T cells and antigen-presenting APCs has to be strengthened[10]. Amongst APCs, DCs are most frequently the targets of immunotherapy protocols since they are probably the most immunogenic [10, 11].

To activate T cells during antigen presentation, these T cells have to receive at least three different signals from APCs (Figure 4) [10, 12-16]. The first signal, or signal 1, is the direct recognition of the peptide-major histocompatibility molecule complex (p-MHC) by the TCR. However, this interaction is not sufficient to confer T cells with effector activities. For this, a second co-stimulatory signal (signal 2) has to be co-delivered together with p-MHC recogni‐ tion. This signal 2 is the consequence of the integration of activatory and inhibitory interac‐ tions between ligands/receptors on the surface of DCs and T cells (Figure 4)[16, 17]. For example, CD80 binding to CD28 is strongly activatory, while CD80 binding with CTLA-4, or PD-L1 with PD-1, are strongly inhibitory[16, 17]. Apart from these two signals, T cells re‐ quire a third signal which drives their differentiation into distinct subtypes that will regulate different types of immune responses [13, 18]. This third signal is usually provided by differ‐ ent combination of cytokines present within the immunological synapse (Figure 4). For ex‐ ample, the presence of high levels of IL12 will polarise T cell differentation towards a Th1 type (cellular cytotoxic immunity). On the other hand, high levels of IL10 will drive polari‐ sation towards Th2 (antibody responses).

strategy is particularly important to generate T cells with high affinity TCRs towards tu‐

**Figure 3.** The HIV-1 genome is shown at the top of the figure. All structural, accessory and enzymatic genes are indicat‐ ed throughout the genome. The two LTRs are shown as present in an integrated provirus. The three functional regions of the HIV LTR are shown on top of the 5- LTR. Numbers indicate nucleotide positions. The HIV genome is splitted in three different plasmids to engineer a gene vector. The transfer (vector) plasmid is indicated, with only the HIV-1 LTRs con‐ taining an internal promoter driving the expression of a gene of interest. In the packaging plasmid, only the Gag-Pro-Pol and Rev, Tat genes have been retained. This increases biosafety. Transcription of these genes takes place under the control of the cytomegalovirus promoter (CMV), as indicated in the figure. Lastly, a third plasmid encoding an enve‐

For the elimination of cancer cells and chronic infections such as HIV, hepatitits B and ma‐ laria, a strong, effective T cell response is required. To initiate these strong T cell responses, the interaction between antigen-specific T cells and antigen-presenting APCs has to be strengthened[10]. Amongst APCs, DCs are most frequently the targets of immunotherapy

To activate T cells during antigen presentation, these T cells have to receive at least three different signals from APCs (Figure 4) [10, 12-16]. The first signal, or signal 1, is the direct recognition of the peptide-major histocompatibility molecule complex (p-MHC) by the TCR. However, this interaction is not sufficient to confer T cells with effector activities. For this, a second co-stimulatory signal (signal 2) has to be co-delivered together with p-MHC recogni‐ tion. This signal 2 is the consequence of the integration of activatory and inhibitory interac‐ tions between ligands/receptors on the surface of DCs and T cells (Figure 4)[16, 17]. For example, CD80 binding to CD28 is strongly activatory, while CD80 binding with CTLA-4, or

lope glycoprotein is shown on the bottom of the figure. This envelope pseudotypes the lentivector particle.

**2. Genetic modification of DCs with lentivectors**

protocols since they are probably the most immunogenic [10, 11].

mour-associated antigens.

322 Gene Therapy - Tools and Potential Applications

**Figure 4.** In this scheme, DCs (left) present antigens to specific CD8 and CD4 T cells (as indicated) in the context of MHC class I (I) or class II (II) molecules. These T cells receive further stimuli by co-stimulation through ligand-receptor interactions between the DC and T cells (as indicated in the figure). Simultaneously, activated DCs secrete cytokines and chemokines (indicated in the figure as spheres) that will drive T cell activation, proliferation and differentation into either cytotoxic CD8 T cells or T helper cells, as shown on the right.

An ideal immunotherapeutic approach would be to use lentiviral vectors to deliver the antigen of interest together with the three signals required for the desired T cell polarisation. Lentivec‐ tors have been extensively used for this purpose, because they are particularly effective in transducing DCs without affecting their functionality, unlike other vectors such as those based on adenoviruses [8, 19-26]. In fact, the stable integration of the lentivector genome allows longterm, sustained transgene expression[1, 9]. In addition, the expressed transgene is processed and its antigen peptides loaded in MHC I and MHC II molecules [27]. This is of the outmost im‐ portance for immunotherapy, since expressed proteins can be processed and loaded onto MHC-II molecules through several pathways. While secreted proteins can enter the endocytic pathway and membrane proteins can be recycled towards the endosomal pathway, cytoplas‐ mic proteins can still enter the MHC II pathway by autophagy[28]. Nevertheless, to improve MHC II loading of peptides from cytoplasmic proteins, endocytic localisation sequences can be fused to the transgene, with the lysosomal-associated membrane protein 1 or with the aminoterminal portion of the MHC II invariant chain [29-32].

The use of lentivectors to express whole trangenes rather than antigen petides circumvents the necessity of designing specific peptide/protein vaccines for loading into specific MHC genotypes [27]. Thus, lentivectors expressing model antigens have been extensively used as a proof of principle. Amongst others, the antigens chicken ovalbumin (OVA), tumour-tu‐ mour associated antigens such as MELAN-A, tyrosinase related protein (Trp), NY-ESO or antigens from infectious agents have been expressed in DCs. These modified DCs induced strong activation and proliferation of antigen-specific T cells. [17, 33-38].

#### **3. Lentivector immunogenicity**

Lentivectors have been extensively used in vaccination protocols, due to their capacity of raising strong transgene-specific immune responses [9, 17, 30, 38-40]. Interestingly, some re‐ ports suggest that lentivectors are incapable of inducing DC maturation *in vitro*, suggesting that some components of the lentivector preparations provide signals 2 and 3 through a mechanisms not well understood [40, 41].

ure 5). Lentivectors have thus been used to express the Kaposi's sarcoma-associated herpes virus FLICE-like inhibitory protein (vFLIP), a constitutive activator of NF-κB by direct asso‐ ciation and activation of NF-κB essential modulator (NEMO) [50-53]. In fact, vFLIP expres‐ sion has resulted to be a strong adjuvant when expressed in DCs, leading to strong DC maturation and effective CD4 and CD8 T cell responses. Lentivector expression of vFLIP sig‐ nificantly improves anti-tumour activities in a lymphoma mouse model and anti-parasitic efficacy in an OVA-expressing leishmania model [38, 54]. Lentivectors have also been used to inhibit negative regulators of NF-κB activation, such as the ubiqutin ligase A20. Lentivec‐ tors have successfully delivered to DCs short hairpin RNAs (shRNAs) targeting A20. The abrogation of A20 expression caused DC maturation, effective CD8 cell responses and inihi‐

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 325

Other molecular activators of mitogen activated protein kinases (MAPKs), activated after TLR engagement, have also been co-expressed in DCs with antigens of interest (Figure 5). MAPKs are mainly divided in three groups, ERK, p38 and JNK. While ERK is associated to survival and immune suppression, p38 and JNK are thought to stimulate DC maturation and inflammation (Figure 5). Constitutive p38 activation was achieved by expressing the MKK6 EE mutant using lentivectors, and it resulted in CD80, CD40 and ICAM-I up-regula‐ tion without significant secretion of pro-inflammatory cytokines [17, 40, 41]. A similar result was achieved by JNK1 activation, following expression of the MKK7-JNK1 fusion gene in DCs. Interestingly, although a full DC maturation phenotype was not achieved *ex vivo*, coexpression of these MAPK activators with an OVA-containing transgene or MELAN-A in‐ duced significant antigen-specific CD4 and CD8 T cell responses. Moreover, these lentivectors improved survival in a murine tumour model for lymphoma, both with inte‐

Other molecules have been applied for DC maturation. For example, CD40 ligand expres‐ sion achieved human DC activation and up-regulated the expression of CD83, CD80, MHC-I

bition of regulatory T cells (Tregs) [55, 56].

grating and non-integrating lentivectors [38].

**Figure 5.** Intracellular signalling pathways regulating DC functions.

DC maturation is a complex, step-wise process in which they up-regulate the surface expres‐ sion of co-stimulatory molecules such as CD80, CD83, CD86, CD40, adhesion molecules such as ICAM-1 and also the expression of MHC molecules. In general terms, DC matura‐ tion can be triggered by recognition of pathogen-derived molecules by specific receptors on the DC surface, such as the family of toll-like receptors (TLRs) [42, 43]. DCs can also mature through the exposure of pro-inflammatory cytokines by a process called cytokine priming [13-15]. Matured DCs can effectively provide strong signals 1 and 2, leading to efficient T cell activation and proliferation. Thus, their administration *in vivo* induces DC maturation and production of type I interferon that can provide signal 3 [9, 39, 44, 45].

The capacity of lentivectors to induce DC maturation after vaccination is probably caused by either specific components of the lentivector particle or by contaminants present in the lenti‐ vector preparation. As a matter of fact, lentivector particles resemble viruses and therefore, some components have the potential to stimulate immune responses such as the RNA genome or the cDNA [46]. These are ligands for TLR7 and TLR9, respectively [41, 45]. In addition to specific components of the lentivector particle, contaminants can also alter their immunosti‐ mulatory properties. In fact, most lentivector preparations pseudotyped with VSV G contain VSV-G tubulo-vesicular structures enclosing plasmid DNA that stimulate TLR9 *in vitro*, lead‐ ing to type I IFN production by pDCs [47]. In addition, foetal calf serum (FCS) contributes to immunogenicity by providing T cell epitopes with adjuvant capacities [48].

#### **4. Control of DC maturation by expression of molecular activators with lentivectors**

Lentivector preparations can induce DC maturation in vivo. However, in some circumstan‐ ces this is not enough for effective therapeutic activities. This is the case for cancer immuno‐ therapy, in which breaking tolerance towards TAAs is still a medical challenge. One possible solution is to co-express TAAs with molecular activators of DCs using lentivectors, particularly using activators of signalling cascades belonging to the TLR pathways.

This has been firstly achieved by over-expressing adaptor molecules, which associate with TLR cytoplasmic tails. These adaptor molecules recruit activatory protein kinases leading to DC maturation. Thus, lentivectors have been used to express MYD88 or TRIF1 in mouse myeloid DCs, which also increases secretion of pro-inflammatory cytokines IL-6, IL-12 and IFN-α, which enhanced T cell cytotoxicity [49]. The NF-κB pathway has also been an attrac‐ tive target because it controls transcription of the majority of pro-inflammatory genes (Fig‐ ure 5). Lentivectors have thus been used to express the Kaposi's sarcoma-associated herpes virus FLICE-like inhibitory protein (vFLIP), a constitutive activator of NF-κB by direct asso‐ ciation and activation of NF-κB essential modulator (NEMO) [50-53]. In fact, vFLIP expres‐ sion has resulted to be a strong adjuvant when expressed in DCs, leading to strong DC maturation and effective CD4 and CD8 T cell responses. Lentivector expression of vFLIP sig‐ nificantly improves anti-tumour activities in a lymphoma mouse model and anti-parasitic efficacy in an OVA-expressing leishmania model [38, 54]. Lentivectors have also been used to inhibit negative regulators of NF-κB activation, such as the ubiqutin ligase A20. Lentivec‐ tors have successfully delivered to DCs short hairpin RNAs (shRNAs) targeting A20. The abrogation of A20 expression caused DC maturation, effective CD8 cell responses and inihi‐ bition of regulatory T cells (Tregs) [55, 56].

**3. Lentivector immunogenicity**

324 Gene Therapy - Tools and Potential Applications

mechanisms not well understood [40, 41].

**lentivectors**

Lentivectors have been extensively used in vaccination protocols, due to their capacity of raising strong transgene-specific immune responses [9, 17, 30, 38-40]. Interestingly, some re‐ ports suggest that lentivectors are incapable of inducing DC maturation *in vitro*, suggesting that some components of the lentivector preparations provide signals 2 and 3 through a

DC maturation is a complex, step-wise process in which they up-regulate the surface expres‐ sion of co-stimulatory molecules such as CD80, CD83, CD86, CD40, adhesion molecules such as ICAM-1 and also the expression of MHC molecules. In general terms, DC matura‐ tion can be triggered by recognition of pathogen-derived molecules by specific receptors on the DC surface, such as the family of toll-like receptors (TLRs) [42, 43]. DCs can also mature through the exposure of pro-inflammatory cytokines by a process called cytokine priming [13-15]. Matured DCs can effectively provide strong signals 1 and 2, leading to efficient T cell activation and proliferation. Thus, their administration *in vivo* induces DC maturation

The capacity of lentivectors to induce DC maturation after vaccination is probably caused by either specific components of the lentivector particle or by contaminants present in the lenti‐ vector preparation. As a matter of fact, lentivector particles resemble viruses and therefore, some components have the potential to stimulate immune responses such as the RNA genome or the cDNA [46]. These are ligands for TLR7 and TLR9, respectively [41, 45]. In addition to specific components of the lentivector particle, contaminants can also alter their immunosti‐ mulatory properties. In fact, most lentivector preparations pseudotyped with VSV G contain VSV-G tubulo-vesicular structures enclosing plasmid DNA that stimulate TLR9 *in vitro*, lead‐ ing to type I IFN production by pDCs [47]. In addition, foetal calf serum (FCS) contributes to

**4. Control of DC maturation by expression of molecular activators with**

Lentivector preparations can induce DC maturation in vivo. However, in some circumstan‐ ces this is not enough for effective therapeutic activities. This is the case for cancer immuno‐ therapy, in which breaking tolerance towards TAAs is still a medical challenge. One possible solution is to co-express TAAs with molecular activators of DCs using lentivectors,

This has been firstly achieved by over-expressing adaptor molecules, which associate with TLR cytoplasmic tails. These adaptor molecules recruit activatory protein kinases leading to DC maturation. Thus, lentivectors have been used to express MYD88 or TRIF1 in mouse myeloid DCs, which also increases secretion of pro-inflammatory cytokines IL-6, IL-12 and IFN-α, which enhanced T cell cytotoxicity [49]. The NF-κB pathway has also been an attrac‐ tive target because it controls transcription of the majority of pro-inflammatory genes (Fig‐

particularly using activators of signalling cascades belonging to the TLR pathways.

and production of type I interferon that can provide signal 3 [9, 39, 44, 45].

immunogenicity by providing T cell epitopes with adjuvant capacities [48].

Other molecular activators of mitogen activated protein kinases (MAPKs), activated after TLR engagement, have also been co-expressed in DCs with antigens of interest (Figure 5). MAPKs are mainly divided in three groups, ERK, p38 and JNK. While ERK is associated to survival and immune suppression, p38 and JNK are thought to stimulate DC maturation and inflammation (Figure 5). Constitutive p38 activation was achieved by expressing the MKK6 EE mutant using lentivectors, and it resulted in CD80, CD40 and ICAM-I up-regula‐ tion without significant secretion of pro-inflammatory cytokines [17, 40, 41]. A similar result was achieved by JNK1 activation, following expression of the MKK7-JNK1 fusion gene in DCs. Interestingly, although a full DC maturation phenotype was not achieved *ex vivo*, coexpression of these MAPK activators with an OVA-containing transgene or MELAN-A in‐ duced significant antigen-specific CD4 and CD8 T cell responses. Moreover, these lentivectors improved survival in a murine tumour model for lymphoma, both with inte‐ grating and non-integrating lentivectors [38].

**Figure 5.** Intracellular signalling pathways regulating DC functions.

Other molecules have been applied for DC maturation. For example, CD40 ligand expres‐ sion achieved human DC activation and up-regulated the expression of CD83, CD80, MHC-I and induced IL-12 secretion [57]. This strategy increased CD4 and CD8 responses towards influenza epitopes and the TAA gp100. Co-expression of DC-promoting cytokines such as GM-CSF- and IL-4 using lentivectors resulted in long-lasting immunity against melanoma when co-expressed with TAAs Trp-2 and Mart-1 [58].

In this scheme, the main signalling pathways triggered after the engagement of a wide range of receptors on the DC surface (see the indicated receptors embedded in the mem‐ brane) with their ligands. Engagement of these receptors starts a complicated cascade of sig‐ nalling pathways that will converge in a few, well-characterised ones, the NK-κB, MAPKs and interferon regulatory factors (IRFs) (as indicated below the membrane). Some of these pathways, such as NK-κB, MAPKs p38 and JNK1 are pro-inflammatory and lead to DC ma‐ turation. Others, such as ERK, are clearly immunosuppressive.

#### **5. Control of DC maturation by inhibiting negative co-stimulation using lentivectors**

**Figure 6.** A scheme of the TCR is shown embedded in the cellular membrane. Both α and β chains are shown as indi‐ cated, subdivided in variable and constant regions (V, C). The other CD3 chains that associate with the TCR are also included in the figure. On the bottom, a lentivector co-expressing α and β TCR chains is shown, under the control of the spleen focus forming virus promoter (SFFV) and an internal ribosome entry site (IRES) [67]. This particular lentivec‐ tor is self-inactivating (SIN) and presents a deletion of viral enhancers in the 3' LTR. When this construct is integrated,

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 327

As mentioned above, a major issue with cancer immunotherapy is that most TAA-specific T cells may have been eliminated during thymic clonal deletion. Thus, even if effective and strong DC maturation is achieved, no effective responses will be achieved due to lack of TAA-specific T cells. To circumvent this, TAA-specific T cells can be generated by lentivec‐ tor transduction *in vitro*, and adoptively transfered in patients (Figure 6) [62]. Clinical effica‐ cy has been reported for melanoma, synovial cell sarcoma, colorectal, neuroblastoma and

T cells are largely refractory to transduction by VSV G-pseudotyped lentivectors, and they require some level of T cell stimulation [67]. Treatment with IL-2 and IL-7 allows lentivector transduction and preserves a functional T cell repertoire [68, 69]. As an example, Wilms tu‐ mour antigen (WT1)-specific T cells were generated by lentivector expression of a WT1-spe‐ cific TCR in the presence of IL-15 and IL-21. These modified T cells were multifunctional and exhibited the expected antigen specificity [67]. This approach of T cell modification is rather promising. In a clinical trial with 15 terminally sick melanoma patients, 2 showed complete regression and long-term survival after transfer of T cells expressing a MART-1 specific TCR using γ-retrovirus vectors [70]. Interestingly, it has been recently demonstrated that entivectors pseudotyped with measles virus H/F glycoproteins effectively transduce quiescent adult T cells in the absence of any exogenous stimulus, whether cytokines or anti-CD3/anti-CD28 stimulation. In fact, transduction with these lentivectors did not affect T cells

lymphoma, but using γ-retrovirus vectors instead of lentivectors [63-66].

the 5' LTR disappears and it is replaced with the deleted version.

in any way [17, 71-73].

DC maturation can also up-regulate molecules that provide negative stimulation to T cells, such as programmed cell death receptor ligand 1 (PD-L1) and PD-L2, the ligands for the PD-1 receptor on T cells. Negative co-stimulation is part of a regulatory mechanism that controls the activation state of T cells following antigen presentation [17, 59, 60]. Thus, inter‐ ference with negative co-stimulation could in principle reinforce T cell activation and en‐ hance cytotoxic activities. Therefore, lentivectors have been used to deliver shRNAs in DC against PD-L1. PD-L1 silencing in antigen-presenting DCs hyperactivated T cells by pre‐ venting the up-regulation of Casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase. This strategy co- accelerated anti-tumour immune responses, particularly if combined with a p38 activa‐ tor or dominant negative mutant of MEK1, the upstream kinase of ERK [17, 59].

#### **6. Lentivectors and cancer immunotherapy**

Lentivectors are particularly promising in cancer immunotherapy, for which conventional im‐ munization is largely ineffective due to two major barriers. Firstly, TAAs are generally selfproteins to which there is strong immunological tolerance. Secondly, that tumours are strongly immune-suppressive and they use several mechanisms to avoid immune responses [41].

Lentivectors can be used in cancer immunotherapy in two different ways. In the first one, DCs can be generated *ex vivo* from the patient, followed by lentivector transduction and *in vivo* administration. Thus, cellular vaccination with transduced DCs expressing HLA-Cw3 induced activation and proliferation of CD8 T cells in a mouse model [37]. Similarly, lenti‐ vector transduction was shown to be superior to peptide pulsing in inducing OVA-specific T cell responses [61], protected mice from OVA-expressing tumour cells and significantly in‐ hibited tumour growth. The second strategy is direct lentivector vaccination, taking advant‐ age of their intrinsic immuno-stimulatory capacities and their reduced cost [24, 26, 35].

and induced IL-12 secretion [57]. This strategy increased CD4 and CD8 responses towards influenza epitopes and the TAA gp100. Co-expression of DC-promoting cytokines such as GM-CSF- and IL-4 using lentivectors resulted in long-lasting immunity against melanoma

In this scheme, the main signalling pathways triggered after the engagement of a wide range of receptors on the DC surface (see the indicated receptors embedded in the mem‐ brane) with their ligands. Engagement of these receptors starts a complicated cascade of sig‐ nalling pathways that will converge in a few, well-characterised ones, the NK-κB, MAPKs and interferon regulatory factors (IRFs) (as indicated below the membrane). Some of these pathways, such as NK-κB, MAPKs p38 and JNK1 are pro-inflammatory and lead to DC ma‐

**5. Control of DC maturation by inhibiting negative co-stimulation using**

DC maturation can also up-regulate molecules that provide negative stimulation to T cells, such as programmed cell death receptor ligand 1 (PD-L1) and PD-L2, the ligands for the PD-1 receptor on T cells. Negative co-stimulation is part of a regulatory mechanism that controls the activation state of T cells following antigen presentation [17, 59, 60]. Thus, inter‐ ference with negative co-stimulation could in principle reinforce T cell activation and en‐ hance cytotoxic activities. Therefore, lentivectors have been used to deliver shRNAs in DC against PD-L1. PD-L1 silencing in antigen-presenting DCs hyperactivated T cells by pre‐ venting the up-regulation of Casitas B-lymphoma (Cbl)-b E3 ubiquitin ligase. This strategy co- accelerated anti-tumour immune responses, particularly if combined with a p38 activa‐

Lentivectors are particularly promising in cancer immunotherapy, for which conventional im‐ munization is largely ineffective due to two major barriers. Firstly, TAAs are generally selfproteins to which there is strong immunological tolerance. Secondly, that tumours are strongly immune-suppressive and they use several mechanisms to avoid immune responses [41].

Lentivectors can be used in cancer immunotherapy in two different ways. In the first one, DCs can be generated *ex vivo* from the patient, followed by lentivector transduction and *in vivo* administration. Thus, cellular vaccination with transduced DCs expressing HLA-Cw3 induced activation and proliferation of CD8 T cells in a mouse model [37]. Similarly, lenti‐ vector transduction was shown to be superior to peptide pulsing in inducing OVA-specific T cell responses [61], protected mice from OVA-expressing tumour cells and significantly in‐ hibited tumour growth. The second strategy is direct lentivector vaccination, taking advant‐ age of their intrinsic immuno-stimulatory capacities and their reduced cost [24, 26, 35].

tor or dominant negative mutant of MEK1, the upstream kinase of ERK [17, 59].

**6. Lentivectors and cancer immunotherapy**

when co-expressed with TAAs Trp-2 and Mart-1 [58].

326 Gene Therapy - Tools and Potential Applications

**lentivectors**

turation. Others, such as ERK, are clearly immunosuppressive.

**Figure 6.** A scheme of the TCR is shown embedded in the cellular membrane. Both α and β chains are shown as indi‐ cated, subdivided in variable and constant regions (V, C). The other CD3 chains that associate with the TCR are also included in the figure. On the bottom, a lentivector co-expressing α and β TCR chains is shown, under the control of the spleen focus forming virus promoter (SFFV) and an internal ribosome entry site (IRES) [67]. This particular lentivec‐ tor is self-inactivating (SIN) and presents a deletion of viral enhancers in the 3' LTR. When this construct is integrated, the 5' LTR disappears and it is replaced with the deleted version.

As mentioned above, a major issue with cancer immunotherapy is that most TAA-specific T cells may have been eliminated during thymic clonal deletion. Thus, even if effective and strong DC maturation is achieved, no effective responses will be achieved due to lack of TAA-specific T cells. To circumvent this, TAA-specific T cells can be generated by lentivec‐ tor transduction *in vitro*, and adoptively transfered in patients (Figure 6) [62]. Clinical effica‐ cy has been reported for melanoma, synovial cell sarcoma, colorectal, neuroblastoma and lymphoma, but using γ-retrovirus vectors instead of lentivectors [63-66].

T cells are largely refractory to transduction by VSV G-pseudotyped lentivectors, and they require some level of T cell stimulation [67]. Treatment with IL-2 and IL-7 allows lentivector transduction and preserves a functional T cell repertoire [68, 69]. As an example, Wilms tu‐ mour antigen (WT1)-specific T cells were generated by lentivector expression of a WT1-spe‐ cific TCR in the presence of IL-15 and IL-21. These modified T cells were multifunctional and exhibited the expected antigen specificity [67]. This approach of T cell modification is rather promising. In a clinical trial with 15 terminally sick melanoma patients, 2 showed complete regression and long-term survival after transfer of T cells expressing a MART-1 specific TCR using γ-retrovirus vectors [70]. Interestingly, it has been recently demonstrated that entivectors pseudotyped with measles virus H/F glycoproteins effectively transduce quiescent adult T cells in the absence of any exogenous stimulus, whether cytokines or anti-CD3/anti-CD28 stimulation. In fact, transduction with these lentivectors did not affect T cells in any way [17, 71-73].

### **7. Lentivector gene immunotherapy for the treatment of autoimmune disorders**

The organism is in permanent direct contact with many substances and commensal organ‐ isms in mucosal areas and in peripheral tissues. In these situations, inducible Tregs differen‐ tiate from naïve CD4 T cells after antigen presentation by tolerogenic DCs. These regulatory T cell types are usually classified in Tr1 (CD4 CD25 IL10 or TGF-β) and Th3 (CD4 CD25 Foxp3) cells [78-82]. Therefore, DCs can also be converted in tolerogenic by expression of immunomodulatory genes with lentivectors. This strategy opens up the application of lenti‐

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 329

DCs can induce immunological tolerance through a number of mechanisms. It is generally accepted that antigen presentation by immature DCs is poorly immunogenic, and results in Treg differentiation, T cell apoptosis and T cell anergy [83-86]. These immature tolerogenic DCs express low levels of co-stimulatory molecules CD80, CD86, CD83, CD40 and MHC molecules [10, 40, 41, 78, 87]. Resident mucosal DCs are intrinsically tolerogenic independ‐ ently on their maturation phenotype as a consequence of the presence of retinoic-acid [88]. In addition, these DCs become strongly immunosuppressive due to contact with TLR ago‐ nists from commensal microbiota [88-90]. DCs can also become strongly immunosuppres‐ sive after treatment with lectin ligands or exposure to immunosuppressive cytokines such as IL-10, IL-4 or TGF-β[78, 87, 89-92]. Tolerogenic DCs usually express high levels of these im‐ munosuppressive cytokines, even if they are phenotypically mature [10, 40, 78, 87, 89-93]. In this situation, they provide strong signals 1 and 2 to T cells, together with a simultaneous strong tolerogenic signal 3. For example, in the presence of bioactive TGF-β, strong antigen presentation leads to differentiation to antigen-specific Foxp3 Tregs, while secretion of IL-10

Tolerogenic DCs can also up-regulate molecules that provide an inhibitory signal to T cells, such as PD-L1 (or B7-H1), a member of the B7 co-stimulatory molecules [17, 96]. PD-L1 ex‐ pression in DCs regulates T cell activities during antigen presentation and prevents T cell hyperactivation [17]. In addition, PD-L1-CD80 binding on T cells induces antigen-specific Treg differentiation [97]. Other members of the B7 family are immunosuppressive [98]. Im‐ munosuppressive DCs also up-regulate aminoacid-metabolising enzymes, such as arginase or indoleamine 2,3-dioxygenase (IDO) [99-104]. It is thought that these enzymes deplete T

Lentivectors can be used to confer tolerogenic activities to DCs by expression of immunore‐ gulatory genes together with antigens of interest. The first strategy that was tested experi‐ mental was the expression of potent immunosuppressive cytokines. This approach was used with γ-retroviral vectors for inflammatory diseases [95, 105, 106], 105, 106]. Lentivectors have been applied in an experimental model of asthma by expressing IL-10, leading to ex‐ pansion of IL-10-expressing Foxp3 Tregs with potent anti-inflammatory properties [107]. Al‐ ternatively, small immunosuppressive peptides can also be delivered with lentivectors, such as the vasointestinal peptide (VIP). Intraperitoneal administration of VIP-encoding lentivec‐

vectors for the treatment of autoimmune disorders

usually results in Tr1 differentiation [91, 93-95].

cells of essential aminoacids.

**9. Induction of tolerogenic DCs using lentivectors**

It is relatively "straightforward" to achieve immune stimulation using lentivectors. Howev‐ er, the induction of immune suppression or tolerance with lentivectors is rather challenging. Nevertheless, the induction and maintenance of immunological tolerance is critical for ho‐ meostasis. The organism is permanently and closely in contact with a very wide range of an‐ tigens of many origins. A large majority of them are innocuous and do not pose a direct threat. Thus, the immune system must not respond to these antigens, as an immune re‐ sponse is associated with significant collateral tissue damage. The immune system should be activated only if a real threat appears. Therefore, the immune system possesses several tol‐ erogenic mechanisms in place to keep immunological homeostasis. As mentioned before, a key one is clonal deletion of auto-reactive T cells in the thymus [74]. However, there is a sig‐ nificant number of auto-reactive T cells that escape from clonal deletion. Many of them will differentiate towards natural Foxp3 CD4 regulatory T cells [74-77].

In addition to clonal deletion and differentiation of natural Tregs, there are a number of tol‐ erogenic mechanisms in place that regulate immune responses towards peripheral antigens. The organism is in permanent direct contact with many substances and commensal organ‐ isms in mucosal areas and in peripheral tissues. In these situations, inducible Tregs differen‐ tiate from naïve CD4 T cells after antigen presentation by tolerogenic DCs. These regulatory T cell types are usually classified in Tr1 (CD4 CD25 IL10 or TGF-β) and Th3 (CD4 CD25 Foxp3) cells [78-82]. Therefore, DCs can also be converted in tolerogenic by expression of immunomodulatory genes with lentivectors. This strategy opens up the application of lenti‐ vectors for the treatment of autoimmune disorders.

#### **8. Induction of tolerogenic DCs using lentivectors**

It is relatively "straightforward" to achieve immune stimulation using lentivectors. Howev‐ er, the induction of immune suppression or tolerance with lentivectors is rather challenging. However, the induction and maintenance of immunological tolerance is critical for homeo‐ stasis. The organism is permanently and closely in contact with a very wide range of anti‐ gens of many origins. A large majority of them are innocuous and do not pose a direct threat. Thus, the immune system must not respond to these antigens, as an immune re‐ sponse is associated with significant collateral tissue damage. The immune system should be activated only if a real threat appears. Therefore, the immune system possesses several tol‐ erogenic mechanisms in place to keep immunological homeostasis. As mentioned before, a key one is clonal deletion of auto-reactive T cells in the thymus [74]. However, there is a sig‐ nificant number of auto-reactive T cells that escape from clonal deletion. Many of them will differentiate towards natural Foxp3 CD4 regulatory T cells [74-77].

In addition to clonal deletion and differentiation of natural Tregs, there are a number of tol‐ erogenic mechanisms in place that regulate immune responses towards peripheral antigens. The organism is in permanent direct contact with many substances and commensal organ‐ isms in mucosal areas and in peripheral tissues. In these situations, inducible Tregs differen‐ tiate from naïve CD4 T cells after antigen presentation by tolerogenic DCs. These regulatory T cell types are usually classified in Tr1 (CD4 CD25 IL10 or TGF-β) and Th3 (CD4 CD25 Foxp3) cells [78-82]. Therefore, DCs can also be converted in tolerogenic by expression of immunomodulatory genes with lentivectors. This strategy opens up the application of lenti‐ vectors for the treatment of autoimmune disorders

#### **9. Induction of tolerogenic DCs using lentivectors**

**7. Lentivector gene immunotherapy for the treatment of autoimmune**

differentiate towards natural Foxp3 CD4 regulatory T cells [74-77].

vectors for the treatment of autoimmune disorders.

**8. Induction of tolerogenic DCs using lentivectors**

differentiate towards natural Foxp3 CD4 regulatory T cells [74-77].

It is relatively "straightforward" to achieve immune stimulation using lentivectors. Howev‐ er, the induction of immune suppression or tolerance with lentivectors is rather challenging. Nevertheless, the induction and maintenance of immunological tolerance is critical for ho‐ meostasis. The organism is permanently and closely in contact with a very wide range of an‐ tigens of many origins. A large majority of them are innocuous and do not pose a direct threat. Thus, the immune system must not respond to these antigens, as an immune re‐ sponse is associated with significant collateral tissue damage. The immune system should be activated only if a real threat appears. Therefore, the immune system possesses several tol‐ erogenic mechanisms in place to keep immunological homeostasis. As mentioned before, a key one is clonal deletion of auto-reactive T cells in the thymus [74]. However, there is a sig‐ nificant number of auto-reactive T cells that escape from clonal deletion. Many of them will

In addition to clonal deletion and differentiation of natural Tregs, there are a number of tol‐ erogenic mechanisms in place that regulate immune responses towards peripheral antigens. The organism is in permanent direct contact with many substances and commensal organ‐ isms in mucosal areas and in peripheral tissues. In these situations, inducible Tregs differen‐ tiate from naïve CD4 T cells after antigen presentation by tolerogenic DCs. These regulatory T cell types are usually classified in Tr1 (CD4 CD25 IL10 or TGF-β) and Th3 (CD4 CD25 Foxp3) cells [78-82]. Therefore, DCs can also be converted in tolerogenic by expression of immunomodulatory genes with lentivectors. This strategy opens up the application of lenti‐

It is relatively "straightforward" to achieve immune stimulation using lentivectors. Howev‐ er, the induction of immune suppression or tolerance with lentivectors is rather challenging. However, the induction and maintenance of immunological tolerance is critical for homeo‐ stasis. The organism is permanently and closely in contact with a very wide range of anti‐ gens of many origins. A large majority of them are innocuous and do not pose a direct threat. Thus, the immune system must not respond to these antigens, as an immune re‐ sponse is associated with significant collateral tissue damage. The immune system should be activated only if a real threat appears. Therefore, the immune system possesses several tol‐ erogenic mechanisms in place to keep immunological homeostasis. As mentioned before, a key one is clonal deletion of auto-reactive T cells in the thymus [74]. However, there is a sig‐ nificant number of auto-reactive T cells that escape from clonal deletion. Many of them will

In addition to clonal deletion and differentiation of natural Tregs, there are a number of tol‐ erogenic mechanisms in place that regulate immune responses towards peripheral antigens.

**disorders**

328 Gene Therapy - Tools and Potential Applications

DCs can induce immunological tolerance through a number of mechanisms. It is generally accepted that antigen presentation by immature DCs is poorly immunogenic, and results in Treg differentiation, T cell apoptosis and T cell anergy [83-86]. These immature tolerogenic DCs express low levels of co-stimulatory molecules CD80, CD86, CD83, CD40 and MHC molecules [10, 40, 41, 78, 87]. Resident mucosal DCs are intrinsically tolerogenic independ‐ ently on their maturation phenotype as a consequence of the presence of retinoic-acid [88]. In addition, these DCs become strongly immunosuppressive due to contact with TLR ago‐ nists from commensal microbiota [88-90]. DCs can also become strongly immunosuppres‐ sive after treatment with lectin ligands or exposure to immunosuppressive cytokines such as IL-10, IL-4 or TGF-β[78, 87, 89-92]. Tolerogenic DCs usually express high levels of these im‐ munosuppressive cytokines, even if they are phenotypically mature [10, 40, 78, 87, 89-93]. In this situation, they provide strong signals 1 and 2 to T cells, together with a simultaneous strong tolerogenic signal 3. For example, in the presence of bioactive TGF-β, strong antigen presentation leads to differentiation to antigen-specific Foxp3 Tregs, while secretion of IL-10 usually results in Tr1 differentiation [91, 93-95].

Tolerogenic DCs can also up-regulate molecules that provide an inhibitory signal to T cells, such as PD-L1 (or B7-H1), a member of the B7 co-stimulatory molecules [17, 96]. PD-L1 ex‐ pression in DCs regulates T cell activities during antigen presentation and prevents T cell hyperactivation [17]. In addition, PD-L1-CD80 binding on T cells induces antigen-specific Treg differentiation [97]. Other members of the B7 family are immunosuppressive [98]. Im‐ munosuppressive DCs also up-regulate aminoacid-metabolising enzymes, such as arginase or indoleamine 2,3-dioxygenase (IDO) [99-104]. It is thought that these enzymes deplete T cells of essential aminoacids.

Lentivectors can be used to confer tolerogenic activities to DCs by expression of immunore‐ gulatory genes together with antigens of interest. The first strategy that was tested experi‐ mental was the expression of potent immunosuppressive cytokines. This approach was used with γ-retroviral vectors for inflammatory diseases [95, 105, 106], 105, 106]. Lentivectors have been applied in an experimental model of asthma by expressing IL-10, leading to ex‐ pansion of IL-10-expressing Foxp3 Tregs with potent anti-inflammatory properties [107]. Al‐ ternatively, small immunosuppressive peptides can also be delivered with lentivectors, such as the vasointestinal peptide (VIP). Intraperitoneal administration of VIP-encoding lentivec‐ tors in mice effectively inhibited the development of experimental collagen-induced arthri‐ tis. This was achieved by a markedly reduction of pro-inflammatory cytokine secretion and the expansion of Foxp3 Tregs [108]. The administration of genetically modified VIP-express‐ ing DCs also showed significant therapeutic effects in EAE and in the coecal ligation and puncture (CLP) model [109].

most potent and targeted immunotherapeutic approaches are required to break the natural tolerance towards TAAs. The targeted co-delivery of immunomodulatory genes with anti‐ gens of interests to DCs has opened the application of gene therapy for immunotherapy. Lentivectors exhibit a remarkable transduction capacity of DCs and also T cells, and thus, they are ideal tools to achieve immunodulation. In this way, the immune system can be strongly and specifically activated for the treatment of cancer and infectious diseases, but it can on the other hand be strongly immunosuppressed. This makes it possible the induction

Ines Dufait is funded by an ERASMUS scholarship. Alessio Lanna is funded by an Universi‐ ty College London "bench-to-bedside" PhD scholarship. Christopher Bricogne is funded by an University College London MB-PhD scholarship. The Structural Genomics Consortium Oxfod is a registered UK charity (number 1097737) that receives funds from the Canadian Institutes of Health Research, The Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Insitute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundations, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Foundation for Strategic Research and the Wellcome Trust. Karine Breckpot is funded by the Fund for Scientific Research- Flandes. David Escors is funded by an Arthritis Research UK

, Alessio Lanna2

, Grazyna Kochan4

1 Department of Physiology–Immunology, Medical School, Free University of Brussels, Bel‐

2 Division of Infection and Immunity, Rayne Institute, University College London, United

4 Structural Genomics Consortium Oxford, University of Oxford, Old Road Campus Re‐

, Roberta Laranga2

, Karine Breckpot1

, Antonella Padella2

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 331

and

,

of immunological tolerance and treatment of autoimmune disorders.

**Acknowledgements**

**Author details**

Christopher Bricogne3

search Building, United Kingdom

Ines Dufait1

gium

Kingdom

David Escors2\*

Career Development Fellowship (18433).

, Therese Liechtenstein2

\*Address all correspondence to: rekades@ucl.ac.uk

, Frederick Arce3

3 UCL Cancer Institute, University College London, United Kingdom

DCs can also be reprogrammed by direct modulation tolerogenic signalling pathways with‐ in DCs (Figure 5). Therefore, lentivector expression of a constitutively active MEK1 mutant resulted in sustained MAPK ERK phosphorylation, resulting in immunological tolerance [40, 90, 110-114]. These genetically modified DCs exhibit an immature phenotype with low levels of CD40 and secretion of bioactive TGF-β[40, 78]. Antigen presentation by these ERKactivated DCs differentiated antigen-specific Foxp3 Tregs both *ex vivo* and *in vivo* in a mouse model [78]. Direct lentivector vaccination encoding the ERK activator effectively controlled antigen-induced inflammatory arthritis in a mouse model [78].

Similarly, lentivector expression of a constitutively active IRF3 mutant induced high expres‐ sion levels of IL-10, and expanded antigen-specific Foxp3 Tregs which inhibited immune re‐ sponses (Figure 5) [40]. Activation of endogenous negative feedback mechanisms of DC maturation pathways has also been applied to induce immune suppression. In this way, by over-expressing the suppressor of cytokine signalling 3 (SOCS-3) in DCs, pro-inflammatory signalling pathways were severly impaired [115]. These genetically modified DCs signifi‐ cantly decreased secretion of pro-inflammatory cytokines IFN-γ, IL-12 and IL-23, and showed an enhanced IL-10 production, which effectively inhibited effectively inhibit experi‐ mental autoimmune encephalomyelitis (EAE) in mice [115].

An alternative strategy to generate tolerogenic DCs is the inhibition of pro-inflammatory sig‐ nalling pathways instead of activating immunosuppressive pathways. As NF-κB is a critical inflammatory signalling pathway, its inhibition in promising for the induction of immunologi‐ cal tolerance [41]. To achieve this, Rel-B was silenced by the delivery of a shRNA targeted to Rel-B [116]. In this way, its inhibition could effectively prevent DC maturation after engage‐ ment with TLR ligands, and it was sufficient to treat autoimmune myasthenia gravis in a mouse model [116]. In an analogous manner, lentivectors have also been applied to silence B cell activating factor (BAFF) in the inflamed joint [117, 118], which was very effective for the treatment of experimental collagen-induced arthritis [119] without the need of targeting the ar‐ thritogenic antigen. These lentivectors were directly injected in the inflamed joint, where they preferentially transduced resident DCs. BAFF silencing in these DCs inhibited their matura‐ tion, and most importantly, inhibited differentiation of pathogenic Th17 [119].

#### **10. Conclussions**

Classical immunotherapeutic strategies for the treatment of cancer and infectious diseases rely on either administration of the antigen peptides together with adjuvants, or the inocula‐ tion with attenuated strains of pathogenic agents. This approach has been largely successful for the treatment of a wide range of infectious agents. However, for cancer immunotherapy, most potent and targeted immunotherapeutic approaches are required to break the natural tolerance towards TAAs. The targeted co-delivery of immunomodulatory genes with anti‐ gens of interests to DCs has opened the application of gene therapy for immunotherapy. Lentivectors exhibit a remarkable transduction capacity of DCs and also T cells, and thus, they are ideal tools to achieve immunodulation. In this way, the immune system can be strongly and specifically activated for the treatment of cancer and infectious diseases, but it can on the other hand be strongly immunosuppressed. This makes it possible the induction of immunological tolerance and treatment of autoimmune disorders.

#### **Acknowledgements**

tors in mice effectively inhibited the development of experimental collagen-induced arthri‐ tis. This was achieved by a markedly reduction of pro-inflammatory cytokine secretion and the expansion of Foxp3 Tregs [108]. The administration of genetically modified VIP-express‐ ing DCs also showed significant therapeutic effects in EAE and in the coecal ligation and

DCs can also be reprogrammed by direct modulation tolerogenic signalling pathways with‐ in DCs (Figure 5). Therefore, lentivector expression of a constitutively active MEK1 mutant resulted in sustained MAPK ERK phosphorylation, resulting in immunological tolerance [40, 90, 110-114]. These genetically modified DCs exhibit an immature phenotype with low levels of CD40 and secretion of bioactive TGF-β[40, 78]. Antigen presentation by these ERKactivated DCs differentiated antigen-specific Foxp3 Tregs both *ex vivo* and *in vivo* in a mouse model [78]. Direct lentivector vaccination encoding the ERK activator effectively controlled

Similarly, lentivector expression of a constitutively active IRF3 mutant induced high expres‐ sion levels of IL-10, and expanded antigen-specific Foxp3 Tregs which inhibited immune re‐ sponses (Figure 5) [40]. Activation of endogenous negative feedback mechanisms of DC maturation pathways has also been applied to induce immune suppression. In this way, by over-expressing the suppressor of cytokine signalling 3 (SOCS-3) in DCs, pro-inflammatory signalling pathways were severly impaired [115]. These genetically modified DCs signifi‐ cantly decreased secretion of pro-inflammatory cytokines IFN-γ, IL-12 and IL-23, and showed an enhanced IL-10 production, which effectively inhibited effectively inhibit experi‐

An alternative strategy to generate tolerogenic DCs is the inhibition of pro-inflammatory sig‐ nalling pathways instead of activating immunosuppressive pathways. As NF-κB is a critical inflammatory signalling pathway, its inhibition in promising for the induction of immunologi‐ cal tolerance [41]. To achieve this, Rel-B was silenced by the delivery of a shRNA targeted to Rel-B [116]. In this way, its inhibition could effectively prevent DC maturation after engage‐ ment with TLR ligands, and it was sufficient to treat autoimmune myasthenia gravis in a mouse model [116]. In an analogous manner, lentivectors have also been applied to silence B cell activating factor (BAFF) in the inflamed joint [117, 118], which was very effective for the treatment of experimental collagen-induced arthritis [119] without the need of targeting the ar‐ thritogenic antigen. These lentivectors were directly injected in the inflamed joint, where they preferentially transduced resident DCs. BAFF silencing in these DCs inhibited their matura‐

Classical immunotherapeutic strategies for the treatment of cancer and infectious diseases rely on either administration of the antigen peptides together with adjuvants, or the inocula‐ tion with attenuated strains of pathogenic agents. This approach has been largely successful for the treatment of a wide range of infectious agents. However, for cancer immunotherapy,

tion, and most importantly, inhibited differentiation of pathogenic Th17 [119].

antigen-induced inflammatory arthritis in a mouse model [78].

mental autoimmune encephalomyelitis (EAE) in mice [115].

puncture (CLP) model [109].

330 Gene Therapy - Tools and Potential Applications

**10. Conclussions**

Ines Dufait is funded by an ERASMUS scholarship. Alessio Lanna is funded by an Universi‐ ty College London "bench-to-bedside" PhD scholarship. Christopher Bricogne is funded by an University College London MB-PhD scholarship. The Structural Genomics Consortium Oxfod is a registered UK charity (number 1097737) that receives funds from the Canadian Institutes of Health Research, The Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Insitute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundations, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Foundation for Strategic Research and the Wellcome Trust. Karine Breckpot is funded by the Fund for Scientific Research- Flandes. David Escors is funded by an Arthritis Research UK Career Development Fellowship (18433).

#### **Author details**

Ines Dufait1 , Therese Liechtenstein2 , Alessio Lanna2 , Roberta Laranga2 , Antonella Padella2 , Christopher Bricogne3 , Frederick Arce3 , Grazyna Kochan4 , Karine Breckpot1 and David Escors2\*

\*Address all correspondence to: rekades@ucl.ac.uk

1 Department of Physiology–Immunology, Medical School, Free University of Brussels, Bel‐ gium

2 Division of Infection and Immunity, Rayne Institute, University College London, United Kingdom

3 UCL Cancer Institute, University College London, United Kingdom

4 Structural Genomics Consortium Oxford, University of Oxford, Old Road Campus Re‐ search Building, United Kingdom

#### **References**

[1] Escors, D., & Breckpot, K. (2010). Lentiviral Vectors in Gene Therapy: Their Current Status and Future Potential. *Arch Immunol Ther Exp*, 58(2), 107-119.

[15] Curtsinger, J., Lins, D., & Mescher, M. (2003). Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of ef‐

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 333

[16] Nurieva, R., Thomas, S., Nguyen, T., Martin-Orozco, N., Wang, Y., Kaja, M. K., Yu, X. Z., & Dong, C. (2006). T-cell tolerance or function is determined by combinatorial

[17] Karwacz, K., Bricogne, C., Macdonald, D., Arce, F., Bennett, C. L., Collins, M., & Es‐ cors, D. (2011). PD-L1 co-stimulation contributes to ligand-induced T cell receptor

[18] Curtsinger, J. M., Schmidt, C. S., Mondino, A., Lins, D. C., Kedl, R. M., Jenkins, M. K., & Mescher, M. F. (1999). Inflammatory cytokines provide a third signal for activation

[19] Copreni, E., Castellani, S., Palmieri, L., Penzo, M., & Conese, M. (2008). Involvement of glycosaminoglycans in vesicular stomatitis virus G glycoprotein pseudotyped len‐ tiviral vector-mediated gene transfer into airway epithelial cells. *J Gene Med*, 10(12),

[20] Burns, J. C., Matsubara, T., Lozinski, G., Yee, J. K., Friedmann, T., Washabaugh, C. H., & Tsonis, P. A. (1994). Pantropic retroviral vector-mediated gene transfer, inte‐ gration, and expression in cultured newt limb cells. *Developmental biology*, 165(1),

[21] Bouard, D., Alazard-Dany, D., & Cosset, F. L. (2009). Viral vectors: from virology to

[22] Strang, B. L., Ikeda, Y., Cosset, F. L., Collins, M. K., & Takeuchi, Y. (2004). Characteri‐ zation of HIV-1 vectors with gammaretrovirus envelope glycoproteins produced

[23] Miller, A. D., Garcia, J. V., von, Suhr. N., Lynch, C. M., Wilson, C., & Eiden, M. V. (1991). Construction and properties of retrovirus packaging cells based on gibbon

[24] Vanden, Driessche. T., Thorrez, L., Naldini, L., Follenzi, A., Moons, L., Berneman, Z., Collen, D., & Chuah, M. K. (2002). Lentiviral vectors containing the human immuno‐ deficiency virus type-1 central polypurine tract can efficiently transduce nondividing

[25] Faix, P. H., Feldman, S. A., Overbaugh, J., & Eiden, M. V. (2002). Host range and re‐ ceptor binding properties of vectors bearing feline leukemia virus subgroup B enve‐ lopes can be modulated by envelope sequences outside of the receptor binding

[26] Esslinger, C., Chapatte, L., Finke, D., Miconnet, I., Guillaume, P., Levy, F., & Mac, Donald. H. R. (2003). In vivo administration of a lentiviral vaccine targets DCs and

induces efficient CD8(+) T cell responses. *J Clin Invest*, 111(11), 1673-1681.

hepatocytes and antigen-presenting cells in vivo. *Blood*, 100(3), 813-22.

transgene expression. *British journal of pharmacology*, 157(2), 153-165.

from stable packaging cells. *Gene Ther*, 11(7), 591-598.

ape leukemia virus. *J Virol*, 65(5), 2220-2224.

domain. *J Virol*, 76(23), 12369-75.

down-modulation on CD8(+) T cells. *EMBO molecular medicine*, 3(10), 581-92.

of naive CD4+ and CD8+ T cells. *Journal of immunology*, 162(6), 3256-62.

fector function. *Journal of experimental medicine*, 197, 1141-1151.

costimulatory signals. *Embo J.*, 25(11), 2623-2633.

1294-1302.

285-289.


[15] Curtsinger, J., Lins, D., & Mescher, M. (2003). Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and development of ef‐ fector function. *Journal of experimental medicine*, 197, 1141-1151.

**References**

*istry*, 63, 133-173.

332 Gene Therapy - Tools and Potential Applications

67(16), 2717-2747.

66(5), 2814-20.

449(7161), 419-26.

es. *Cell*, 76(2), 275-85.

[1] Escors, D., & Breckpot, K. (2010). Lentiviral Vectors in Gene Therapy: Their Current

[2] Vogt, V. M., & Simon, M. N. (1999). Mass determination of rous sarcoma virus viri‐ ons by scanning transmission electron microscopy. *J Virol*, 73(8), 7050-7055.

[3] Katz, R. A., & Skalka, A. M. (1994). The retroviral enzymes. *Annual review of biochem‐*

[4] Herschhorn, A., & Hizi, A. (2010). Retroviral reverse transcriptases. *Cell Mol Life Sci*,

[5] Charneau, P., Alizon, M., & Clavel, F. (1992). A second origin of DNA plus-strand synthesis is required for optimal human immunodeficiency virus replication. *J Virol*,

[6] Naldini, L., Blomer, U., Gage, F. H., Trono, D., & Verma, I. M. (1996). Efficient trans‐ fer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector. *Proc Natl Acad Sci U S A*, 93(21), 11382-11388.

[7] Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., & Trono, D. (1996). In vivo gene delivery and stable transduction of nondividing cells

[8] Yee, J. K., Friedmann, T., & Burns, J. C. (1994). Generation of high-titer pseudotyped retroviral vectors with very broad host range. *Methods in cell biology*, 43, Pt A, 99-112.

[9] Breckpot, K., Escors, D., Arce, F., Lopes, L., Karwacz, K., Van Lint, S., Keyaerts, M., & Collins, M. (2010). HIV-1 lentiviral vector immunogenicity is mediated by Toll-like

[10] Liechtenstein, T., Dufait, I., Lanna, A., Breckpot, K., & Escors, D. (2012). Modulating co-stimulation during antigen presentation to enhance cancer immunotherapy. *Im‐*

[11] Steinman, R. M., & Banchereau, J. (2007). Taking dendritic cells into medicine. *Nature*,

[12] Janeway, C. A., Jr., & Bottomly, K. (1994). Signals and signs for lymphocyte respons‐

[13] Curtsinger, J. M., Johnson, C. M., & Mescher, M. F. (2003). CD8 T cell clonal expan‐ sion and development of effector function require prolonged exposure to antigen,

[14] Curtsinger, J. M., Lins, D. C., & Mescher, M. F. (2003). Signal 3 determines tolerance versus full activation of naive CD8 T cells: dissociating proliferation and develop‐

costimulation, and signal 3 cytokine. *Journal of immunology*, 171(10), 5165-71.

ment of effector function. *The Journal of experimental medicine*, 197(9), 1141-51.

by a lentiviral vector. *Science*, 272(5259), 263-267.

receptor 3 (TLR3) and TLR7. *J. Virol.*, 84, 5627-5636.

*mun., Endoc. & Metab. Agents in Med. Chem.*, 12, 00.

Status and Future Potential. *Arch Immunol Ther Exp*, 58(2), 107-119.


[27] Collins, M. K., & Cerundolo, V. (2004). Gene therapy meets vaccine development. *Trends Biotechnol*, 22(12), 623-626.

[39] Goold, H. D., Escors, D., Conlan, T. J., Chakraverty, R., & Bennett, C. L. (2011). Con‐ ventional dendritic cells are required for the activation of helper-dependent CD8 T cell responses to a model antigen after cutaneous vaccination with lentiviral vectors.

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 335

[40] Escors, D., Lopes, L., Lin, R., Hiscott, J., Akira, S., Davis, R. J., & Collins, M. K. (2008). Targeting dendritic cell signalling to regulate the response to immunisation. *Blood*,

[41] Breckpot, K., & Escors, D. (2009). Dendritic Cells for Active Anti-cancer Immunother‐ apy: Targeting Activation Pathways Through Genetic Modification. *Endocrine, meta‐*

[42] Pasare, C., & Medzhitov, R. (2005). Toll-like receptors: linking innate and adaptive

[43] Takeda, K., & Akira, S. (2005). Toll-like receptors in innate immunity. *Int Immunol*,

[44] Brown, B. D., Sitia, G., Annoni, A., Hauben, E., Sergi, Sergi. L., Zingale, A., Roncaro‐ lo, M. G., Guidotti, L. G., & Naldini, L. (2006). In vivo administration of lentiviral vectors triggers a type I interferon response that restricts hepatocyte gene transfer

[45] Rossetti, M., Gregori, S., Hauben, E., Brown, B. D., Sergi, L. S., Naldini, L., & Roncar‐ olo, M. G. (2011). HIV-1-derived lentiviral vectors directly activate plasmacytoid den‐ dritic cells, which in turn induce the maturation of myeloid dendritic cells. *Hum Gene*

[46] Harman, A. N., Wilkinson, J., Bye, C. R., Bosnjak, L., Stern, J. L., Nicholle, M., Lai, J., & Cunningham, A. L. (2006). HIV induces maturation of monocyte-derived dendritic

[47] Pichlmair, A., Diebold, S. S., Gschmeissner, S., Takeuchi, Y., Ikeda, Y., Collins, M. K., & Reis e Sousa, C. (2007). Tubulovesicular structures within vesicular stomatitis virus G protein-pseudotyped lentiviral vector preparations carry DNA and stimulate anti‐

[48] Bao, L., Guo, H., Huang, X., Tammana, S., Wong, M., Mc Ivor, R. S., & Zhou, X. (2009). High-titer lentiviral vectors stimulate fetal calf serum-specific human CD4 T-

[49] Akazawa, T., Shingai, M., Sasai, M., Ebihara, T., Inoue, N., Matsumoto, M., & Seya, T. (2007). Tumor immunotherapy using bone marrow-derived dendritic cells overex‐

[50] Bagneris, C., Ageichik, A. V., Cronin, N., Wallace, B., Collins, M., Boshoff, C., Waks‐ man, G., & Barrett, T. (2008). Crystal structure of a vFlip-IKKgamma complex: in‐ sights into viral activation of the IKK signalosome. *Molecular cell*, 30(5), 620-31.

cell responses: implications in human gene therapy. *Gene Ther*, 16(6), 788-795.

pressing Toll-like receptor adaptors. *FEBS Lett*, 581(18), 3334-3340.

cells and Langerhans cells. *J Immunol*, 177(10), 7103-7113.

viral responses via Toll-like receptor 9. *J Virol*, 81(2), 539-47.

J Immunol, , 186(8), 4565-4572.

*bolic & immune disorders drug targets*, 9, 328-343.

immunity. *Adv Exp Med Biol*, 560, 11-18.

and promotes vector clearance. *Blood*.

111(6), 3050-3061.

17(1), 1-14.

*Ther*, 22(2), 177-188.


[39] Goold, H. D., Escors, D., Conlan, T. J., Chakraverty, R., & Bennett, C. L. (2011). Con‐ ventional dendritic cells are required for the activation of helper-dependent CD8 T cell responses to a model antigen after cutaneous vaccination with lentiviral vectors. J Immunol, , 186(8), 4565-4572.

[27] Collins, M. K., & Cerundolo, V. (2004). Gene therapy meets vaccine development.

[28] Paludan, C., Schmid, D., Landthaler, M., Vockerodt, M., Kube, D., Tuschl, T., & Munz, C. (2005). Endogenous MHC class II processing of a viral nuclear antigen after

[29] Gregers, T. F., Fleckenstein, B., Vartdal, F., Roepstorff, P., Bakke, O., & Sandlie, I. (2003). MHC class II loading of high or low affinity peptides directed by Ii/peide fu‐ sion constructs: implications for T cell activation. *International immunology*, 15(11),

[30] Rowe, H. M., Lopes, L., Ikeda, Y., Bailey, R., Barde, I., Zenke, M., Chain, B. M., & Col‐ lins, M. K. (2006). Immunization with a lentiviral vector stimulates both CD4 and

[31] Sanderson, S., Frauwirth, K., & Shastri, N. (1995). Expression of endogenous peptidemajor histocompatibility complex class II complexes derived from invariant chainantigen fusion proteins. *Proceedings of the National Academy of Sciences of the United*

[32] Wu, T. C., Guarnieri, F. G., Staveley-O'Carroll, K. F., Viscidi, R. P., Levitsky, H. I., Hedrick, L., Cho, K. R., August, J. T., & Pardoll, D. M. (1995). Engineering an intracel‐ lular pathway for major histocompatibility complex class II presentation of antigens. *Proceedings of the National Academy of Sciences of the United States of America*, 92(25),

[33] Miller, D. G., & Miller, A. D. (1994). A family of retroviruses that utilize related phos‐

[34] Lopes, L., Fletcher, K., Ikeda, Y., & Collins, M. (2006). Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy. *Cancer Immunol*

[35] Palmowski, M. J., Lopes, L., Ikeda, Y., Salio, M., Cerundolo, V., & Collins, M. K. (2004). Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an ef‐

[36] Christodoulopoulos, I., & Cannon, P. M. (2001). Sequences in the cytoplasmic tail of the gibbon ape leukemia virus envelope protein that prevent its incorporation into

[37] Rasko, J. E., Battini, J. L., Gottschalk, R. J., Mazo, I., & Miller, A. D. (1999). The RD114/ simian type D retrovirus receptor is a neutral amino acid transporter. *Proc Natl Acad*

[38] Karwacz, K., Mukherjee, S., Apolonia, L., Blundell, M. P., Bouma, G., Escors, D., Col‐ lins, M. K., & Thrasher, A. J. (2009). Nonintegrating lentivector vaccines stimulate prolonged T-cell and antibody responses and are effective in tumor therapy. *J Virol*,

phate transporters for cell entry. *J Virol*, 68(12), 8270-8276.

fective CTL response. *J Immunol*, 172(3), 1582-7.

lentivirus vectors. *J Virol*, 75(9), 4129-4138.

CD8 T cell responses to an ovalbumin transgene. *Mol Ther*, 13(2), 310-9.

*Trends Biotechnol*, 22(12), 623-626.

334 Gene Therapy - Tools and Potential Applications

1291-1299.

11671-11675.

autophagy. *Science*, 307(5709), 593-596.

*States of America*, 92(16), 7217-7221.

*Immunother*, 55(8), 1011-1016.

*Sci U S A*, 96(5), 2129-2134.

83(7), 3094-103.


[51] Efklidou, S., Bailey, R., Field, N., Noursadeghi, M., & Collins, M. K. (2008). vFLIP from KSHV inhibits anoikis of primary endothelial cells. *J Cell Sci*, 121, Pt 4, 450-7.

[63] Kochenderfer, J. N., Yu, Z., Frasheri, D., Restifo, N. P., & Rosenberg, S. A. (2010). Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. *Blood*,

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 337

[64] Parkhurst, M. R., Yang, J. C., Langan, R. C., Dudley, M. E., Nathan, D. A., Feldman, S. A., Davis, J. L., Morgan, R. A., Merino, M. J., Sherry, R. M., Hughes, M. S., Kammula, U. S., Phan, G. Q., Lim, R. M., Wank, S. A., Restifo, N. P., Robbins, P. F., Laurencot, C. M., & Rosenberg, S. A. (2011). T cells targeting carcinoembryonic antigen can medi‐ ate regression of metastatic colorectal cancer but induce severe transient colitis. *Mol*

[65] Pule, M. A., Savoldo, B., Myers, G. D., Rossig, C., Russell, H. V., Dotti, G., Huls, M. H., Liu, E., Gee, A. P., Mei, Z., Yvon, E., Weiss, H. L., Liu, H., Rooney, C. M., Heslop, H. E., & Brenner, M. K. (2008). Virus-specific T cells engineered to coexpress tumorspecific receptors: persistence and antitumor activity in individuals with neuroblas‐

[66] Till, B. G., Jensen, M. C., Wang, J., Chen, E. Y., Wood, B. L., Greisman, H. A., Qian, X., James, S. E., Raubitschek, A., Forman, S. J., Gopal, A. K., Pagel, J. M., Lindgren, C. G., Greenberg, P. D., Riddell, S. R., & Press, O. W. (2008). Adoptive immunotherapy for indolent non-Hodgkin lymphoma and mantle cell lymphoma using genetically

[67] Perro, M., Tsang, J., Xue, S. A., Escors, D., Cesco-Gaspere, M., Pospori, C., Gao, L., Hart, D., Collins, M., Stauss, H., & Morris, E. C. (2010). Generation of multi-function‐ al antigen-specific human T-cells by lentiviral TCR gene transfer. *Gene Ther*, 17,

[68] Cavalieri, S., Cazzaniga, S., Geuna, M., Magnani, Z., Bordignon, C., Naldini, L., & Bo‐ nini, C. (2003). Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence. *Blood*, 102(2), 497-505.

[69] Ducrey-Rundquist, O., Guyader, M., & Trono, D. (2002). Modalities of interleukin-7 induced human immunodeficiency virus permissiveness in quiescent T lymphocytes.

[70] Morgan, R. A., Dudley, M. E., Wunderlich, J. R., Hughes, M. S., Yang, J. C., Sherry, R. M., Royal, R. E., Topalian, S. L., Kammula, U. S., Restifo, N. P., Zheng, Z., Nahvi, A., de Vries, C. R., Rogers-Freezer, L. J., Mavroukakis, S. A., & Rosenberg, S. A. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes.

[71] Frecha, C., Costa, C., Negre, D., Gauthier, E., Russell, S. J., Cosset, F. L., & Verhoeyen, E. (2008). Stable transduction of quiescent T cells without induction of cycle progres‐ sion by a novel lentiviral vector pseudotyped with measles virus glycoproteins.

modified autologous CD 20-specific T cells. *Blood*, 112(6), 2261-2271.

116(19), 3875-3886.

*Ther*, 19(3), 620-626.

721-732.

*J Virol*, 76(18), 9103-9111.

Science , 314(5796), 126-129.

*Blood*, 112(13), 4843-4852.

toma. *Nat Med*, 14(11), 1264-1270.


[63] Kochenderfer, J. N., Yu, Z., Frasheri, D., Restifo, N. P., & Rosenberg, S. A. (2010). Adoptive transfer of syngeneic T cells transduced with a chimeric antigen receptor that recognizes murine CD19 can eradicate lymphoma and normal B cells. *Blood*, 116(19), 3875-3886.

[51] Efklidou, S., Bailey, R., Field, N., Noursadeghi, M., & Collins, M. K. (2008). vFLIP from KSHV inhibits anoikis of primary endothelial cells. *J Cell Sci*, 121, Pt 4, 450-7. [52] Field, N., Low, W., Daniels, M., Howell, S., Daviet, L., Boshoff, C., & Collins, M. (2003). KSHV vFLIP binds to IKK-gamma to activate IKK. *J Cell Sci*, 116, Pt 18,

[53] Shimizu, A., Baratchian, M., Takeuchi, Y., Escors, D., Macdonald, D., Barrett, T., Bag‐ neris, C., Collins, M., & Noursadeghi, M. (2011). Kaposi's sarcoma-associated herpes‐ virus vFLIP and human T cell lymphotropic virus type 1 Tax oncogenic proteins activate IkappaB kinase subunit gamma by different mechanisms independent of the

[54] Rowe, H. M., Lopes, L., Brown, N., Efklidou, S., Smallie, T., Karrar, S., Kaye, P. M., & Collins, M. K. (2009). Expression of vFLIP in a lentiviral vaccine vector activates NF- {kappa}B, matures dendritic cells, and increases CD8+ T-cell responses. *J Virol*, 83(4),

[55] Breckpot, K., Aerts-Toegaert, C., Heirman, C., Peeters, U., Beyaert, R., Aerts, J. L., & Thielemans, K. (2009). Attenuated expression of A20 markedly increases the efficacy of double-stranded RNA-activated dendritic cells as an anti-cancer vaccine. *J Immu‐*

[56] Song, X. T., Evel-Kabler, K., Shen, L., Rollins, L., Huang, X. F., & Chen, S. Y. (2008). A20 is an antigen presentation attenuator, and its inhibition overcomes regulatory T

[57] Koya, R. C., Kasahara, N., Favaro, P. M., Lau, R., Ta, H. Q., Weber, J. S., & Stripecke, R. (2003). Potent maturation of monocyte-derived dendritic cells after CD40L lentivi‐

[58] Koya, R. C., Kimura, T., Ribas, A., Rozengurt, N., Lawson, G. W., Faure-Kumar, E., Wang, H. J., Herschman, H., Kasahara, N., & Stripecke, R. (2007). Lentiviral vectormediated autonomous differentiation of mouse bone marrow cells into immunologi‐

[59] Karwacz, K., Arce, F., Bricogne, C., Kochan, G., & Escors, D. (2012). PD-L1 co-stimu‐ lation, ligand-induced TCR down-modulation and anti-tumor immunotherapy. *On‐*

[60] Escors, D., Bricogne, C., Arce, F., Kochan, G., & Karwacz, K. (2011). On the Mecha‐ nism of T cell receptor down-modulation and its physiological significance. *The jour‐*

[61] He, Y., Zhang, J., Mi, Z., Robbins, P., & Falo, L. D., Jr. (2005). Immunization with len‐ tiviral vector-transduced dendritic cells induces strong and long-lasting T cell re‐

[62] Park, T. S., Rosenberg, S. A., & Morgan, R. A. (2011). Treating cancer with genetically

physiological cytokine-induced pathways. *J Virol*, 85(14), 7444-8.

cell-mediated suppression. *Nat Med, 14* [3], 258-265.

cally potent dendritic cell vaccines. *Mol Ther*, 15(5), 971-80.

sponses and therapeutic immunity. *J Immunol*, 174(6), 3808-17.

ral gene delivery. *J Immunother*, 26(5), 451-60.

3721-3728.

336 Gene Therapy - Tools and Potential Applications

1555-62.

*nol, 182* [2], 860-870.

*coimmunology*, 1(1), 86-88.

*nal of bioscience and medicine*, 1(1).

engineered T cells. *Trends Biotechnol*.


[72] Frecha, C., Costa, C., Levy, C., Negre, D., Russell, S. J., Maisner, A., Salles, G., Peng, K. W., Cosset, F. L., & Verhoeyen, E. (2009). Efficient and stable transduction of rest‐ ing B lymphocytes and primary chronic lymphocyte leukemia cells using measles vi‐ rus gp displaying lentiviral vectors. *Blood*, 114(15), 3173-3180.

[85] Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M., Ravetch, J. V., Steinman, R. M., & Nussenzweig, M. C. (2001). Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. *J Exp Med*, 194(6),

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 339

[86] Kretschmer, K., Apostolou, I., Hawiger, D., Khazaie, K., Nussenzweig, M. C., & von Boehmer, H. (2005). Inducing and expanding regulatory T cell populations by for‐

[87] Rutella, S., Danese, S., & Leone, G. (2006). Tolerogenic dendritic cells: cytokine mod‐

[88] Manicassamy, S., Ravindran, R., Deng, J., Oluoch, H., Denning, T. L., Kasturi, S. P., Rosenthal, K. M., Evavold, B. D., & Pulendran, B. (2009). Toll-like receptor 2-depend‐ ent induction of vitamin A-metabolizing enzymes in dendritic cells promotes T regu‐

[89] Ilarregui, J. M., Croci, D. O., Bianco, G. A., Toscano, M. A., Salatino, M., Vermeulen, M. E., Geffner, J. R., & Rabinovich, G. A. (2009). Tolerogenic signals delivered by dendritic cells to T cells through a galectin-1-driven immunoregulatory circuit in‐

[90] Dillon, S., Agrawal, S., Banerjee, K., Letterio, J., Denning, T. L., Oswald-Richter, K., Kasprowicz, D. J., Kellar, K., Pare, J., van Dyke, T., Ziegler, S., Unutmaz, D., & Pulen‐ dran, B. (2006). Yeast zymosan, a stimulus for TLR2 and dectin-1, induces regulatory antigen-presenting cells and immunological tolerance. *J Clin Invest*, 116(4), 916-928.

[91] Corinti, S., Albanesi, C., la Sala, A., Pastore, S., & Girolomoni, G. (2001). Regulatory activity of autocrine IL-10 on dendritic cell functions. *J Immunol*, 166(7), 4312-8.

[92] Ghiringhelli, F., Puig, P. E., Roux, S., Parcellier, A., Schmitt, E., Solary, E., Kroemer, G., Martin, F., Chauffert, B., & Zitvogel, L. (2005). Tumor cells convert immature myeloid dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulato‐

[93] Saraiva, M., & O'Garra, A. (2010). The regulation of IL-10 production by immune

[94] Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., & Muller, W. (1993). Interleukin-10-

[95] Takayama, T., Nishioka, Y., Lu, L., Lotze, M. T., Tahara, H., & Thomson, A. W. (1998). Retroviral delivery of viral interleukin-10 into myeloid dendritic cells mark‐ edly inhibits their allostimulatory activity and promotes the induction of T-cell hypo‐

[96] Sakuishi, K., Apetoh, L., Sullivan, J. M., Blazar, B. R., Kuchroo, V. K., & Anderson, A. C. (2010). Targeting Tim-3 and PD-1 pathways to reverse T cell exhaustion and re‐

deficient mice develop chronic enterocolitis. *Cell*, 75(2), 263-274.

latory responses and inhibits autoimmunity. *Nat Med*, 15(4), 401-409.

volving interleukin 27 and interleukin 10. . Nat Immunol , 10(9), 981-991.

769-779.

eign antigen. *Nat Immunol*, 6(12), 1219-27.

ulation comes of age. *Blood*, 108(5), 1435-40.

ry T cell proliferation. *J Exp Med*, 202(7), 919-929.

responsiveness. *Transplantation*, 66(12), 1567-74.

store anti-tumor immunity. *J Exp Med*, 207(10), 2187-2194.

cells. *Nature reviews*, 10(3), 170-181.


[85] Hawiger, D., Inaba, K., Dorsett, Y., Guo, M., Mahnke, K., Rivera, M., Ravetch, J. V., Steinman, R. M., & Nussenzweig, M. C. (2001). Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. *J Exp Med*, 194(6), 769-779.

[72] Frecha, C., Costa, C., Levy, C., Negre, D., Russell, S. J., Maisner, A., Salles, G., Peng, K. W., Cosset, F. L., & Verhoeyen, E. (2009). Efficient and stable transduction of rest‐ ing B lymphocytes and primary chronic lymphocyte leukemia cells using measles vi‐

[73] Frecha, C., Levy, C., Cosset, F. L., & Verhoeyen, E. (2010). Advances in the field of lentivector-based transduction of T and B lymphocytes for gene therapy. *Mol Ther*,

[74] Griesemer, A. D., Sorenson, E. C., & Hardy, M. A. (2010). The role of the thymus in

[75] Hori, S., Nomura, T., & Sakaguchi, S. (2003). Control of regulatory T cell develop‐

[76] Sakaguchi, S. (2003). The origin of FOXP 3-expressing CD4+ regulatory T cells: thy‐

[77] Sakaguchi, S., Yamaguchi, T., Nomura, T., & Ono, M. (2008). Regulatory T cells and

[78] Arce, F., Breckpot, K., Stephenson, H., Karwacz, K., Ehrenstein, M. R., Collins, M., & Escors, D. (2011). Selective ERK activation differentiates mouse and human tolero‐ genic dendritic cells, expands antigen-specific regulatory T cells, and suppresses ex‐

[79] Mahnke, K., Qian, Y., Knop, J., & Enk, A. H. (2003). Induction of CD4+/CD25+ regula‐ tory T cells by targeting of antigens to immature dendritic cells. *Blood*, 101(12),

[80] O'Garra, A., & Vieira, P. (2004). Regulatory T cells and mechanisms of immune sys‐

[81] O'Garra, A., Vieira, P. L., Vieira, P., & Goldfeld, A. E. (2004). IL-10-producing and naturally occurring CD4+ Tregs: limiting collateral damage. *J Clin Invest*, 114(10),

[82] Peng, Y., Laouar, Y., Li, M. O., Green, E. A., & Flavell, R. A. (2004). TGF-beta regu‐ lates in vivo expansion of Foxp3-expressing CD4+CD25+ regulatory T cells responsi‐ ble for protection against diabetes. *Proc Natl Acad Sci U S A*, 101(13), 4572-4577. [83] Bonifaz, L., Bonnyay, D., Mahnke, K., Rivera, M., Nussenzweig, M. C., & Steinman, R. M. (2002). Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8+ T cell tolerance. *J Exp Med*, 196(12),

[84] Dhodapkar, M. V., & Steinman, R. M. (2002). Antigen-bearing immature dendritic cells induce peptide-specific CD8(+) regulatory T cells in vivo in humans. *Blood*,

ment by the transcription factor Foxp3. *Science*, 299(5609), 1057-1061.

perimental inflammatory arthritis. *Arthritis and rheumatism*, 63, 84-95.

rus gp displaying lentiviral vectors. *Blood*, 114(15), 3173-3180.

tolerance. . Transplantation , 90(5), 465-474.

mus or periphery. *J Clin Invest*, 112(9), 1310-1312.

immune tolerance. *Cell*, 133(5), 775-787.

tem control. *Nat Med*, 10(8), 801-5.

18(10), 1748-1757.

338 Gene Therapy - Tools and Potential Applications

4862-9.

1372-1378.

1627-38.

100(1), 174-177.


[97] Wang, L., Pino-Lagos, K., de Vries, V. C., Guleria, I., Sayegh, M. H., & Noelle, R. J. (2008). Programmed death 1 ligand signaling regulates the generation of adaptive Foxp3+CD4+ regulatory T cells. *Proc Natl Acad Sci U S A*, 105(27), 9331-9336.

testinal peptide complementary DNA as gene therapy for collagen-induced arthritis.

Lentiviral Vectors in Immunotherapy http://dx.doi.org/10.5772/50717 341

[109] Toscano, M. G., Delgado, M., Kong, W., Martin, F., Skarica, M., & Ganea, D. (2010). Dendritic cells transduced with lentiviral vectors expressing VIP differentiate into

[110] Agrawal, A., Dillon, S., Denning, T. L., & Pulendran, B. (2006). ERK1-/- mice exhibit Th1 cell polarization and increased susceptibility to experimental autoimmune ence‐

[111] Anastasaki, C., Estep, A. L., Marais, R., Rauen, K. A., & Patton, E. E. (2009). Kinaseactivating and kinase-impaired cardio-facio-cutaneous syndrome alleles have activi‐ ty during zebrafish development and are sensitive to small molecule inhibitors.

[112] Caparros, E., Munoz, P., Sierra-Filardi, E., Serrano-Gomez, D., Puig-Kroger, A., Ro‐ driguez-Fernandez, J. L., Mellado, M., Sancho, J., Zubiaur, M., & Corbi, A. L. (2006). DC-SIGN ligation on dendritic cells results in ERK and PI3K activation and modu‐

[113] Pages, G., Brunet, A., L'Allemain, G., & Pouyssegur, J. (1994). Constitutive mutant and putative regulatory serine phosphorylation site of mammalian MAP kinase kin‐

[114] Raingeaud, J., Whitmarsh, A. J., Barrett, T., Derijard, B., & Davis, R. J. (1996). MKK3 and MKK6-regulated gene expression is mediated by the 38 mitogen-activated pro‐

[115] Li, Y., Chu, N., Rostami, A., & Zhang, G. X. (2006). Dendritic cells transduced with SOCS-3 exhibit a tolerogenic/DC2 phenotype that directs type 2 Th cell differentia‐

[116] Zhang, Y., Yang, H., Xiao, B., Wu, M., Zhou, W., Li, J., Li, G., & Christadoss, P. (2009). Dendritic cells transduced with lentiviral-mediated RelB-specific ShRNAs inhibit the development of experimental autoimmune myasthenia gravis. *Molecular immunology*,

[117] Yang, M., Sun, L., Wang, S., Ko, K. H., Xu, H., Zheng, B. J., Cao, X., & Lu, L. (2010). Novel function of B cell-activating factor in the induction of IL-10-producing regula‐

[118] Batten, M., Groom, J., Cachero, T. G., Qian, F., Schneider, P., Tschopp, J., Browning, J. L., & Mackay, F. (2000). BAFF mediates survival of peripheral immature B lympho‐

[119] Lai, Kwan., Lam, Q., King, Hung., Ko, O., Zheng, B. J., & Lu, L. (2008). Local BAFF gene silencing suppresses Th17-cell generation and ameliorates autoimmune arthri‐

tein kinase signal transduction pathway. *Mol Cell Biol*, 16(3), 1247-1255.

VIP-secreting tolerogenic-like DCs. *Mol Ther,* , 18(5), 1035-1045.

*Arthritis and rheumatism*, 58(4), 1026-1037.

phalomyelitis. *J Immunol*, 176(10), 5788-5796.

*Human molecular genetics*, 18(14), 2543-2554.

ase (MEK1). *Embo J*, 13(13), 3003-3010.

46(4), 657-667.

lates cytokine production. *Blood*, 107(10), 3950-3958.

tion in vitro and in vivo. *J Immunol*, 177(3), 1679-88.

tory B cells. . J Immunol , 184(7), 3321-3325.

tis. *Proc Natl Acad Sci U S A*, 105(39), 14993-14998.

cytes. *J Exp Med*, 192(10), 1453-1466.


testinal peptide complementary DNA as gene therapy for collagen-induced arthritis. *Arthritis and rheumatism*, 58(4), 1026-1037.

[109] Toscano, M. G., Delgado, M., Kong, W., Martin, F., Skarica, M., & Ganea, D. (2010). Dendritic cells transduced with lentiviral vectors expressing VIP differentiate into VIP-secreting tolerogenic-like DCs. *Mol Ther,* , 18(5), 1035-1045.

[97] Wang, L., Pino-Lagos, K., de Vries, V. C., Guleria, I., Sayegh, M. H., & Noelle, R. J. (2008). Programmed death 1 ligand signaling regulates the generation of adaptive

[98] Sica, G. L., Choi, I. H., Zhu, G., Tamada, K., Wang, S. D., Tamura, H., Chapoval, A. I., Flies, D. B., Bajorath, J., & Chen, L. (2003). B7-H4, a molecule of the B7 family, nega‐

[99] Belladonna, M. L., Orabona, C., Grohmann, U., & Puccetti, P. (2009). TGF-beta and kynurenines as the key to infectious tolerance. *Trends in molecular medicine*, 15(2),

[100] Cobbold, S. P., Adams, E., Farquhar, C. A., Nolan, K. F., Howie, D., Lui, K. O., Fair‐ child, P. J., Mellor, A. L., Ron, D., & Waldmann, H. (2009). Infectious tolerance via the consumption of essential amino acids and mTOR signaling. *Proc Natl Acad Sci U S A*,

[101] Fallarino, F., Vacca, C., Orabona, C., Belladonna, M. L., Bianchi, R., Marshall, B., Ke‐ skin, D. B., Mellor, A. L., Fioretti, M. C., Grohmann, U., & Puccetti, P. (2002). Func‐ tional expression of indoleamine 2,3-dioxygenase by murine CD8 alpha(+) dendritic

[102] Mellor, A. L., & Munn, D. H. (2004). IDO expression by dendritic cells: tolerance and

[103] Munder, M. (2009). Arginase: an emerging key player in the mammalian immune

[104] Norian, L. A., Rodriguez, P. C., O'Mara, L. A., Zabaleta, J., Ochoa, A. C., Cella, M., & Allen, P. M. (2009). Tumor-infiltrating regulatory dendritic cells inhibit CD8+ T cell

[105] Lee, W. C., Zhong, C., Qian, S., Wan, Y., Gauldie, J., Mi, Z., Robbins, P. D., Thomson, A. W., & Lu, L. (1998). Phenotype, function, and in vivo migration and survival of allogeneic dendritic cell progenitors genetically engineered to express TGF-beta.

[106] Morita, Y., Yang, J., Gupta, R., Shimizu, K., Shelden, E. A., Endres, J., Mule, J. J., Mc Donagh, K. T., & Fox, D. A. (2001). Dendritic cells genetically engineered to express

IL-4 inhibit murine collagen-induced arthritis. *J Clin Invest*, 107(10), 1275-1284.

[107] Henry, E., Desmet, C. J., Garze, V., Fievez, L., Bedoret, D., Heirman, C., Faisca, P., Jas‐ par, F. J., Gosset, P., Jacquet, A. P., Desmecht, D., Thielemans, K., Lekeux, P., Moser, M., & Bureau, F. (2008). Dendritic cells genetically engineered to express IL-10 induce long-lasting antigen-specific tolerance in experimental asthma. *J Immunol*, 181(10),

[108] Delgado, M., Toscano, M. G., Benabdellah, K., Cobo, M., O'Valle, F., Gonzalez-Rey, E., & Martin, F. (2008). In vivo delivery of lentiviral vectors expressing vasoactive in‐

Foxp3+CD4+ regulatory T cells. *Proc Natl Acad Sci U S A*, 105(27), 9331-9336.

tively regulates T cell immunity. *Immunity*, 18(6), 849-861.

tryptophan catabolism. *Nature reviews*, 4(10), 762-74.

system. *British journal of pharmacology*, 158(3), 638-651.

function via L-arginine metabolism. *Cancer Res*, 69(7), 3086-3094.

41-9.

106(29), 12055-12060.

340 Gene Therapy - Tools and Potential Applications

cells. *Int Immunol*, 14(1), 65-68.

*Transplantation*, 66(12), 1810-1817.

7230-7242.


**Chapter 14**

**Targeted Lentiviral Vectors: Current Applications and**

About two decades ago recombinant human immunodeficiency virus type 1 (HIV-1) was proposed as a blueprint for the development of lentiviral vectors (LVs) (Naldini, Blomer et al. 1996). Lentiviral vectors exhibit several characteristics that make them favorable tools for gene therapy, including sustained gene delivery through vector integration, transduction of both dividing and non-dividing cells, applicability to different target cell types, absence of expression of viral proteins after transduction, delivery of complex genetic elements, low genotoxicity and the relative ease of vector manipulation and production (Cattoglio, Facchi‐ ni et al. 2007; Bauer, Dao et al. 2008). This is reflected in the numerous applications such as: transgene (tg) overexpression (Lopez-Ornelas, Mejia-Castillo et al. 2011), persistent gene si‐ lencing (Wang, Hu et al. 2012), immunization (Breckpot, Emeagi et al. 2008), generation of transgenic animals (Baup, Fraga et al. 2010), *in vivo* imaging (Roet, Eggers et al. 2012), induc‐ tion of pluripotent cells, stem cell modification (Sanchez-Danes, Consiglio et al. 2012), line‐ age tracking and site-directed gene editing (Lombardo, Genovese et al. 2007) as well as

Recombinant LVs can be derived from primate as well as non-primate lentiviruses such as HIV-1 and simian immunodeficiency virus (SIV) next to the equine infectious anemia virus, caprine arthritis-encephalitis virus, maedi-visna virus, feline immunodeficiency virus (FIV) and bovine immunodeficiency virus respectively (Escors and Breckpot 2010). They are all members of the *Retroviridae* family with 'retro' referring to their capacity to retro-transcribe their diploid single stranded (ss) RNA genome into a double stranded (ds) DNA copy that is

> © 2013 Goyvaerts et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

many applications targeting cancer cells (Petrigliano, Virk et al. 2009).

**Future Potential**

Karine Breckpot

**1. Introduction**

http://dx.doi.org/10.5772/52770

Cleo Goyvaerts, Therese Liechtenstein, Christopher Bricogne, David Escors and

Additional information is available at the end of the chapter

### **Targeted Lentiviral Vectors: Current Applications and Future Potential**

Cleo Goyvaerts, Therese Liechtenstein, Christopher Bricogne, David Escors and Karine Breckpot

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52770

#### **1. Introduction**

About two decades ago recombinant human immunodeficiency virus type 1 (HIV-1) was proposed as a blueprint for the development of lentiviral vectors (LVs) (Naldini, Blomer et al. 1996). Lentiviral vectors exhibit several characteristics that make them favorable tools for gene therapy, including sustained gene delivery through vector integration, transduction of both dividing and non-dividing cells, applicability to different target cell types, absence of expression of viral proteins after transduction, delivery of complex genetic elements, low genotoxicity and the relative ease of vector manipulation and production (Cattoglio, Facchi‐ ni et al. 2007; Bauer, Dao et al. 2008). This is reflected in the numerous applications such as: transgene (tg) overexpression (Lopez-Ornelas, Mejia-Castillo et al. 2011), persistent gene si‐ lencing (Wang, Hu et al. 2012), immunization (Breckpot, Emeagi et al. 2008), generation of transgenic animals (Baup, Fraga et al. 2010), *in vivo* imaging (Roet, Eggers et al. 2012), induc‐ tion of pluripotent cells, stem cell modification (Sanchez-Danes, Consiglio et al. 2012), line‐ age tracking and site-directed gene editing (Lombardo, Genovese et al. 2007) as well as many applications targeting cancer cells (Petrigliano, Virk et al. 2009).

Recombinant LVs can be derived from primate as well as non-primate lentiviruses such as HIV-1 and simian immunodeficiency virus (SIV) next to the equine infectious anemia virus, caprine arthritis-encephalitis virus, maedi-visna virus, feline immunodeficiency virus (FIV) and bovine immunodeficiency virus respectively (Escors and Breckpot 2010). They are all members of the *Retroviridae* family with 'retro' referring to their capacity to retro-transcribe their diploid single stranded (ss) RNA genome into a double stranded (ds) DNA copy that is

© 2013 Goyvaerts et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. integrated in the genome of the infected host cell (Figure 1A). Since LVs are most often de‐ rived from HIV-1, the generation of recombinant LVs has been accompanied by several safety concerns such as the generation of replication-competent lentiviruses (RCLs). Another poten‐ tial biosafety concern is the induction of insertional mutagenesis, a major genotoxic problem that emerged in gene therapy clinical trials using their γ-retroviral counterparts (Manilla, Re‐ bello et al. 2005). Generally, LVs are produced by transiently transfecting HEK 293 or 293T cells with plasmids encoding structural and functional sequences, imperative for proper LV particle generation. Over the last decades, vector development has largely focused on the de‐ sign of these plasmids. Firstly, only critical viral structural and functional sequences are pro‐ vided and secondly, these sequences are divided over a certain number of individual plasmids either in *cis* (encoded by the LV) or *trans* (packaged as a protein within the LV parti‐ cle), with a minimal overlap between viral sequences. This led to a LV production procedure where at least three different plasmids are used: (1) a packaging plasmid which provides all viral structural and enzymatic sequences (encoded by *gag* and *pol*) in *trans* to generate a func‐ tional particle, (2) a transfer plasmid providing the expression cassette in *cis,* cloned into the non-coding remains of the original lentiviral genome (Figure 1B, adapted from (Delenda 2004)) including a packaging signal and the two long terminal repeats (LTRs) of which the promoter activity has been deleted from the 3' LTR and (3) an envelope plasmid encoding an envelope glycoprotein (gp) consisting of a transmembranary domain (TM) and a receptorbinding domain (SU) that determines the LVs' tropism (Figure 1A).

Besides this division over different plasmids, other important construct optimization steps have been implemented. While in the first generation LV packaging plasmids the entire *gag* and pol genes were encoded together with all accessory regulatory and viru‐ lence genes, the second generation was multiply attenuated by removal of the four viru‐ lence genes*,* but not the regulatory genes *tat* and *rev (Zufferey, Nagy et al. 1997)*. In the third generation, the *rev* gene is expressed from a separate plasmid and the *tat* gene is removed by insertion of a strong constitutive promoter replacing the U3 region in the 5' LTR of the transfer plasmid (Dull, Zufferey et al. 1998). A major improvement was ach‐ ieved with the development of SIN or self-inactivating LVs where a deletion in the U3 region of the 3' LTR of the transfer plasmid abolished the production of full-length vec‐ tor RNA in transduced cells. This not only minimizes the risk for RCLs, but also re‐ duces the chance that the viral LTR enhancers interfere with the expression cassette, which minimizes aberrant expression of adjacent cellular coding regions. Subsequently these and many other optimization steps paved the way towards a more effective and

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safer version of the lentiviral gene delivery vehicle (Romano, Claudio et al. 2003).

or transductional targeting, and targeted integration of the proviral DNA.

In addition to packaging and transfer plasmid optimization, also the envelope plasmid was modified by replacing the natural HIV-1 envelope gp with an alternative gp gene, most often the gp of vesicular stomatitis virus (VSV.G). This concept is called pseudo‐ typing and VSV.G endowed the LV particle with an increased stability and broad cellu‐ lar tropism (to most if not all mammalian cells). However, it became clear that for numerous *in vivo* applications, a broad tropism may not be desirable. First, the tg that is encoded could be toxic to many cell types, *e.g.* pro-apoptotic or suicide genes, so a strin‐ gent control over the induction of tg expression in time and/or place is a necessity (Uch, Gerolami et al. 2003; Seo, Kim et al. 2009). A second point of concern is the risk for in‐ sertional mutagenesis; the more cells get infected, the higher this risk becomes. Although it has been demonstrated that LVs intrinsically exhibit low genotoxicity, clonal expansion and dominance of transduced hematopoietic progenitors have been reported in a clinical trial in which hematopoietic stem cells were genetically modified with a LV that ex‐ pressed the β-globin gene for treatment of β-thalassemia (Fehse and Roeder 2008; Cavaz‐ zana-Calvo, Payen et al. 2010). Thirdly, while a broad tropism LV is favorable in antitumor immunotherapy to efficiently transduce antigen-presenting cells (APCs) which can induce an antigen specific immune response (Palmowski, Lopes et al. 2004), this is not desirable when a genetic disorder has to be restored as in this case the tg may not be‐ come an immunological target (Annoni, Battaglia et al. 2007). Finally, during production of pantropic viruses encoding oncogenes, narrow tropism vectors would be more valua‐ ble due to biosafety level handling requirements and safety issues (Barrilleaux and Knoepfler 2011). Therefore, in view of safety as well as applicability aspects, four main targeting strategies can be brought forward: targeted gene expression or transcriptional targeting, targeted gene translation or microRNA based (de)targeting, targeted infection

**Figure 1. Schematic representation of an HIV-1 particle (A) and its genome (B).** The diploid ssRNA genome of HIV-1 is stabilized by structural nucleocapsid proteins and together with the enzymatic proteins reverse transcriptase, protease and integrase packaged in a nucleocapsid structure, which in turn is enclosed by capsid proteins. This nucleo‐ capsid is surrounded by a matrix protein layer and a producer cell derived phospholipid bilayer in which the envelope proteins consisting of an SU and TM part, are embedded (A). All proviral genes (*gag, pol, pro, vif, vpr, vpu, ref, tat, env* en *nef*) are flanked by two identical LTRs that consist of three regions: U3, R and U5. Within the U3 region, all proviral transcriptional control elements are situated such as the promoter and several enhancer sequences. Ψ represents the packaging signal. At the 3' end of the pol gene the central polypurine tract (red) and central termination sequence (green) are located. Both ensure the formation of a triple stranded DNA flap, crucial for nuclear entry of the pre-inte‐ gration complex in non-dividing cells (B).

Besides this division over different plasmids, other important construct optimization steps have been implemented. While in the first generation LV packaging plasmids the entire *gag* and pol genes were encoded together with all accessory regulatory and viru‐ lence genes, the second generation was multiply attenuated by removal of the four viru‐ lence genes*,* but not the regulatory genes *tat* and *rev (Zufferey, Nagy et al. 1997)*. In the third generation, the *rev* gene is expressed from a separate plasmid and the *tat* gene is removed by insertion of a strong constitutive promoter replacing the U3 region in the 5' LTR of the transfer plasmid (Dull, Zufferey et al. 1998). A major improvement was ach‐ ieved with the development of SIN or self-inactivating LVs where a deletion in the U3 region of the 3' LTR of the transfer plasmid abolished the production of full-length vec‐ tor RNA in transduced cells. This not only minimizes the risk for RCLs, but also re‐ duces the chance that the viral LTR enhancers interfere with the expression cassette, which minimizes aberrant expression of adjacent cellular coding regions. Subsequently these and many other optimization steps paved the way towards a more effective and safer version of the lentiviral gene delivery vehicle (Romano, Claudio et al. 2003).

integrated in the genome of the infected host cell (Figure 1A). Since LVs are most often de‐ rived from HIV-1, the generation of recombinant LVs has been accompanied by several safety concerns such as the generation of replication-competent lentiviruses (RCLs). Another poten‐ tial biosafety concern is the induction of insertional mutagenesis, a major genotoxic problem that emerged in gene therapy clinical trials using their γ-retroviral counterparts (Manilla, Re‐ bello et al. 2005). Generally, LVs are produced by transiently transfecting HEK 293 or 293T cells with plasmids encoding structural and functional sequences, imperative for proper LV particle generation. Over the last decades, vector development has largely focused on the de‐ sign of these plasmids. Firstly, only critical viral structural and functional sequences are pro‐ vided and secondly, these sequences are divided over a certain number of individual plasmids either in *cis* (encoded by the LV) or *trans* (packaged as a protein within the LV parti‐ cle), with a minimal overlap between viral sequences. This led to a LV production procedure where at least three different plasmids are used: (1) a packaging plasmid which provides all viral structural and enzymatic sequences (encoded by *gag* and *pol*) in *trans* to generate a func‐ tional particle, (2) a transfer plasmid providing the expression cassette in *cis,* cloned into the non-coding remains of the original lentiviral genome (Figure 1B, adapted from (Delenda 2004)) including a packaging signal and the two long terminal repeats (LTRs) of which the promoter activity has been deleted from the 3' LTR and (3) an envelope plasmid encoding an envelope glycoprotein (gp) consisting of a transmembranary domain (TM) and a receptor-

**Figure 1. Schematic representation of an HIV-1 particle (A) and its genome (B).** The diploid ssRNA genome of HIV-1 is stabilized by structural nucleocapsid proteins and together with the enzymatic proteins reverse transcriptase, protease and integrase packaged in a nucleocapsid structure, which in turn is enclosed by capsid proteins. This nucleo‐ capsid is surrounded by a matrix protein layer and a producer cell derived phospholipid bilayer in which the envelope proteins consisting of an SU and TM part, are embedded (A). All proviral genes (*gag, pol, pro, vif, vpr, vpu, ref, tat, env* en *nef*) are flanked by two identical LTRs that consist of three regions: U3, R and U5. Within the U3 region, all proviral transcriptional control elements are situated such as the promoter and several enhancer sequences. Ψ represents the packaging signal. At the 3' end of the pol gene the central polypurine tract (red) and central termination sequence (green) are located. Both ensure the formation of a triple stranded DNA flap, crucial for nuclear entry of the pre-inte‐

binding domain (SU) that determines the LVs' tropism (Figure 1A).

gration complex in non-dividing cells (B).

344 Gene Therapy - Tools and Potential Applications

In addition to packaging and transfer plasmid optimization, also the envelope plasmid was modified by replacing the natural HIV-1 envelope gp with an alternative gp gene, most often the gp of vesicular stomatitis virus (VSV.G). This concept is called pseudo‐ typing and VSV.G endowed the LV particle with an increased stability and broad cellu‐ lar tropism (to most if not all mammalian cells). However, it became clear that for numerous *in vivo* applications, a broad tropism may not be desirable. First, the tg that is encoded could be toxic to many cell types, *e.g.* pro-apoptotic or suicide genes, so a strin‐ gent control over the induction of tg expression in time and/or place is a necessity (Uch, Gerolami et al. 2003; Seo, Kim et al. 2009). A second point of concern is the risk for in‐ sertional mutagenesis; the more cells get infected, the higher this risk becomes. Although it has been demonstrated that LVs intrinsically exhibit low genotoxicity, clonal expansion and dominance of transduced hematopoietic progenitors have been reported in a clinical trial in which hematopoietic stem cells were genetically modified with a LV that ex‐ pressed the β-globin gene for treatment of β-thalassemia (Fehse and Roeder 2008; Cavaz‐ zana-Calvo, Payen et al. 2010). Thirdly, while a broad tropism LV is favorable in antitumor immunotherapy to efficiently transduce antigen-presenting cells (APCs) which can induce an antigen specific immune response (Palmowski, Lopes et al. 2004), this is not desirable when a genetic disorder has to be restored as in this case the tg may not be‐ come an immunological target (Annoni, Battaglia et al. 2007). Finally, during production of pantropic viruses encoding oncogenes, narrow tropism vectors would be more valua‐ ble due to biosafety level handling requirements and safety issues (Barrilleaux and Knoepfler 2011). Therefore, in view of safety as well as applicability aspects, four main targeting strategies can be brought forward: targeted gene expression or transcriptional targeting, targeted gene translation or microRNA based (de)targeting, targeted infection or transductional targeting, and targeted integration of the proviral DNA.

#### **2. Transcriptional targeting**

Most often a strong constitutive promoter with or without enhancer sequences is used to drive the LV encoded tg. These include the cytomegalovirus (CMV), spleen focus forming virus (SFFV), human polypeptide chain elongation factor-1alpha (EF-1alpha), phosphogly‐ cerate kinase (PGK) and ubiquitin C promoters (Kim, Kim et al. 2007; Gilham, Lie et al. 2010; Li, Husic et al. 2010). Although these promoters generally induce strong and ubiquitous ex‐ pression of the tg, they present some disadvantages. A first drawback is that they are more prone to promoter inactivation than cell-specific promoters. In addition, they are more po‐ tent in terms of activating the host-cell defense machinery and increasing the long-distance effects of insertional mutagenesis caused by their enhancer sequences (Liu, Wang et al. 2004; Stein, Ott et al. 2010; Singhal, Deng et al. 2011). These downsides resulted in the develop‐ ment of various strategies to allow cell-specific tg expression by incorporating cell type spe‐ cific regulatory elements and/or promoter(s) in the expression cassette of the LV. Because of the availability of a large number of endogenous cellular promoters, targeted expression can be achieved to potentially any cell type or tissue. In addition, its advantage over unselective expression has been demonstrated in numerous studies (Di Nunzio, Maruggi et al. 2008; Kerns, Ryu et al. 2010; Cao, Sodhi et al. 2011). This is exemplified by a study where LV en‐ coding iduronidase under the control of the hepatocyte specific albumin gene promoter was injected intravenously to treat mucopolysaccharidosis type I. While the same LV with a CMV promoter resulted in the induction of an immune response that diminished the tg ex‐ pression over time, the albumin gene promoter enabled stable and prolonged tg expression with a partial correction of the pathology (Di Domenico, Di Napoli et al. 2006). In addition to hepatocyte specific targeting, an ever-growing list of cell-type specific promoters has been used for the specific expression in tissues such as the erythroid lineage, endothelial cells, myocardial cells, retinal cells, B cells, epidermal, hematopoietic stem cells, *etcetera* (Hanawa, Persons et al. 2002; De Palma, Venneri et al. 2003; Semple-Rowland, Eccles et al. 2007; Di Nunzio, Maruggi et al. 2008; Leuci, Gammaitoni et al. 2009; Kerns, Ryu et al. 2010; Semple-Rowland, Coggin et al. 2010; Cao, Sodhi et al. 2011; Lee, Fan et al. 2011; Friedrich, Nopora et al. 2012).

targeted LVs and subsequent exclusive tg expression in cancer cells is the ultimate goal. Metastatic prostate cancer, for example, has been transcriptionally targeted in various ways (1) using a prostate-specific antigen (PSA) promoter to drive the expression of diphtheria toxin A, (2) using the prostate-stem cell antigen (PSCA) promoter to drive the expression of the Herpes Simplex Virus thymidine kinase (HSV-TK) suicide gene, or (3) combining the prostate-specific promoter ARR2PB and a short DNA sequence in the 5'-untranslated region that is recognized by the translation initiation factor eIF4E, often overexpressed in malignant cells, to drive the expression of the HSV-TK suicide gene (Yu, Chen et al. 2001; Zheng, Chen et al. 2003; Yu, Scott et al. 2006; Kimura, Koya et al. 2007; Petrigliano, Virk et al. 2009). Addi‐ tionally, the tumor vasculature has been transcriptionally targeted using the endothelial spe‐ cific Tie2 promoter to drive the conditionally toxic nitroreductase and subsequently diminish tumor growth (De Palma, Venneri et al. 2003). Another cancer cell type specific tar‐ geting strategy to limit tg expression to hepatocarcinoma was applied by Uch et al. They constructed a LV expressing HSV-TK under the control of the rat alpha-fetoprotein promot‐ er elements (Uch, Gerolami et al. 2003). Besides cancer cell type specific strategies, also more generalized cancer targeting strategies have been developed. For example, as the human te‐ lomerase reverse transcriptase (hTERT) is expressed in most malignant tumors, its promoter has been used to drive the expression of the cytosine deaminase gene together with a green fluorescent protein (GFP) reporter gene. It was demonstrated that hTERT-positive tumors could be visualized after intratumoral injection of the LV in tumor-bearing nude mice and, more importantly, that significant tumor growth suppression was observed after delivery of the pro-drug 5-fluorocytosine (Yu, Li et al. 2011). Besides avoidance of toxic tg expression in a non-tumor cell, tumor specific gene therapy is also interesting for targeted imaging. For example, the use of the chimeric promoter EIIAPA containing the alpha-fetoprotein promot‐ er and hepatitis B virus enhancer II was used to control the downstream expression of luci‐ ferase genes to subsequently assay the selective transcriptional activity by bioluminescence

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As LVs efficiently infect non-dividing cells, they provide suitable platforms for tg delivery to multiple mammalian neuronal cell types. It has been shown that stereotactic injection of LVs in the brain parenchyma leads to transduction of the striatum, hippocampus and thala‐ mus (Watson, Kobinger et al. 2002). Moreover, transcriptional targeting has proven to be a reliable technique to unravel the complexity of the nervous system by neuron and brain spe‐ cific assessment of the effects of therapeutic proteins and RNA interference, or to investigate neuronal gene expression (Hioki, Kameda et al. 2007; Gascon, Paez-Gomez et al. 2008; Kuro‐ da, Kutner et al. 2008; Peviani, Kurosaki et al. 2012). Regulatory sequences of rat neuron spe‐ cific enolase, human glial fibrillary acidic protein and myelin basic protein have already been exploited to obtain LV-mediated selective gene targeting of neurons, astrocytes and oli‐ godendrocytes, respectively (Jakobsson, Ericson et al. 2003; McIver, Lee et al. 2005). This has led to applications like subregional tg expression in the hippocampus using the hybrid hEF1alfa/HTLV promoter or neuron specific synapsin I promoter or targeting the central se‐ rotonergic neurons using a two-step transcriptional amplification strategy co-expressing the tryptophan hydroxylase-2 gene promoter with the chimeric enhancer GAL4/p65 (Kuroda, Kutner et al. 2008; Benzekhroufa, Liu et al. 2009). Next to the central nervous system, Bend‐

imaging (Hsieh, Chen et al. 2011).

Besides the advantage of increased and prolonged expression levels when expressed in the target cell of choice, targeted expression can also be a necessity when the tg causes undesira‐ ble damage in non-target cells. For the treatment of Mpl-deficient aplastic anemia, for exam‐ ple, targeted transfer to hematopoietic stem cells is inevitable since ectopic Mpl expression causes lethal adverse reactions (Heckl, Wicke et al. 2011). The same holds true for toxin, proapoptotic or suicide gene encoding LVs used in anti-tumor therapy (Zheng, Chen et al. 2003; Hsieh, Chen et al. 2011). LVs are excellent candidates to modulate the tumor and its environ‐ ment since they transduce both dividing cells such as most cancer cells but also non- or very slowly dividing cells such as cancer stem cells. Furthermore LVs are able to integrate in the genome of transduced cells, potentially generating clonal populations of genetically modi‐ fied cancer cells, which may then spread throughout the tumor mass (Steffens, Tebbets et al. 2004). Vector targeting can be attempted by local vector delivery, although this raises practi‐ cal concerns for non-solid and metastatic tumor cells. Consequently, systemic delivery of a targeted LVs and subsequent exclusive tg expression in cancer cells is the ultimate goal. Metastatic prostate cancer, for example, has been transcriptionally targeted in various ways (1) using a prostate-specific antigen (PSA) promoter to drive the expression of diphtheria toxin A, (2) using the prostate-stem cell antigen (PSCA) promoter to drive the expression of the Herpes Simplex Virus thymidine kinase (HSV-TK) suicide gene, or (3) combining the prostate-specific promoter ARR2PB and a short DNA sequence in the 5'-untranslated region that is recognized by the translation initiation factor eIF4E, often overexpressed in malignant cells, to drive the expression of the HSV-TK suicide gene (Yu, Chen et al. 2001; Zheng, Chen et al. 2003; Yu, Scott et al. 2006; Kimura, Koya et al. 2007; Petrigliano, Virk et al. 2009). Addi‐ tionally, the tumor vasculature has been transcriptionally targeted using the endothelial spe‐ cific Tie2 promoter to drive the conditionally toxic nitroreductase and subsequently diminish tumor growth (De Palma, Venneri et al. 2003). Another cancer cell type specific tar‐ geting strategy to limit tg expression to hepatocarcinoma was applied by Uch et al. They constructed a LV expressing HSV-TK under the control of the rat alpha-fetoprotein promot‐ er elements (Uch, Gerolami et al. 2003). Besides cancer cell type specific strategies, also more generalized cancer targeting strategies have been developed. For example, as the human te‐ lomerase reverse transcriptase (hTERT) is expressed in most malignant tumors, its promoter has been used to drive the expression of the cytosine deaminase gene together with a green fluorescent protein (GFP) reporter gene. It was demonstrated that hTERT-positive tumors could be visualized after intratumoral injection of the LV in tumor-bearing nude mice and, more importantly, that significant tumor growth suppression was observed after delivery of the pro-drug 5-fluorocytosine (Yu, Li et al. 2011). Besides avoidance of toxic tg expression in a non-tumor cell, tumor specific gene therapy is also interesting for targeted imaging. For example, the use of the chimeric promoter EIIAPA containing the alpha-fetoprotein promot‐ er and hepatitis B virus enhancer II was used to control the downstream expression of luci‐ ferase genes to subsequently assay the selective transcriptional activity by bioluminescence imaging (Hsieh, Chen et al. 2011).

**2. Transcriptional targeting**

346 Gene Therapy - Tools and Potential Applications

al. 2012).

Most often a strong constitutive promoter with or without enhancer sequences is used to drive the LV encoded tg. These include the cytomegalovirus (CMV), spleen focus forming virus (SFFV), human polypeptide chain elongation factor-1alpha (EF-1alpha), phosphogly‐ cerate kinase (PGK) and ubiquitin C promoters (Kim, Kim et al. 2007; Gilham, Lie et al. 2010; Li, Husic et al. 2010). Although these promoters generally induce strong and ubiquitous ex‐ pression of the tg, they present some disadvantages. A first drawback is that they are more prone to promoter inactivation than cell-specific promoters. In addition, they are more po‐ tent in terms of activating the host-cell defense machinery and increasing the long-distance effects of insertional mutagenesis caused by their enhancer sequences (Liu, Wang et al. 2004; Stein, Ott et al. 2010; Singhal, Deng et al. 2011). These downsides resulted in the develop‐ ment of various strategies to allow cell-specific tg expression by incorporating cell type spe‐ cific regulatory elements and/or promoter(s) in the expression cassette of the LV. Because of the availability of a large number of endogenous cellular promoters, targeted expression can be achieved to potentially any cell type or tissue. In addition, its advantage over unselective expression has been demonstrated in numerous studies (Di Nunzio, Maruggi et al. 2008; Kerns, Ryu et al. 2010; Cao, Sodhi et al. 2011). This is exemplified by a study where LV en‐ coding iduronidase under the control of the hepatocyte specific albumin gene promoter was injected intravenously to treat mucopolysaccharidosis type I. While the same LV with a CMV promoter resulted in the induction of an immune response that diminished the tg ex‐ pression over time, the albumin gene promoter enabled stable and prolonged tg expression with a partial correction of the pathology (Di Domenico, Di Napoli et al. 2006). In addition to hepatocyte specific targeting, an ever-growing list of cell-type specific promoters has been used for the specific expression in tissues such as the erythroid lineage, endothelial cells, myocardial cells, retinal cells, B cells, epidermal, hematopoietic stem cells, *etcetera* (Hanawa, Persons et al. 2002; De Palma, Venneri et al. 2003; Semple-Rowland, Eccles et al. 2007; Di Nunzio, Maruggi et al. 2008; Leuci, Gammaitoni et al. 2009; Kerns, Ryu et al. 2010; Semple-Rowland, Coggin et al. 2010; Cao, Sodhi et al. 2011; Lee, Fan et al. 2011; Friedrich, Nopora et

Besides the advantage of increased and prolonged expression levels when expressed in the target cell of choice, targeted expression can also be a necessity when the tg causes undesira‐ ble damage in non-target cells. For the treatment of Mpl-deficient aplastic anemia, for exam‐ ple, targeted transfer to hematopoietic stem cells is inevitable since ectopic Mpl expression causes lethal adverse reactions (Heckl, Wicke et al. 2011). The same holds true for toxin, proapoptotic or suicide gene encoding LVs used in anti-tumor therapy (Zheng, Chen et al. 2003; Hsieh, Chen et al. 2011). LVs are excellent candidates to modulate the tumor and its environ‐ ment since they transduce both dividing cells such as most cancer cells but also non- or very slowly dividing cells such as cancer stem cells. Furthermore LVs are able to integrate in the genome of transduced cells, potentially generating clonal populations of genetically modi‐ fied cancer cells, which may then spread throughout the tumor mass (Steffens, Tebbets et al. 2004). Vector targeting can be attempted by local vector delivery, although this raises practi‐ cal concerns for non-solid and metastatic tumor cells. Consequently, systemic delivery of a

As LVs efficiently infect non-dividing cells, they provide suitable platforms for tg delivery to multiple mammalian neuronal cell types. It has been shown that stereotactic injection of LVs in the brain parenchyma leads to transduction of the striatum, hippocampus and thala‐ mus (Watson, Kobinger et al. 2002). Moreover, transcriptional targeting has proven to be a reliable technique to unravel the complexity of the nervous system by neuron and brain spe‐ cific assessment of the effects of therapeutic proteins and RNA interference, or to investigate neuronal gene expression (Hioki, Kameda et al. 2007; Gascon, Paez-Gomez et al. 2008; Kuro‐ da, Kutner et al. 2008; Peviani, Kurosaki et al. 2012). Regulatory sequences of rat neuron spe‐ cific enolase, human glial fibrillary acidic protein and myelin basic protein have already been exploited to obtain LV-mediated selective gene targeting of neurons, astrocytes and oli‐ godendrocytes, respectively (Jakobsson, Ericson et al. 2003; McIver, Lee et al. 2005). This has led to applications like subregional tg expression in the hippocampus using the hybrid hEF1alfa/HTLV promoter or neuron specific synapsin I promoter or targeting the central se‐ rotonergic neurons using a two-step transcriptional amplification strategy co-expressing the tryptophan hydroxylase-2 gene promoter with the chimeric enhancer GAL4/p65 (Kuroda, Kutner et al. 2008; Benzekhroufa, Liu et al. 2009). Next to the central nervous system, Bend‐ otti et al. recently focused on selective tg expression in mouse spinal cord motor neurons us‐ ing motor neuron specific regulatory sequences derived from the promoter of the homeobox gene Hb9 (Benzekhroufa, Liu et al. 2009; Peviani, Kurosaki et al. 2012). However, neuron specific gene expression is not always very efficient and therefore several groups have at‐ tempted to improve the endogenous promoters using extra enhancers or artificial transcrip‐ tional activators such as the bidirectional promoter. For the latter, Liu et al. based their bidirectional promoter on the transcriptional activity of the human synapsin-1 promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter of 65 basepairs (bp) derived from the CMV promoter was joined upstream of both promoters, which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator while the downstream cassette drove the expression of the gene of interest (Liu, Paton et al. 2008).

which immunological tolerance was induced. Furthermore, this tg was only expressed in CD11c+ cells derived from the spleen, lymph nodes as well as the thymus (Zhang, Zou et al. 2009). Dresch et al. made use of the DC-STAMP promoter to engineer bone mar‐ row-targeted LVs. Therefore, e*x vivo* transduced hematopoietic stem cells (HSC) were in‐ jected in lethally irradiated mice to make HSC chimeric animals (Dresch, Edelmann et al. 2008). When GFP expression was analyzed in the leukocyte population isolated from the

cytes. Furthermore, tg expression could only be detected in CD11c+ cells in the thymus. While the previous two tolerance inducing studies could be explained by the fact that undifferentiated DC precursors were transduced, Kimura et al. intravenously injected LVs encoding Trp2 driven by the MHCII promoter and also observed persistent tg ex‐ pression selectively in the CD11c, CD11b and CD19+ MHCII+ cells of the spleen without CD8+ T cell responses against Trp2 in contrast to a CMV carrying construct (Kimura, Koya et al. 2007; Dresch, Edelmann et al. 2008). The induction of tolerance in this study might be explained by the activation status of the transduced APCs. Induction of tg spe‐ cific effector T cells requires fully activated APCs. Since, DC activation by LVs was shown to be dose-dependent, the LV titers used in these studies could explain the tol‐ erogenic instead of stimulatory outcome (Breckpot, Emeagi et al. 2007; Breckpot, Escors

Finally, also controllable or inducible tg expression can be a prerequisite. Reasons to use tg regulation are: to maintain appropriate levels of a gene product within the therapeu‐ tic range, to modulate, stop or resume tg expression in response to disease evolution, or in response to an endogenous molecule as *e.g.* the secretion of insulin induced by hyper‐ glycemia. For human gene therapy, several ligand dependent transcription regulatory systems have been developed. For clinical applications, such systems need to be safe, specific, highly inducible, reversible and only show dose dependent activation with low basal activity while their ligands need to be bioavailable and low in immunogenicity (Toniatti, Bujard et al. 2004). One of the first and most widely used ligands is Tetracylin (Tet) or its more potent analog Doxycycline (Dox) (Efrat, Fusco-DeMane et al. 1995; Reis‐ er, Lai et al. 2000). In contrast to the bacterial lac repressor/operator or the Cre-loxP re‐ combinase system, it is applicable *in vivo* and reversible (Deuschle, Hipskind et al. 1990; Lakso, Sauer et al. 1992). The original bacterial Tet system is based on a Tet repressor protein (TetR) that inhibits the expression of the bacterial Tet resistance genes by bind‐ ing to cognate operator sequences (TetO) in their regulatory regions. Upon the addition of Tet, the repressor is inactivated by allosteric change, allowing gene transcription (Gos‐ sen and Bujard 1992). The artificial Tet-off system is based on the generation of a hybrid transactivator (tTA) by fusion of the TetR to the transcription activation domain of the HSV VP16 protein. This fusion product will bind and activate transcription at promoters that include TetO while the presence of Dox impairs this binding, resulting in the shut off of gene expression (Furth, St Onge et al. 1994) (Figure 2A, adapted from (Ramezani and Hawley 2002). In contrast, the reverse Tet transactivator (rtTA), generated by ran‐

CD8+ DCs, CD11b+

Targeted Lentiviral Vectors: Current Applications and Future Potential

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CD11b+ mono‐

spleen, the main DC subpopulations such as CD11b<sup>−</sup>

et al. 2010).

plasmacytoid DCs were GFP positive next to a small percentage of CD11c<sup>−</sup>

To fulfill the high expectations of gene therapy, both efficient delivery and sustained ex‐ pression of the therapeutic gene are essential requirements. However, one of the major barriers to stable gene transfer by LVs is the development of innate and adaptive im‐ mune responses to the delivery vector and the transferred therapeutic tg. It became clear that *in vivo* administered broad tropism LVs efficiently transduce APCs and that these play a major role in the induction of tg specific immune responses (Annoni, Battaglia et al. 2007; Vandendriessche, Thorrez et al. 2007). Consequently transcriptional targeting can be applied to avoid tg expression in APCs. Brown et al. demonstrated stable GFP production by modified cells *in vivo* when tg expression was prevented in APCs (Brown, Venneri et al. 2006). Another study combined the hepatocyte specific enhanced transthyr‐ etin promoter with an APC-detargeting microRNA strategy, and showed the induction of GFP-specific regulatory T cells and the promotion of immunological tolerance (Anno‐ ni, Brown et al. 2009 ). Moreover, Matrai et al. demonstrated that hepatocyte-targeted ex‐ pression by an integrase-defective LV (IDLV) induced tolerance to coagulation factor IX with prevention of the induction of neutralizing antibodies in mice (Matrai, Cantore et al. 2011). In contrast to gene therapy, immunotherapy pursuits the induction of a tg-spe‐ cific immune response where APC-specific transduction is imperative. Therefore, LVs that drive tg expression *via* an APC-specific promoter have been developed. For instance Cui et al. used the HLA-DR promoter to target human MHC class II+ cells like dendritic cells (DCs, CD83+ ) and macrophages (CD14+ ). They demonstrated the induction of an al‐ logeneic T cell response *in vitro* (Cui, Golob et al. 2002). The dectin-2 promoter was used to target the expression of the human melanoma antigen NY-ESO-1 to murine APCs. Af‐ ter intravenous injection of the targeted LVs, selective tg expression in dectin-2+ splenic myeloid and plasmacytoid DCs as well as in F4/80+ macrophages was reported. Further‐ more CD11c+ draining lymph node residing DCs were targeted after subcutaneous injec‐ tion which resulted in strong NY-ESO-1 specific CD8+ and CD4+ T cell responses (Lopes, Dewannieux et al. 2008). On the other hand, DC-induced tg specific tolerance has also been achieved after the use of a DC-specific promoter. When LVs carrying a CD11c pro‐ moter were used to make DC-specific transgenic mice by injecting the purified virus into the perivitelline space of single-cell embryos, the tg became an autologous antigen to which immunological tolerance was induced. Furthermore, this tg was only expressed in CD11c+ cells derived from the spleen, lymph nodes as well as the thymus (Zhang, Zou et al. 2009). Dresch et al. made use of the DC-STAMP promoter to engineer bone mar‐ row-targeted LVs. Therefore, e*x vivo* transduced hematopoietic stem cells (HSC) were in‐ jected in lethally irradiated mice to make HSC chimeric animals (Dresch, Edelmann et al. 2008). When GFP expression was analyzed in the leukocyte population isolated from the spleen, the main DC subpopulations such as CD11b<sup>−</sup> CD8+ DCs, CD11b+ CD8<sup>−</sup> DCs, and plasmacytoid DCs were GFP positive next to a small percentage of CD11c<sup>−</sup> CD11b+ mono‐ cytes. Furthermore, tg expression could only be detected in CD11c+ cells in the thymus. While the previous two tolerance inducing studies could be explained by the fact that undifferentiated DC precursors were transduced, Kimura et al. intravenously injected LVs encoding Trp2 driven by the MHCII promoter and also observed persistent tg ex‐ pression selectively in the CD11c, CD11b and CD19+ MHCII+ cells of the spleen without CD8+ T cell responses against Trp2 in contrast to a CMV carrying construct (Kimura, Koya et al. 2007; Dresch, Edelmann et al. 2008). The induction of tolerance in this study might be explained by the activation status of the transduced APCs. Induction of tg spe‐ cific effector T cells requires fully activated APCs. Since, DC activation by LVs was shown to be dose-dependent, the LV titers used in these studies could explain the tol‐ erogenic instead of stimulatory outcome (Breckpot, Emeagi et al. 2007; Breckpot, Escors et al. 2010).

otti et al. recently focused on selective tg expression in mouse spinal cord motor neurons us‐ ing motor neuron specific regulatory sequences derived from the promoter of the homeobox gene Hb9 (Benzekhroufa, Liu et al. 2009; Peviani, Kurosaki et al. 2012). However, neuron specific gene expression is not always very efficient and therefore several groups have at‐ tempted to improve the endogenous promoters using extra enhancers or artificial transcrip‐ tional activators such as the bidirectional promoter. For the latter, Liu et al. based their bidirectional promoter on the transcriptional activity of the human synapsin-1 promoter and the compact glial fibrillary acidic protein (GfaABC1D) promoter. In the opposite orientation, a minimal core promoter of 65 basepairs (bp) derived from the CMV promoter was joined upstream of both promoters, which were flanked with two gene expression cassettes. The 5' cassette transcribed the artificial transcriptional activator while the downstream cassette

To fulfill the high expectations of gene therapy, both efficient delivery and sustained ex‐ pression of the therapeutic gene are essential requirements. However, one of the major barriers to stable gene transfer by LVs is the development of innate and adaptive im‐ mune responses to the delivery vector and the transferred therapeutic tg. It became clear that *in vivo* administered broad tropism LVs efficiently transduce APCs and that these play a major role in the induction of tg specific immune responses (Annoni, Battaglia et al. 2007; Vandendriessche, Thorrez et al. 2007). Consequently transcriptional targeting can be applied to avoid tg expression in APCs. Brown et al. demonstrated stable GFP production by modified cells *in vivo* when tg expression was prevented in APCs (Brown, Venneri et al. 2006). Another study combined the hepatocyte specific enhanced transthyr‐ etin promoter with an APC-detargeting microRNA strategy, and showed the induction of GFP-specific regulatory T cells and the promotion of immunological tolerance (Anno‐ ni, Brown et al. 2009 ). Moreover, Matrai et al. demonstrated that hepatocyte-targeted ex‐ pression by an integrase-defective LV (IDLV) induced tolerance to coagulation factor IX with prevention of the induction of neutralizing antibodies in mice (Matrai, Cantore et al. 2011). In contrast to gene therapy, immunotherapy pursuits the induction of a tg-spe‐ cific immune response where APC-specific transduction is imperative. Therefore, LVs that drive tg expression *via* an APC-specific promoter have been developed. For instance Cui et al. used the HLA-DR promoter to target human MHC class II+ cells like dendritic

logeneic T cell response *in vitro* (Cui, Golob et al. 2002). The dectin-2 promoter was used to target the expression of the human melanoma antigen NY-ESO-1 to murine APCs. Af‐ ter intravenous injection of the targeted LVs, selective tg expression in dectin-2+ splenic myeloid and plasmacytoid DCs as well as in F4/80+ macrophages was reported. Further‐ more CD11c+ draining lymph node residing DCs were targeted after subcutaneous injec‐ tion which resulted in strong NY-ESO-1 specific CD8+ and CD4+ T cell responses (Lopes, Dewannieux et al. 2008). On the other hand, DC-induced tg specific tolerance has also been achieved after the use of a DC-specific promoter. When LVs carrying a CD11c pro‐ moter were used to make DC-specific transgenic mice by injecting the purified virus into the perivitelline space of single-cell embryos, the tg became an autologous antigen to

). They demonstrated the induction of an al‐

drove the expression of the gene of interest (Liu, Paton et al. 2008).

348 Gene Therapy - Tools and Potential Applications

) and macrophages (CD14+

cells (DCs, CD83+

Finally, also controllable or inducible tg expression can be a prerequisite. Reasons to use tg regulation are: to maintain appropriate levels of a gene product within the therapeu‐ tic range, to modulate, stop or resume tg expression in response to disease evolution, or in response to an endogenous molecule as *e.g.* the secretion of insulin induced by hyper‐ glycemia. For human gene therapy, several ligand dependent transcription regulatory systems have been developed. For clinical applications, such systems need to be safe, specific, highly inducible, reversible and only show dose dependent activation with low basal activity while their ligands need to be bioavailable and low in immunogenicity (Toniatti, Bujard et al. 2004). One of the first and most widely used ligands is Tetracylin (Tet) or its more potent analog Doxycycline (Dox) (Efrat, Fusco-DeMane et al. 1995; Reis‐ er, Lai et al. 2000). In contrast to the bacterial lac repressor/operator or the Cre-loxP re‐ combinase system, it is applicable *in vivo* and reversible (Deuschle, Hipskind et al. 1990; Lakso, Sauer et al. 1992). The original bacterial Tet system is based on a Tet repressor protein (TetR) that inhibits the expression of the bacterial Tet resistance genes by bind‐ ing to cognate operator sequences (TetO) in their regulatory regions. Upon the addition of Tet, the repressor is inactivated by allosteric change, allowing gene transcription (Gos‐ sen and Bujard 1992). The artificial Tet-off system is based on the generation of a hybrid transactivator (tTA) by fusion of the TetR to the transcription activation domain of the HSV VP16 protein. This fusion product will bind and activate transcription at promoters that include TetO while the presence of Dox impairs this binding, resulting in the shut off of gene expression (Furth, St Onge et al. 1994) (Figure 2A, adapted from (Ramezani and Hawley 2002). In contrast, the reverse Tet transactivator (rtTA), generated by ran‐ dom mutagenesis of tTA, requires Dox to bind to cognate operator sequences and acti‐ vate transcription resulting in the inducible Tet-on system (Figure 2B).

mediated Tet-on inducible system based on co-transduction of two LVs to drive the expres‐ sion of a pro-apoptotic gene by the promoter of matrix-metalloproteinase-2 (MMP-2), which is highly expressed in several cancer cell lines. The first LV expressed a rtTA under the con‐ trol of the MMP-2 promoter, whereas the second LV expressed the pro-apoptotic gene Bax, under the control of the tetracycline-responsive element (Seo, Kim et al. 2009). While most Dox inducible systems are based on the co-transduction of two LVs, all-in-one vectors have also been described recently (Ogueta, Yao et al. 2001; Barde, Zanta-Boussif et al. 2006; Her‐ old, van den Brandt et al. 2008; Wiederschain, Wee et al. 2009; Benabdellah, Cobo et al. 2011). Furthermore, an extra Dox-regulated system based on the original TetR protein was developed in 1998. It serves as an alternative to the tTA- and rtTA-based systems because the latter were accompanied by secondary effects due to expression of the transactivator do‐ mains. Benabdellah et al. made use of the Dox-responsive cassette driving the expression of eGFP and the SFFV promoter expressing high amounts of the TetR protein in an all-in one vector system. This LV efficiently produced Dox-regulated cell lines, including primary hu‐ man fibroblasts and human mesenchymal stem cells. However, a major concern using Dox remains the possibility to develop resistance to the antibioticum Tet, and although it seems a non-immunogenic system in several mouse strains, studies with intramuscularly delivered Tet-on activators in non-human primates did elicit a cellular and humoral response (Latta-

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

351

Besides the Tet on/off systems, a plethora of inducible systems has been examined both *in vitro* and *in vivo.* An interesting strategy is based on the use of small molecules with distinct binding surfaces for two different polypeptides to modulate the activity of dimerizer-regu‐ lated systems. The prototype molecule is rapamycin, which mediates the heterodimer for‐ mation between two molecules (FK506-binding protein and FKBP rapamycin binding) that are coupled to a DNA binding domain (DBD) and transcription activation domain (AD) re‐ spectively (Pollock, Issner et al. 2000). The rapamycin inducible system has low basal activi‐ ty because of the physical separation of the DBD and AD molecules, the ligand has a short half-life of about 4.5 hrs although the induced gene expression lasts for days due to the strong stability of the DBD-AD assembled complex (Toniatti, Bujard et al. 2004). Tian et al. used a variant of this system to engineer LVs that produce a fusion protein between the fur‐ in-cleavable proinsulin and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes. Epidermal keratinocytes in culture, in stratified bioengi‐ neered epidermis as well as implanted in diabetic athymic mice released insulin within max‐ imally 1 hr after addition of rapamycin. Secretion slowed or stopped within 2-3 hrs after removal of the inducing agent. Even in diabetic animals with severe hyperglycemia, de‐ creased serum glucose levels to normal levels were reported (Tian, Lei et al. 2008). The ma‐ jor disadvantage of this technique is the immunosuppressing activity of rapamycin and the only partial oral availability, which renders this system impractical for clinical applications.

Another strategy is based on the fact that heterologous proteins can be made hormone re‐ sponsive by fusing them with the hormone-binding domain of steroid receptors. The bestcharacterized system is regulated by mifepristone or RU486, a synthetic progesterone antagonist. Prototypically the RU486-binding chimera known as GeneSwitch® consists of the

Mahieu, Rolland et al. 2002).

**Figure 2. Representation of the artificial Tet-off (A) and Tet-on system (B).** While the Dox binding transactivator (tTA) binds to the tetracycline-responsive promoter element (TRE) and stimulates tg transcription in the absence of Dox(A), the mutant reverse Tet-controlled transactivator (rtTA) binds to the TRE in the presence of Dox and stimulates transcription(B).

However, the *in vivo* applicability of the Tet system remained limited due to leakiness and insufficient induction levels. Therefore the Tet-on system has been optimized *e.g.* by isolat‐ ing novel rtTA variants and incorporating a Dox-dependent trans-silencer called tTS which consists of the KRAB (Kruppel-Associated Box) trans-repressing domain of the human Kid-1 protein fused to the wild type TetR. This tTS has been used by the group of Szulc et al. to develop a LV-based conditional gene expression system for drug-controllable expression of inhibitory short hairpin RNAs (shRNAs), and reported on a robust and versatile system that governed the tight control over the tg expression both *in vitro* as well as *in vivo* among oth‐ ers to generate transgenic mice (Szulc, Wiznerowicz et al. 2006). Moreover, Dox is orally bio‐ available, has a half-life of 14-22 hrs and has an excellent tissue penetration. Therefore numerous groups have used both the Tet-on and Tet-off system within LV-based gene re‐ porter and therapeutic applications (Blomer, Naldini et al. 1997; Bahi, Boyer et al. 2004; Blesch, Conner et al. 2005; Liu, Wang et al. 2008; Hioki, Kuramoto et al. 2009; Adriani, Boyer et al. 2010). This is exemplified by a study of Seo et al. who developed an oncolytic LV- mediated Tet-on inducible system based on co-transduction of two LVs to drive the expres‐ sion of a pro-apoptotic gene by the promoter of matrix-metalloproteinase-2 (MMP-2), which is highly expressed in several cancer cell lines. The first LV expressed a rtTA under the con‐ trol of the MMP-2 promoter, whereas the second LV expressed the pro-apoptotic gene Bax, under the control of the tetracycline-responsive element (Seo, Kim et al. 2009). While most Dox inducible systems are based on the co-transduction of two LVs, all-in-one vectors have also been described recently (Ogueta, Yao et al. 2001; Barde, Zanta-Boussif et al. 2006; Her‐ old, van den Brandt et al. 2008; Wiederschain, Wee et al. 2009; Benabdellah, Cobo et al. 2011). Furthermore, an extra Dox-regulated system based on the original TetR protein was developed in 1998. It serves as an alternative to the tTA- and rtTA-based systems because the latter were accompanied by secondary effects due to expression of the transactivator do‐ mains. Benabdellah et al. made use of the Dox-responsive cassette driving the expression of eGFP and the SFFV promoter expressing high amounts of the TetR protein in an all-in one vector system. This LV efficiently produced Dox-regulated cell lines, including primary hu‐ man fibroblasts and human mesenchymal stem cells. However, a major concern using Dox remains the possibility to develop resistance to the antibioticum Tet, and although it seems a non-immunogenic system in several mouse strains, studies with intramuscularly delivered Tet-on activators in non-human primates did elicit a cellular and humoral response (Latta-Mahieu, Rolland et al. 2002).

dom mutagenesis of tTA, requires Dox to bind to cognate operator sequences and acti‐

**Figure 2. Representation of the artificial Tet-off (A) and Tet-on system (B).** While the Dox binding transactivator (tTA) binds to the tetracycline-responsive promoter element (TRE) and stimulates tg transcription in the absence of Dox(A), the mutant reverse Tet-controlled transactivator (rtTA) binds to the TRE in the presence of Dox and stimulates

However, the *in vivo* applicability of the Tet system remained limited due to leakiness and insufficient induction levels. Therefore the Tet-on system has been optimized *e.g.* by isolat‐ ing novel rtTA variants and incorporating a Dox-dependent trans-silencer called tTS which consists of the KRAB (Kruppel-Associated Box) trans-repressing domain of the human Kid-1 protein fused to the wild type TetR. This tTS has been used by the group of Szulc et al. to develop a LV-based conditional gene expression system for drug-controllable expression of inhibitory short hairpin RNAs (shRNAs), and reported on a robust and versatile system that governed the tight control over the tg expression both *in vitro* as well as *in vivo* among oth‐ ers to generate transgenic mice (Szulc, Wiznerowicz et al. 2006). Moreover, Dox is orally bio‐ available, has a half-life of 14-22 hrs and has an excellent tissue penetration. Therefore numerous groups have used both the Tet-on and Tet-off system within LV-based gene re‐ porter and therapeutic applications (Blomer, Naldini et al. 1997; Bahi, Boyer et al. 2004; Blesch, Conner et al. 2005; Liu, Wang et al. 2008; Hioki, Kuramoto et al. 2009; Adriani, Boyer et al. 2010). This is exemplified by a study of Seo et al. who developed an oncolytic LV-

transcription(B).

vate transcription resulting in the inducible Tet-on system (Figure 2B).

350 Gene Therapy - Tools and Potential Applications

Besides the Tet on/off systems, a plethora of inducible systems has been examined both *in vitro* and *in vivo.* An interesting strategy is based on the use of small molecules with distinct binding surfaces for two different polypeptides to modulate the activity of dimerizer-regu‐ lated systems. The prototype molecule is rapamycin, which mediates the heterodimer for‐ mation between two molecules (FK506-binding protein and FKBP rapamycin binding) that are coupled to a DNA binding domain (DBD) and transcription activation domain (AD) re‐ spectively (Pollock, Issner et al. 2000). The rapamycin inducible system has low basal activi‐ ty because of the physical separation of the DBD and AD molecules, the ligand has a short half-life of about 4.5 hrs although the induced gene expression lasts for days due to the strong stability of the DBD-AD assembled complex (Toniatti, Bujard et al. 2004). Tian et al. used a variant of this system to engineer LVs that produce a fusion protein between the fur‐ in-cleavable proinsulin and the self-dimerization mutant of FK506-binding protein to yield bioactive insulin in keratinocytes. Epidermal keratinocytes in culture, in stratified bioengi‐ neered epidermis as well as implanted in diabetic athymic mice released insulin within max‐ imally 1 hr after addition of rapamycin. Secretion slowed or stopped within 2-3 hrs after removal of the inducing agent. Even in diabetic animals with severe hyperglycemia, de‐ creased serum glucose levels to normal levels were reported (Tian, Lei et al. 2008). The ma‐ jor disadvantage of this technique is the immunosuppressing activity of rapamycin and the only partial oral availability, which renders this system impractical for clinical applications.

Another strategy is based on the fact that heterologous proteins can be made hormone re‐ sponsive by fusing them with the hormone-binding domain of steroid receptors. The bestcharacterized system is regulated by mifepristone or RU486, a synthetic progesterone antagonist. Prototypically the RU486-binding chimera known as GeneSwitch® consists of the GAL4 DBD from *Saccharomyces cerevisiae* fused to the ligand-binding domain of a mutant progesterone receptor and the activation domain of the p65 subunit of human NF-κB (Abruzzese, Godin et al. 2000; Sirin and Park 2003). Upon ligand binding the GeneSwitch® protein binds to GAL4 upstream activating sequences in the promoter driving the expres‐ sion of the tg of interest. An advantage of the GeneSwitch® system is that the majority of its components are modified human proteins with no impact on cell viability. Furthermore, us‐ age of a mifepristone-inducible (auto-inducible) promoter to regulate expression of the chi‐ meric transactivator dramatically reduced basal expression of the tg in the absence of the inducer, thereby improving the dynamic range of *in vivo* tg regulation (Shinoda, Hieda et al. 2009). In addition, although mifepristone has anti-progesterone and -glucocorticoid activi‐ ties, the concentration needed for ligand-inducible transactivation of the target gene is much lower than the concentration producing an anti-progesterone effect in humans. However, it is thought that the lower dosage may still affect the ovarian cycle and exert a contraceptive activity. Therefore the search for other inducers that are unable to interact with endogenous progesterone would be more appropriate for clinical use (Sarkar 2002). As an alternative ste‐ roid-receptor based inducible system, the glucocorticosteroid responsive element (GRE5) was cloned into a LV (LV-GRE-IL10) encoding interleukin-10 (IL-10). Expression of IL-10 by LV-GRE-IL-10 appeared rapidly, was sustained and inducible in both ovine and human cor‐ neas in the presence of dexamethasone (Parker, Brereton et al. 2009). Another alternative can be the steroid hormone ecdysone, which plays a fundamental role during insect molting and metamorphosis. Ecdysteroids are considered safe because they are found in large amounts as phytoecdysteroids in vegetables, present in the human diet without detrimental effects. Mouse hematopoietic progenitors transduced with LVs containing an ecdysone-regulated GFP expression cassette efficiently turned GFP expression on and off in transplanted ani‐ mals with low basal activity (Xu, Mizuguchi et al. 2003; Galimi, Saez et al. 2005). Possibly, several other systems will be developed to control tg expression after LV transduction. Po‐ tential systems could be based on the cell-cell communication quorum sensing process (Neddermann, Gargioli et al. 2003) or the naturally evolved mechanisms of antibiotic resist‐ ance to pristinamycin, a composite streptogramin antibiotic or erythromicin, a member of the macrolide antibiotics (Fussenegger, Morris et al. 2000; Roberts 2002).

transcriptional regulation process, do not lie within the scope of this book chapter but are reviewed elsewhere (Nelson, Kiriakidou et al. 2003; Bartel 2004). A brief description together with a schematic representation is depicted in Figure 3 (adapted from http://www.micro‐

Targeted Lentiviral Vectors: Current Applications and Future Potential

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353

**Figure 3. miRNA-based post-transcriptional gene silencing.** Briefly, endogenous miRNA genes are transcribed by RNA polymerase II to pri-miRNA precursor molecules in the nucleus. These are processed to pre-miRNA by a special‐ ized enzymatic pathway called Pasha/Drosha and will release the pre-miRNA in short hairpin RNA (shRNA). Then, these pre-miRNAs are exported to the cytoplasm where Dicer degrades most of the shRNA, leaving a miRNA duplex which is loaded onto the AGO complex (Argonaut), forming the preRISC (RNA Interference Silencing Complex). Subse‐ quently the miRNA strand is degraded, leaving its complementary miRNA intact within the RISC complex. Then, this complex scans mRNAs and when complementation is found, the mRNA is degraded or the poly-A tail is removed,

In order to limit undesired vector tg expression, LVs encoding target sequences of endoge‐ nous miRNAs have been developed. By incorporating at the 3 'UTR region of the expression cassette one or more copies of a sequence that is perfectly complementary to a miRNA (miR‐ NA tagging), the transgenic mRNA will be degraded or repressed in cells where the comple‐ mentary miRNA is expressed. This new way of controlling tg expression at the level of the

leading to mRNA destabilization or stalled mRNA translation.

rna.ic.cz/mirna4.html).

#### **3. microRNA detargeting**

Recently, the concept of microRNA (miRNA) mediated post-transcriptional tg regulation was introduced in LV-based targeting. miRNAs are 21-22 nucleotide long non-coding frag‐ ments which are partially or extensively complementary to an endogenous mRNA molecule (Lai 2002). In mammals, over 400 different miRNAs have been identified so far, most of which are well conserved among species ranging from plants, worms, insects to humans (Brown and Naldini 2009). Some of these miRNAs are expressed ubiquitously whereas oth‐ ers are only expressed at certain developmental stages or in a certain cell type. Upon bind‐ ing of a miRNA molecule to its complementary target sequence, repression of translation or direct destruction of the mRNA is induced. The detailed mechanisms involved in this posttranscriptional regulation process, do not lie within the scope of this book chapter but are reviewed elsewhere (Nelson, Kiriakidou et al. 2003; Bartel 2004). A brief description together with a schematic representation is depicted in Figure 3 (adapted from http://www.micro‐ rna.ic.cz/mirna4.html).

GAL4 DBD from *Saccharomyces cerevisiae* fused to the ligand-binding domain of a mutant progesterone receptor and the activation domain of the p65 subunit of human NF-κB (Abruzzese, Godin et al. 2000; Sirin and Park 2003). Upon ligand binding the GeneSwitch® protein binds to GAL4 upstream activating sequences in the promoter driving the expres‐ sion of the tg of interest. An advantage of the GeneSwitch® system is that the majority of its components are modified human proteins with no impact on cell viability. Furthermore, us‐ age of a mifepristone-inducible (auto-inducible) promoter to regulate expression of the chi‐ meric transactivator dramatically reduced basal expression of the tg in the absence of the inducer, thereby improving the dynamic range of *in vivo* tg regulation (Shinoda, Hieda et al. 2009). In addition, although mifepristone has anti-progesterone and -glucocorticoid activi‐ ties, the concentration needed for ligand-inducible transactivation of the target gene is much lower than the concentration producing an anti-progesterone effect in humans. However, it is thought that the lower dosage may still affect the ovarian cycle and exert a contraceptive activity. Therefore the search for other inducers that are unable to interact with endogenous progesterone would be more appropriate for clinical use (Sarkar 2002). As an alternative ste‐ roid-receptor based inducible system, the glucocorticosteroid responsive element (GRE5) was cloned into a LV (LV-GRE-IL10) encoding interleukin-10 (IL-10). Expression of IL-10 by LV-GRE-IL-10 appeared rapidly, was sustained and inducible in both ovine and human cor‐ neas in the presence of dexamethasone (Parker, Brereton et al. 2009). Another alternative can be the steroid hormone ecdysone, which plays a fundamental role during insect molting and metamorphosis. Ecdysteroids are considered safe because they are found in large amounts as phytoecdysteroids in vegetables, present in the human diet without detrimental effects. Mouse hematopoietic progenitors transduced with LVs containing an ecdysone-regulated GFP expression cassette efficiently turned GFP expression on and off in transplanted ani‐ mals with low basal activity (Xu, Mizuguchi et al. 2003; Galimi, Saez et al. 2005). Possibly, several other systems will be developed to control tg expression after LV transduction. Po‐ tential systems could be based on the cell-cell communication quorum sensing process (Neddermann, Gargioli et al. 2003) or the naturally evolved mechanisms of antibiotic resist‐ ance to pristinamycin, a composite streptogramin antibiotic or erythromicin, a member of

the macrolide antibiotics (Fussenegger, Morris et al. 2000; Roberts 2002).

Recently, the concept of microRNA (miRNA) mediated post-transcriptional tg regulation was introduced in LV-based targeting. miRNAs are 21-22 nucleotide long non-coding frag‐ ments which are partially or extensively complementary to an endogenous mRNA molecule (Lai 2002). In mammals, over 400 different miRNAs have been identified so far, most of which are well conserved among species ranging from plants, worms, insects to humans (Brown and Naldini 2009). Some of these miRNAs are expressed ubiquitously whereas oth‐ ers are only expressed at certain developmental stages or in a certain cell type. Upon bind‐ ing of a miRNA molecule to its complementary target sequence, repression of translation or direct destruction of the mRNA is induced. The detailed mechanisms involved in this post-

**3. microRNA detargeting**

352 Gene Therapy - Tools and Potential Applications

**Figure 3. miRNA-based post-transcriptional gene silencing.** Briefly, endogenous miRNA genes are transcribed by RNA polymerase II to pri-miRNA precursor molecules in the nucleus. These are processed to pre-miRNA by a special‐ ized enzymatic pathway called Pasha/Drosha and will release the pre-miRNA in short hairpin RNA (shRNA). Then, these pre-miRNAs are exported to the cytoplasm where Dicer degrades most of the shRNA, leaving a miRNA duplex which is loaded onto the AGO complex (Argonaut), forming the preRISC (RNA Interference Silencing Complex). Subse‐ quently the miRNA strand is degraded, leaving its complementary miRNA intact within the RISC complex. Then, this complex scans mRNAs and when complementation is found, the mRNA is degraded or the poly-A tail is removed, leading to mRNA destabilization or stalled mRNA translation.

In order to limit undesired vector tg expression, LVs encoding target sequences of endoge‐ nous miRNAs have been developed. By incorporating at the 3 'UTR region of the expression cassette one or more copies of a sequence that is perfectly complementary to a miRNA (miR‐ NA tagging), the transgenic mRNA will be degraded or repressed in cells where the comple‐ mentary miRNA is expressed. This new way of controlling tg expression at the level of the mRNA product came as a complementary strategy to transcriptional targeting since the lat‐ ter is associated with some disadvantages such as: (1) difficulty to identify and faithfully re‐ constitute a gene's promoter; (2) for integrating LVs, promoters and enhancers can be trapped, leading to aberrant expression (De Palma, Montini et al. 2005), (3) transcription can be promiscuous and (4) only few genes have truly cell-specific transcriptional patterns while several promoters are active in many different cell types or states. Moreover, as miRNAs regulate expression at the post-transcriptional level, copy number and vector integration site have no appreciable effect on their regulation, which ensures consistent control throughout the transduced cell population.

the thymus and subsequent challenge with antigen expressing tumors did not result in tu‐

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355

Furthermore this technology is useful as a mechanism to increase vector safety and efficacy by limiting the expression of a toxic or pro-apoptotic tg to certain target cells. For example Lachmann et al. used the miRNA-150 target sequence to suppress GFP expression in lym‐ phocytes and thereby prevented tg-induced lymphotoxicity (Lachmann, Jagielska et al. 2011). On the other hand unrestrained growth of transduced cells could also be avoided us‐ ing miRNA-based detargeting when growth-promoting gens are replaced (Hawley, Fong et al. 1998). Moreover, miRNA-based regulation could be desirable when targeted gene expres‐ sion is needed to assess the contribution of a particular cell type to physiological processes or for the development of new therapeutic strategies. This is exemplified by the work of Col‐ in et al. who segregated tg expression between neurons and astrocytes following injection into the brain by exploiting the activity of miRNA-124 (Colin, Faideau et al. 2009). Another miRNA-based targeting strategy developed a few years ago was the concept of miRNA sponges, decoys, erasers, antagomirs or knockdowns (Ebert, Neilson et al. 2007; Scherr, Ven‐ turini et al. 2007; Gentner, Schira et al. 2009). Therefore vectors expressing miRNA target sites can effectively saturate an endogenous miRNA and prevent it from regulating its natu‐ ral targets. This technology enables a new way of investigating miRNA biology and has al‐ ready been used to study the role of miRNAs in cancer, cardiac function and hematopoiesis (Scherr, Venturini et al. 2007; Bonci, Coppola et al. 2008; Kumar, Erkeland et al. 2008; Sayed,

Rane et al. 2008; Gentner, Schira et al. 2009; Valastyan, Reinhardt et al. 2009).

fully move towards clinical translation (Brown and Naldini 2009)

**4. Transductional targeting**

A possible concern of miRNA-based detargeting is whether sufficient target knockdown can be achieved for specific applications without escape mutants arising (Kelly, Hadac et al. 2008). In addition, it is highly likely that overexpression of the synthetic target sites will sat‐ urate their corresponding endogenous miRNAs and deregulate expression of natural targets with deleterious consequences. However, the latter has not been reported so far (Brown, Gentner et al. 2007). Moreover, miRNA-based regulation is a very robust system since at low copy vector number miRNA regulation of tg expression remains effective. Apparently, when a threshold miRNA concentration is present, the tg will be suppressed. This robust‐ ness can probably be explained by the perfect complementarity of the target sequence and the endogenous miRNA sequence. Indeed, when imperfectly complementary sites were used, this did result in a detectable decrease in target suppression, although only at very high vector copy numbers. So, although it should be recognized that the knowledge regard‐ ing miRNA biology and function is still limited, this strategy holds great potential to care‐

Although the strategies described above demonstrate cell-specific gene expression, they of‐ ten require broad tropism LVs which does not reduce the risk for RCL formation or inser‐ tional mutagenesis. Therefore transductional targeting of LVs seems a more interesting

mor growth (Papapetrou, Kovalovsky et al. 2009).

Successful outcomes of LV-based gene therapy have long been precluded by the develop‐ ment of tg-specific immunity as a consequence of the direct expression of the tg product by professional APCs. Therefore Brown et al. challenged mice with LVs encoding a target se‐ quence for miRNA-142-3p, a microRNA specifically expressed in the hematopoietic lineag‐ es. Upon injection, they demonstrated a 100-fold suppression of reporter gene expression in intravascular and extravascular hematopoietic lineages, including APCs (Brown, Venneri et al. 2006). One year later, its usefulness was evidenced by the miRNA-142-3p regulated LV mediated stable correction of hemophilia B in mice (Brown, Cantore et al. 2007). Its expres‐ sion leads to reduced tg expression in APCs and subsequently lower anti-tg immune re‐ sponses. Moreover it was demonstrated that *in vivo* delivery of this post-transcriptionally regulated LV induced tg-specific Foxp3+ regulatory CD4+ T cells, which promoted immuno‐ logic tolerance (Annoni, Brown et al. 2009 ). Curiously, they also reported the necessity of a hepatocyte specific promoter for this immunological tolerance. So, these studies showed the impressive potential of miRNA-based detargeting to overcome a major hurdle for clinical gene therapy, however also other factors than tg expression in APCs seem to influence the immunological outcome of a gene transfer procedure. Examples are the type of vector used, the tissue targeted and the presence of inflammation (Brown and Lillicrap 2002; Cao, Furlan-Freguia et al. 2007).

Another reason to pursue stringent tg regulation, is to express the tg in a specific develop‐ mental state. Brown et al. showed that multiple endogenous miRNAs can be used to achieve tg expression patterns that rapidly adjust and sharply discriminate among the myeloid and lymphoid lineage in therapeutically relevant HSCs and their progeny with miRNA-223, or among immature and mature APCs using miRNA-155 (Brown, Gentner et al. 2007). Another example is provided by Gentner et al. who used the miRNA-126 target sequence to detarget tg expression from stem cells and progenitors from the hematopoietic cell lineage in order to avoid expression of the highly toxic GALC in these stages, while inducing GALC expression in mature cells from the hematopoietic lineage to correct globoid cell leukodystrophy (Gent‐ ner, Visigalli et al. 2010). Furthermore the group of Sachdeva et al. used miRNA-292 regulat‐ ed LVs to visualize and segregate differentiating neural progenitors in pluripotent cultures and demonstrated that miRNA-regulated vectors allow a potentially broad use on stem cell applications (Sachdeva, Jonsson et al. 2010). Finally, Sadelain et al. used LVs that encode an‐ tigen specific receptors together with target sites for miRNA-181a to suppress the expression of the receptor in late thymocytes. This avoided clonal deletion of antigen specific T cells in the thymus and subsequent challenge with antigen expressing tumors did not result in tu‐ mor growth (Papapetrou, Kovalovsky et al. 2009).

Furthermore this technology is useful as a mechanism to increase vector safety and efficacy by limiting the expression of a toxic or pro-apoptotic tg to certain target cells. For example Lachmann et al. used the miRNA-150 target sequence to suppress GFP expression in lym‐ phocytes and thereby prevented tg-induced lymphotoxicity (Lachmann, Jagielska et al. 2011). On the other hand unrestrained growth of transduced cells could also be avoided us‐ ing miRNA-based detargeting when growth-promoting gens are replaced (Hawley, Fong et al. 1998). Moreover, miRNA-based regulation could be desirable when targeted gene expres‐ sion is needed to assess the contribution of a particular cell type to physiological processes or for the development of new therapeutic strategies. This is exemplified by the work of Col‐ in et al. who segregated tg expression between neurons and astrocytes following injection into the brain by exploiting the activity of miRNA-124 (Colin, Faideau et al. 2009). Another miRNA-based targeting strategy developed a few years ago was the concept of miRNA sponges, decoys, erasers, antagomirs or knockdowns (Ebert, Neilson et al. 2007; Scherr, Ven‐ turini et al. 2007; Gentner, Schira et al. 2009). Therefore vectors expressing miRNA target sites can effectively saturate an endogenous miRNA and prevent it from regulating its natu‐ ral targets. This technology enables a new way of investigating miRNA biology and has al‐ ready been used to study the role of miRNAs in cancer, cardiac function and hematopoiesis (Scherr, Venturini et al. 2007; Bonci, Coppola et al. 2008; Kumar, Erkeland et al. 2008; Sayed, Rane et al. 2008; Gentner, Schira et al. 2009; Valastyan, Reinhardt et al. 2009).

A possible concern of miRNA-based detargeting is whether sufficient target knockdown can be achieved for specific applications without escape mutants arising (Kelly, Hadac et al. 2008). In addition, it is highly likely that overexpression of the synthetic target sites will sat‐ urate their corresponding endogenous miRNAs and deregulate expression of natural targets with deleterious consequences. However, the latter has not been reported so far (Brown, Gentner et al. 2007). Moreover, miRNA-based regulation is a very robust system since at low copy vector number miRNA regulation of tg expression remains effective. Apparently, when a threshold miRNA concentration is present, the tg will be suppressed. This robust‐ ness can probably be explained by the perfect complementarity of the target sequence and the endogenous miRNA sequence. Indeed, when imperfectly complementary sites were used, this did result in a detectable decrease in target suppression, although only at very high vector copy numbers. So, although it should be recognized that the knowledge regard‐ ing miRNA biology and function is still limited, this strategy holds great potential to care‐ fully move towards clinical translation (Brown and Naldini 2009)

#### **4. Transductional targeting**

mRNA product came as a complementary strategy to transcriptional targeting since the lat‐ ter is associated with some disadvantages such as: (1) difficulty to identify and faithfully re‐ constitute a gene's promoter; (2) for integrating LVs, promoters and enhancers can be trapped, leading to aberrant expression (De Palma, Montini et al. 2005), (3) transcription can be promiscuous and (4) only few genes have truly cell-specific transcriptional patterns while several promoters are active in many different cell types or states. Moreover, as miRNAs regulate expression at the post-transcriptional level, copy number and vector integration site have no appreciable effect on their regulation, which ensures consistent control throughout

Successful outcomes of LV-based gene therapy have long been precluded by the develop‐ ment of tg-specific immunity as a consequence of the direct expression of the tg product by professional APCs. Therefore Brown et al. challenged mice with LVs encoding a target se‐ quence for miRNA-142-3p, a microRNA specifically expressed in the hematopoietic lineag‐ es. Upon injection, they demonstrated a 100-fold suppression of reporter gene expression in intravascular and extravascular hematopoietic lineages, including APCs (Brown, Venneri et al. 2006). One year later, its usefulness was evidenced by the miRNA-142-3p regulated LV mediated stable correction of hemophilia B in mice (Brown, Cantore et al. 2007). Its expres‐ sion leads to reduced tg expression in APCs and subsequently lower anti-tg immune re‐ sponses. Moreover it was demonstrated that *in vivo* delivery of this post-transcriptionally

regulatory CD4+

logic tolerance (Annoni, Brown et al. 2009 ). Curiously, they also reported the necessity of a hepatocyte specific promoter for this immunological tolerance. So, these studies showed the impressive potential of miRNA-based detargeting to overcome a major hurdle for clinical gene therapy, however also other factors than tg expression in APCs seem to influence the immunological outcome of a gene transfer procedure. Examples are the type of vector used, the tissue targeted and the presence of inflammation (Brown and Lillicrap 2002; Cao, Furlan-

Another reason to pursue stringent tg regulation, is to express the tg in a specific develop‐ mental state. Brown et al. showed that multiple endogenous miRNAs can be used to achieve tg expression patterns that rapidly adjust and sharply discriminate among the myeloid and lymphoid lineage in therapeutically relevant HSCs and their progeny with miRNA-223, or among immature and mature APCs using miRNA-155 (Brown, Gentner et al. 2007). Another example is provided by Gentner et al. who used the miRNA-126 target sequence to detarget tg expression from stem cells and progenitors from the hematopoietic cell lineage in order to avoid expression of the highly toxic GALC in these stages, while inducing GALC expression in mature cells from the hematopoietic lineage to correct globoid cell leukodystrophy (Gent‐ ner, Visigalli et al. 2010). Furthermore the group of Sachdeva et al. used miRNA-292 regulat‐ ed LVs to visualize and segregate differentiating neural progenitors in pluripotent cultures and demonstrated that miRNA-regulated vectors allow a potentially broad use on stem cell applications (Sachdeva, Jonsson et al. 2010). Finally, Sadelain et al. used LVs that encode an‐ tigen specific receptors together with target sites for miRNA-181a to suppress the expression of the receptor in late thymocytes. This avoided clonal deletion of antigen specific T cells in

T cells, which promoted immuno‐

the transduced cell population.

354 Gene Therapy - Tools and Potential Applications

regulated LV induced tg-specific Foxp3+

Freguia et al. 2007).

Although the strategies described above demonstrate cell-specific gene expression, they of‐ ten require broad tropism LVs which does not reduce the risk for RCL formation or inser‐ tional mutagenesis. Therefore transductional targeting of LVs seems a more interesting strategy to tackle both safety and efficacy concerns. The concept of swapping the viral enve‐ lope proteins of different viral species is called pseudotyping. Already in 1979, the envelope glycoprotein of the avian retrovirus was used to pseudotype VSV virions in order to selec‐ tively enrich for VSV temperature-sensitive mutants of VSV.G biosynthesis (Lodish and Weiss 1979). Later it was shown that wild type HIV-1 particles which were produced in cells that were infected with another virus, *e.g.* murine leukemia virus (MLV) or VSV, led to the generation of phenotypically mixed virions with an expanded host range (Canivet, Hoffman et al. 1990; Zhu, Chen et al. 1990). These observations introduced the concept of pseudotyp‐ ing and in the early 90's the gp160 sequence of a replication defective HIV-1 derived LV was replaced by a MLV gp (Page, Landau et al. 1990). Later on the natural envelope gp from an MLV-based vector was replaced with the viral attachment protein of VSV (Emi, Friedmann et al. 1991; Burns, Friedmann et al. 1993). Today, most synthetic LVs are pseudotyped with a heterologous envelope protein to increase their stability, infectivity and safety. Notably, the first LVs were not pseudotyped but displayed the native HIV-1 envelope protein at their surface. This limited their tropism to CD4-expressing cells (Dropulic 2011). Interestingly, VSV.G pseudotyped vectors are more stable than their natural counterparts. This allows concentration to higher titers by ultracentrifugation and confers broad tropism, as VSV.G binds to a still unknown ubiquitous membrane component (Cronin, Zhang et al. 2005). This superior transduction efficiency comes in handy for the treatment of genetic disorders such as β-thalassemia and X-linked adenoleukodystrophy (Cartier, Hacein-Bey-Abina et al. 2009; Cavazzana-Calvo, Payen et al. 2010). Nonetheless, VSV.G pseudotyped LVs also present several downsides. Firstly, the VSV gp is cytotoxic when expressed constitutively at high concentrations, which impedes the production of stable packaging cell lines (Lopes, Dewan‐ nieux et al. 2011). In addition, cytotoxicity associated with VSV.G pseudotyped LVs has been observed when *in vivo* administered at high concentration, in comparison with other pseudotypes (Watson, Kobinger et al. 2002). Another critical hurdle for systemic delivery us‐ ing VSV.G pseudotyped LVs is their susceptibility to neutralization by human serum com‐ plement, although this can be bypassed by polyethylene glycol-modification (PEGylation) of the virions (DePolo, Reed et al. 2000; Croyle, Callahan et al. 2004).

(Strang, Takeuchi et al. 2005), and with an astrocyte and oligodendrocyte specific tropism when injected into the mouse brain (Kang, Stein et al. 2002). Another example is provided by the family of the *Baculoviridae* where the gp64 gp ensures high particle stability in addi‐ tion to a hepatocyte specific tropism (Matsui, Hegadorn et al. 2011). LVs pseudotyped with the lymphocytic choriomeningitis virus (LCMV) envelope from the *Arenaviridae* preferential‐ ly transduce cells from the central nervous system such as neural stem cells and progenitor cells, and also to glioma cells and insulin secreting β-cells (Kobinger, Deng et al. 2004; Milet‐ ic, Fischer et al. 2004; Stein, Martins et al. 2005). As there is an increasing interest in the de‐ velopment of gene therapeutic strategies for malignant gliomas, the most frequent primary brain tumors with very poor prognosis, several groups report on the use of LCMV gp pseu‐ dotyped LVs to target almost exclusively astrocytes, the main source of malignant glioma cells (Beyer, Westphal et al. 2002; Miletic, Fischer et al. 2004; Steffens, Tebbets et al. 2004). The H and F envelope proteins from the *Paramyxoviridae* family, such as those derived from measles viruses, provide LVs with the capacity to bind to SLAM and CD46, which confers efficient virus entry, nuclear transport and integration in non-activated B and T lympho‐ cytes. This property is particularly important, since primary unstimulated B and T cells are generally difficult to transduce if not pre-treated to induce progression through the cell cy‐ cle (*e.g.* through stimulation with anti-CD3/anti-CD28 antibodies or cytokines) (Frecha, Levy et al. 2010; Frecha, Levy et al. 2011). To transduce airway epithelial cells efficiently, envelope proteins from several viruses that infect respiratory tissues or cells have been evaluated. For efficient transduction of unconditioned airway epithelial cells from the apical side, enve‐ lopes derived from the ebola virus (*Filoviridae)*, members of the *Paramyxoviridae* such as the respiratory syncytial (RSV) and sendai viruses, and members of the *Orthomyxoviridae* such as the influenza and fowl plaque viruses have been evaluated (Kobinger, Weiner et al. 2001; Nefkens, Garcia et al. 2007; Mitomo, Griesenbach et al. 2010). Surprisingly, it has been re‐ ported that the S protein of the severe acute respiratory syndrome-associated coronavirus (*Coronaviridae)* mediates entry into hepatoma cell lines (Hofmann, Hattermann et al. 2004). Finally, although the vesicular stomatitis, mokola and rabies virus are all derived from the *Rhabdoviridae* family, only LVs pseudotyped with the rabies-G envelope enable retrograde transport to motoneurons of the spinal cord upon intramuscular injection or to the thalamus upon striatal injection. In contrast, VSV.G displaying LVs transduce cells only locally while mokola-pseudotyped LVs preferentially target non-neuronal glial cells (Mazarakis, Azzouz et al. 2001; Azzouz, Ralph et al. 2004; Wong, Azzouz et al. 2004; Colin, Faideau et al. 2009;

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357

Although the use of an existing viral envelope gp seems the most straightforward way to pseudotype LVs, a natural variant with the desired delivery properties is not available for every therapeutic application. Moreover, natural gps can come with limitations such as sen‐ sitivity to neutralization by the host immune response, lack of specificity and/or insufficient transduction efficiency. Also their production and purification can be inefficient (Schaffer, Koerber et al. 2008). Therefore, the development of LVs with customized, user-defined gene delivery properties by molecular engineering of the envelope gps is an alternative strategy to retarget the LV to specific cell-surface receptors. This molecular engineering has become a

collective term for many different strategies, which will be described below.

Calame, Cachafeiro et al. 2011).

An ever-growing list of alternative pantropic as well as ecotropic naturally occurring gps has been evaluated for LV pseudotyping. These vary in origin, tropism, titer, stability, effi‐ ciency of packaging, inactivation by complement, efficiency of cell transduction and induc‐ tion of an immune response (Cronin, Zhang et al. 2005). They can be of retroviral origin such as those from T-lymphotropic virus, maedi-visna virus, MLV, feline endogenous retrovirus and gibbon ape leukemia virus (GALV) (Rasko, Battini et al. 1999; Stitz, Buchholz et al. 2000; Zeilfelder and Bosch 2001; Strang, Ikeda et al. 2004; Sakuma, De Ravin et al. 2010). In gener‐ al, LVs pseudotyped with a γ-retroviral envelope transduce CD34<sup>+</sup> hematopoetic precursors, a property that has been used for the correction of X-linked severe combined immunodefi‐ ciency (SCID) using the GALV or MLV-A envelopes (Cavazzana-Calvo, Hacein-Bey et al. 2000; Gaspar, Parsley et al. 2004). Nonetheless, envelope gps of numerous non-retroviral families have been used as well to pseudotype LVs. A first example is provided by the *Toga‐ viridae* family, where their envelope gps (from alphaviruses such as the Ross River virus) equips the LV with a mouse and human DC-specific tropism when injected intravenously (Strang, Takeuchi et al. 2005), and with an astrocyte and oligodendrocyte specific tropism when injected into the mouse brain (Kang, Stein et al. 2002). Another example is provided by the family of the *Baculoviridae* where the gp64 gp ensures high particle stability in addi‐ tion to a hepatocyte specific tropism (Matsui, Hegadorn et al. 2011). LVs pseudotyped with the lymphocytic choriomeningitis virus (LCMV) envelope from the *Arenaviridae* preferential‐ ly transduce cells from the central nervous system such as neural stem cells and progenitor cells, and also to glioma cells and insulin secreting β-cells (Kobinger, Deng et al. 2004; Milet‐ ic, Fischer et al. 2004; Stein, Martins et al. 2005). As there is an increasing interest in the de‐ velopment of gene therapeutic strategies for malignant gliomas, the most frequent primary brain tumors with very poor prognosis, several groups report on the use of LCMV gp pseu‐ dotyped LVs to target almost exclusively astrocytes, the main source of malignant glioma cells (Beyer, Westphal et al. 2002; Miletic, Fischer et al. 2004; Steffens, Tebbets et al. 2004). The H and F envelope proteins from the *Paramyxoviridae* family, such as those derived from measles viruses, provide LVs with the capacity to bind to SLAM and CD46, which confers efficient virus entry, nuclear transport and integration in non-activated B and T lympho‐ cytes. This property is particularly important, since primary unstimulated B and T cells are generally difficult to transduce if not pre-treated to induce progression through the cell cy‐ cle (*e.g.* through stimulation with anti-CD3/anti-CD28 antibodies or cytokines) (Frecha, Levy et al. 2010; Frecha, Levy et al. 2011). To transduce airway epithelial cells efficiently, envelope proteins from several viruses that infect respiratory tissues or cells have been evaluated. For efficient transduction of unconditioned airway epithelial cells from the apical side, enve‐ lopes derived from the ebola virus (*Filoviridae)*, members of the *Paramyxoviridae* such as the respiratory syncytial (RSV) and sendai viruses, and members of the *Orthomyxoviridae* such as the influenza and fowl plaque viruses have been evaluated (Kobinger, Weiner et al. 2001; Nefkens, Garcia et al. 2007; Mitomo, Griesenbach et al. 2010). Surprisingly, it has been re‐ ported that the S protein of the severe acute respiratory syndrome-associated coronavirus (*Coronaviridae)* mediates entry into hepatoma cell lines (Hofmann, Hattermann et al. 2004). Finally, although the vesicular stomatitis, mokola and rabies virus are all derived from the *Rhabdoviridae* family, only LVs pseudotyped with the rabies-G envelope enable retrograde transport to motoneurons of the spinal cord upon intramuscular injection or to the thalamus upon striatal injection. In contrast, VSV.G displaying LVs transduce cells only locally while mokola-pseudotyped LVs preferentially target non-neuronal glial cells (Mazarakis, Azzouz et al. 2001; Azzouz, Ralph et al. 2004; Wong, Azzouz et al. 2004; Colin, Faideau et al. 2009; Calame, Cachafeiro et al. 2011).

strategy to tackle both safety and efficacy concerns. The concept of swapping the viral enve‐ lope proteins of different viral species is called pseudotyping. Already in 1979, the envelope glycoprotein of the avian retrovirus was used to pseudotype VSV virions in order to selec‐ tively enrich for VSV temperature-sensitive mutants of VSV.G biosynthesis (Lodish and Weiss 1979). Later it was shown that wild type HIV-1 particles which were produced in cells that were infected with another virus, *e.g.* murine leukemia virus (MLV) or VSV, led to the generation of phenotypically mixed virions with an expanded host range (Canivet, Hoffman et al. 1990; Zhu, Chen et al. 1990). These observations introduced the concept of pseudotyp‐ ing and in the early 90's the gp160 sequence of a replication defective HIV-1 derived LV was replaced by a MLV gp (Page, Landau et al. 1990). Later on the natural envelope gp from an MLV-based vector was replaced with the viral attachment protein of VSV (Emi, Friedmann et al. 1991; Burns, Friedmann et al. 1993). Today, most synthetic LVs are pseudotyped with a heterologous envelope protein to increase their stability, infectivity and safety. Notably, the first LVs were not pseudotyped but displayed the native HIV-1 envelope protein at their surface. This limited their tropism to CD4-expressing cells (Dropulic 2011). Interestingly, VSV.G pseudotyped vectors are more stable than their natural counterparts. This allows concentration to higher titers by ultracentrifugation and confers broad tropism, as VSV.G binds to a still unknown ubiquitous membrane component (Cronin, Zhang et al. 2005). This superior transduction efficiency comes in handy for the treatment of genetic disorders such as β-thalassemia and X-linked adenoleukodystrophy (Cartier, Hacein-Bey-Abina et al. 2009; Cavazzana-Calvo, Payen et al. 2010). Nonetheless, VSV.G pseudotyped LVs also present several downsides. Firstly, the VSV gp is cytotoxic when expressed constitutively at high concentrations, which impedes the production of stable packaging cell lines (Lopes, Dewan‐ nieux et al. 2011). In addition, cytotoxicity associated with VSV.G pseudotyped LVs has been observed when *in vivo* administered at high concentration, in comparison with other pseudotypes (Watson, Kobinger et al. 2002). Another critical hurdle for systemic delivery us‐ ing VSV.G pseudotyped LVs is their susceptibility to neutralization by human serum com‐ plement, although this can be bypassed by polyethylene glycol-modification (PEGylation) of

356 Gene Therapy - Tools and Potential Applications

the virions (DePolo, Reed et al. 2000; Croyle, Callahan et al. 2004).

al, LVs pseudotyped with a γ-retroviral envelope transduce CD34<sup>+</sup>

An ever-growing list of alternative pantropic as well as ecotropic naturally occurring gps has been evaluated for LV pseudotyping. These vary in origin, tropism, titer, stability, effi‐ ciency of packaging, inactivation by complement, efficiency of cell transduction and induc‐ tion of an immune response (Cronin, Zhang et al. 2005). They can be of retroviral origin such as those from T-lymphotropic virus, maedi-visna virus, MLV, feline endogenous retrovirus and gibbon ape leukemia virus (GALV) (Rasko, Battini et al. 1999; Stitz, Buchholz et al. 2000; Zeilfelder and Bosch 2001; Strang, Ikeda et al. 2004; Sakuma, De Ravin et al. 2010). In gener‐

a property that has been used for the correction of X-linked severe combined immunodefi‐ ciency (SCID) using the GALV or MLV-A envelopes (Cavazzana-Calvo, Hacein-Bey et al. 2000; Gaspar, Parsley et al. 2004). Nonetheless, envelope gps of numerous non-retroviral families have been used as well to pseudotype LVs. A first example is provided by the *Toga‐ viridae* family, where their envelope gps (from alphaviruses such as the Ross River virus) equips the LV with a mouse and human DC-specific tropism when injected intravenously

hematopoetic precursors,

Although the use of an existing viral envelope gp seems the most straightforward way to pseudotype LVs, a natural variant with the desired delivery properties is not available for every therapeutic application. Moreover, natural gps can come with limitations such as sen‐ sitivity to neutralization by the host immune response, lack of specificity and/or insufficient transduction efficiency. Also their production and purification can be inefficient (Schaffer, Koerber et al. 2008). Therefore, the development of LVs with customized, user-defined gene delivery properties by molecular engineering of the envelope gps is an alternative strategy to retarget the LV to specific cell-surface receptors. This molecular engineering has become a collective term for many different strategies, which will be described below.

A first strategy to alter the tropism of a virally derived gp is by rational point and domain mutagenesis. This is exemplified by the DC-specific targeting strategy from Yang et al. Cer‐ tain subsets of DCs carry the DC-SIGN protein (also known as CD209) on their surface, which is a C-type lectin-like receptor that potentiates rapid binding and endocytosis of ma‐ terials. The sindbis virus envelope gp consists of two integral membrane gps that form a het‐ erodimer and function as one unit. The fusogenic monomer is E1 and needs binding *via* E2 to mediate low pH-dependent fusion. The latter binds to the DC-SIGN receptor, next to the canonical viral receptor heparin sulfate, expressed by many cell types. Since both protein binding sites are physically separated, selective mutation at the E2 monomer is possible, ab‐ rogating the heparin sulfate binding part while leaving the DC-SIGN binding part intact. By pseudotyping a LV with this mutated sindbis virus derived envelope gp, targeted infection of DCs *in vivo* after direct subcutaneous administration was achieved. Moreover, this elicit‐ ed a strong antigen-specific immune response (Yang, Yang et al. 2008; Hu, Dai et al. 2010). Another example is the substitution of the V3-loop region of the SIV envelope gp with the corresponding region of a T cell tropic HIV-1 to create a T-cell targeted MLV vector, pseudo‐ typed with this engineered SIV gp (Steidl, Stitz et al. 2002). A final example is provided by Dylla et al. who diminished the alfa-dystroglycan affinity of the LCMV WE45 strain enve‐ lope gp by a point mutation. When a FIV derived LV was pseudotyped with this point mu‐ tated LCMV gp, their intravenous injection in adult mice yielded low transduction efficiencies in hepatocytes in contrast to abundant liver and cardiomyocyte transduction with the wild type LCMV gp pseudotyped FIVs (Dylla, Xie et al. 2011).

is exemplified by the blockage in trafficking in the producer cells to the plasma membrane of VSV.G when linked to a collagen-binding motif (Guibinga, Hall et al. 2004). Different kinds of ligands such as cytokines and growth factors have been linked to the amino-termi‐ nal region or receptor-binding domain of the envelope gp, most often derived of MLV. This is amongst others exemplified by fusion of the MLV gp to hepatocyte growth factor to target the LV to hepatocytes (Nguyen, Pages et al. 1998), or to the insulin-like growth factor (IGF-I) (Chadwick, Morling et al. 1999). Interestingly, these ligands can elevate the transduction ef‐ ficiency by altering the targets' physiological state. When the fusion product of the MLV gp and IL-2 is used to pseudotype LVs, a 34-fold higher infection efficiency was observed of quiescent IL-2 receptor expressing cells compared to LVs pseudotyped with the wild type MLV gp. This was explained by IL-2 induced activation of the cell cycle from the otherwise barely transducible quiescent cells (Maurice, Mazur et al. 1999). However, a very low to un‐ observable transduction profile is often reported which can be attributed to sequestration of the LV particles at the target cell surface, directing the viral particle to a degradation path‐ way after endocytosis and/or inability of the fusion product to trigger a conformational change essential for viral fusion and subsequent infection (Lavillette, Russell et al. 2001; Ka‐ tane, Takao et al. 2002). In addition to peptides and ligands, also antibodies and their deriva‐ tives can be used. In general, single chain variable fragments or scFvs offer higher specificity than short peptides but as they are larger in size, the chance that they disrupt the process of conformational changes of the gp to mediate membrane fusion increases. Therefore scFvs are most often linked to a spacer peptide that permits proper conformation of both the scFv domain and the envelope gp as exemplified by the fusion of the MLV gp to a scFv against MHC class I (Karavanas, Marin et al. 2002). For LV targeting to APCs, several attempts have been made to couple an anti-MHC class II scFv to an ecotropic gp such as MLV or VSV.G (Dreja and Piechaczyk 2006; Gennari, Lopes et al. 2009). Recently, the use of DARPins or de‐ signed ankyrin repeat proteins has been reported. These can be fused to the H protein of measles virus for example and then be co-displayed with the fusogenic F protein on the sur‐ face of the LV. The advantage is that DARPins can be selected to become high-affinity bind‐ ers to any kind of target molecule thus this seems a promising alternative to scFvs for retargeting LVs (Munch, Muhlebach et al. 2011). So, in general, the use of chimeric envelope proteins for LV targeting has proven to offer tremendous opportunities but at the same time to be a challenge as the function of chimeric gps is often severely compromised which leads to a very inefficient transduction profile (Fielding, Maurice et al. 1998; Dreja and Piechaczyk

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2006; Waehler, Russell et al. 2007; Buchholz, Duerner et al. 2008).

Several solutions have been created to circumvent the problems associated with the forma‐ tion of conformational dysfunctional fusion products. One solution is the inclusion of a pro‐ tease cleavable peptide between the gp and the ligand. This is certainly an interesting strategy for the targeting of tumor cells, as they secrete MMP, which degrade the extracellu‐ lar matrix to metastasize. By linking a proline-rich hinge and an MMP cleavage site to the fusion product of a scFv recognizing carcinoembryonic antigen (CEA) and the MLV gp, se‐ lective targeting of CEA-positive cells after *in vivo* injection of producer cells at the tumor site was observed (Chowdhury, Chester et al. 2004). Taking this hinge region idea one step further, the concept of 'molecular bridges' was introduced where a bispecific linker mole‐

Apart from genetic alterations, chemical modifications can also alter LV tropism. PEGyla‐ tion of VSV.G pseudotyped LVs is one such example where the LVs' tropism is not altered. Nevertheless, chemical modifications can lead to targeted gene delivery vehicles, for exam‐ ple by tagging the MLV vector with galactose to selectively transduce human hepatoma cell lines expressing asialo-gp receptors specific for oligosaccharides with terminal galactose res‐ idues (Neda, Wu et al. 1991). Furthermore, Morizono et al. reported the production of LVs pseudotyped with sindbis virus gps in the presence of deoxymannojirimycin. This modifica‐ tion altered the structures of N-glycans from complex to high mannose structures as it inhib‐ its mannosidase. This led to DC-SIGN specific binding although the gps were genetically modified to prevent interaction with DC-SIGN (Morizono, Ku et al. 2010). Furthermore it was demonstrated that binding of sindbis virus gp to DCs is directly related to the amount of high-mannose structures on the gp (Tai, Froelich et al. 2011). Unfortunately, the effective‐ ness of the chemically modified particles strongly depends on the reaction conditions of the applied modifications.

Other chimeric envelope gps can be generated by covalently fusing a short peptide, a ligand or an antibody to an envelope gp. The advantages of short peptides are that they don't se‐ verely disrupt the original envelope gps' function and that *via* high-throughput library ap‐ proaches, targeted peptides with strong binding affinity and unlimited specificity within the context of a particular gp can be generated (Schaffer, Koerber et al. 2008). However, they can hinder multimerisation of capsid monomers, create fusion products with lower thermosta‐ bility and hinder proper intracellular trafficking of the gp during viral production. The latter is exemplified by the blockage in trafficking in the producer cells to the plasma membrane of VSV.G when linked to a collagen-binding motif (Guibinga, Hall et al. 2004). Different kinds of ligands such as cytokines and growth factors have been linked to the amino-termi‐ nal region or receptor-binding domain of the envelope gp, most often derived of MLV. This is amongst others exemplified by fusion of the MLV gp to hepatocyte growth factor to target the LV to hepatocytes (Nguyen, Pages et al. 1998), or to the insulin-like growth factor (IGF-I) (Chadwick, Morling et al. 1999). Interestingly, these ligands can elevate the transduction ef‐ ficiency by altering the targets' physiological state. When the fusion product of the MLV gp and IL-2 is used to pseudotype LVs, a 34-fold higher infection efficiency was observed of quiescent IL-2 receptor expressing cells compared to LVs pseudotyped with the wild type MLV gp. This was explained by IL-2 induced activation of the cell cycle from the otherwise barely transducible quiescent cells (Maurice, Mazur et al. 1999). However, a very low to un‐ observable transduction profile is often reported which can be attributed to sequestration of the LV particles at the target cell surface, directing the viral particle to a degradation path‐ way after endocytosis and/or inability of the fusion product to trigger a conformational change essential for viral fusion and subsequent infection (Lavillette, Russell et al. 2001; Ka‐ tane, Takao et al. 2002). In addition to peptides and ligands, also antibodies and their deriva‐ tives can be used. In general, single chain variable fragments or scFvs offer higher specificity than short peptides but as they are larger in size, the chance that they disrupt the process of conformational changes of the gp to mediate membrane fusion increases. Therefore scFvs are most often linked to a spacer peptide that permits proper conformation of both the scFv domain and the envelope gp as exemplified by the fusion of the MLV gp to a scFv against MHC class I (Karavanas, Marin et al. 2002). For LV targeting to APCs, several attempts have been made to couple an anti-MHC class II scFv to an ecotropic gp such as MLV or VSV.G (Dreja and Piechaczyk 2006; Gennari, Lopes et al. 2009). Recently, the use of DARPins or de‐ signed ankyrin repeat proteins has been reported. These can be fused to the H protein of measles virus for example and then be co-displayed with the fusogenic F protein on the sur‐ face of the LV. The advantage is that DARPins can be selected to become high-affinity bind‐ ers to any kind of target molecule thus this seems a promising alternative to scFvs for retargeting LVs (Munch, Muhlebach et al. 2011). So, in general, the use of chimeric envelope proteins for LV targeting has proven to offer tremendous opportunities but at the same time to be a challenge as the function of chimeric gps is often severely compromised which leads to a very inefficient transduction profile (Fielding, Maurice et al. 1998; Dreja and Piechaczyk 2006; Waehler, Russell et al. 2007; Buchholz, Duerner et al. 2008).

A first strategy to alter the tropism of a virally derived gp is by rational point and domain mutagenesis. This is exemplified by the DC-specific targeting strategy from Yang et al. Cer‐ tain subsets of DCs carry the DC-SIGN protein (also known as CD209) on their surface, which is a C-type lectin-like receptor that potentiates rapid binding and endocytosis of ma‐ terials. The sindbis virus envelope gp consists of two integral membrane gps that form a het‐ erodimer and function as one unit. The fusogenic monomer is E1 and needs binding *via* E2 to mediate low pH-dependent fusion. The latter binds to the DC-SIGN receptor, next to the canonical viral receptor heparin sulfate, expressed by many cell types. Since both protein binding sites are physically separated, selective mutation at the E2 monomer is possible, ab‐ rogating the heparin sulfate binding part while leaving the DC-SIGN binding part intact. By pseudotyping a LV with this mutated sindbis virus derived envelope gp, targeted infection of DCs *in vivo* after direct subcutaneous administration was achieved. Moreover, this elicit‐ ed a strong antigen-specific immune response (Yang, Yang et al. 2008; Hu, Dai et al. 2010). Another example is the substitution of the V3-loop region of the SIV envelope gp with the corresponding region of a T cell tropic HIV-1 to create a T-cell targeted MLV vector, pseudo‐ typed with this engineered SIV gp (Steidl, Stitz et al. 2002). A final example is provided by Dylla et al. who diminished the alfa-dystroglycan affinity of the LCMV WE45 strain enve‐ lope gp by a point mutation. When a FIV derived LV was pseudotyped with this point mu‐ tated LCMV gp, their intravenous injection in adult mice yielded low transduction efficiencies in hepatocytes in contrast to abundant liver and cardiomyocyte transduction

with the wild type LCMV gp pseudotyped FIVs (Dylla, Xie et al. 2011).

applied modifications.

358 Gene Therapy - Tools and Potential Applications

Apart from genetic alterations, chemical modifications can also alter LV tropism. PEGyla‐ tion of VSV.G pseudotyped LVs is one such example where the LVs' tropism is not altered. Nevertheless, chemical modifications can lead to targeted gene delivery vehicles, for exam‐ ple by tagging the MLV vector with galactose to selectively transduce human hepatoma cell lines expressing asialo-gp receptors specific for oligosaccharides with terminal galactose res‐ idues (Neda, Wu et al. 1991). Furthermore, Morizono et al. reported the production of LVs pseudotyped with sindbis virus gps in the presence of deoxymannojirimycin. This modifica‐ tion altered the structures of N-glycans from complex to high mannose structures as it inhib‐ its mannosidase. This led to DC-SIGN specific binding although the gps were genetically modified to prevent interaction with DC-SIGN (Morizono, Ku et al. 2010). Furthermore it was demonstrated that binding of sindbis virus gp to DCs is directly related to the amount of high-mannose structures on the gp (Tai, Froelich et al. 2011). Unfortunately, the effective‐ ness of the chemically modified particles strongly depends on the reaction conditions of the

Other chimeric envelope gps can be generated by covalently fusing a short peptide, a ligand or an antibody to an envelope gp. The advantages of short peptides are that they don't se‐ verely disrupt the original envelope gps' function and that *via* high-throughput library ap‐ proaches, targeted peptides with strong binding affinity and unlimited specificity within the context of a particular gp can be generated (Schaffer, Koerber et al. 2008). However, they can hinder multimerisation of capsid monomers, create fusion products with lower thermosta‐ bility and hinder proper intracellular trafficking of the gp during viral production. The latter Several solutions have been created to circumvent the problems associated with the forma‐ tion of conformational dysfunctional fusion products. One solution is the inclusion of a pro‐ tease cleavable peptide between the gp and the ligand. This is certainly an interesting strategy for the targeting of tumor cells, as they secrete MMP, which degrade the extracellu‐ lar matrix to metastasize. By linking a proline-rich hinge and an MMP cleavage site to the fusion product of a scFv recognizing carcinoembryonic antigen (CEA) and the MLV gp, se‐ lective targeting of CEA-positive cells after *in vivo* injection of producer cells at the tumor site was observed (Chowdhury, Chester et al. 2004). Taking this hinge region idea one step further, the concept of 'molecular bridges' was introduced where a bispecific linker mole‐ cule recognizes both the viral gp as well as the molecular determinant on the target cell. This concept is based on a bridging system that was introduced more than 20 years ago and where three different linker molecules were involved: two biotinylated antibodies that bound the MLV gp and MHC class I or II proteins on the target cells respectively, and a bridging streptavidin molecule linking both antibodies. This led to the generation of a MLV that was capable of transducing MHC class I and II expressing cells (Roux, Jeanteur et al. 1989). Subsequently, two-protein molecular bridges have been exploited based on the avi‐ din-biotin system. A recent example is provided by O'Leary et al. who used a detoxified re‐ combinant form of the full-length botulinum neurotoxin, fused to core streptavidin that for its part was coupled to a biotinylated LV. This envelope gp construct endowed the LV parti‐ cle with considerable neuron selectivity *in vitro* as well as *in vivo* after injection into the tra‐ chea (O'Leary, Ovsepian et al. 2011). Nowadays, alternative linkers such as ligand-receptor, chemical conjugations and monoclonal antibodies have been exploited to retarget LVs as well (Roux, Jeanteur et al. 1989; Boerger, Snitkovsky et al. 1999). For the latter, the E2 protein of the sindbis gp has been modified to contain the Fc-binding domain (ZZ domain) of pro‐ tein A, making it possible to bind to a monoclonal antibody specific for a target molecule *via* its Fab antigen recognition end (Morizono, Xie et al. 2005). However, doubts are raised about the affinity of the adaptor-virus complex, as this may not be sufficient to prevent dis‐ sociation within the patient's blood. Moreover, complexity ascends as both the virion as the adaptor must be produced, purified and fully characterized for clinical approval. Another alternative possibility is to co-display a chimeric envelope gp together with a wild type gp such as VSV.G to enhance the transduction efficiency (Maurice, Verhoeyen et al. 2002; Ver‐ hoeyen, Dardalhon et al. 2003; Verhoeyen, Wiznerowicz et al. 2005). However, this had also limited success due to partial loss of targeting specificity. Therefore, a final alternative is the usage of a mutated fusogenic but binding-defective envelope gp to mediate fusion upon binding by the chimeric gp. The group of Lin et al. co-expressed the MLV gp fused to solu‐ ble Fms-like tyrosine kinase 3 (Flt3)-ligand together with a binding-defective influenza he‐ magglutinin protein from the fowl plague virus rostock 34 (HAmu). When LVs were pseudotyped with both of these gps, Flt3-targeted transduction was enhanced when com‐ pared to LVs without HAmu and could be competed away by the addition of soluble Flt3 ligand (Lin, Kasahara et al. 2001). Another more straightforward strategy is the use of the E1/E2 heterodimer gp of sindbis virus as the fusion and binding functions are already sepa‐ rated over two different monomers. By mutating the binding E2 monomer, its binding prop‐ erty can be completely abolished. Therefore, this binding defective E2 protein forms an ideal scaffold for cell-specific antibody conjugation to confer specific tropism to an endless list of cell types such as P-gp-expressing melanoma progenitor cells and endothelial cells (Morizo‐ no, Xie et al. 2005; Pariente, Mao et al. 2008). A drawback is that they only induce fusion upon low pH. Therefore alternatives were explored such as the H and F protein of the mea‐ sles virus, which induce fusion without the need for endocytosis (Earp, Delos et al. 2005; Funke, Schneider et al. 2009). This is exemplified by a study were a binding defective form of the H protein was fused to a CD20 specific scFv to pseudotype LVs. When these LVs were used to kill cells in culture, they selectively killed the CD20+ human lymphocytes in co-cul‐ ture with CD20- cells. This demonstrated the ability of these LVs to exclusively transfer a po‐ tentially hazardous therapeutic protein into targeted cell populations with virtual absence of background transduction in non-target cells (Funke, Maisner et al. 2008). Meanwhile, a broad variety of surface antigens has been successfully targeted using this strategy (Blechacz

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

361

A fourth strategy to target LVs is based on two concepts: (1) the separation of binding and fusion functions over two distinct envelope molecules and (2) the ability of LVs to incorpo‐ rate host cell proteins into their envelope as they bud from the plasmamembrane of their producer cells (Chandrashekran, Gordon et al. 2004; Kueng, Leb et al. 2007). Chandrashek‐ ran et al. reported on efficient and specific targeting to human cells expressing stem cell fac‐ tor (SCF) receptor (c-kit) by an ecotropic gp pseudotyped LV which also displayed surface SCF. Another example is the overexpression of the HIV-1 derived primary receptor CD4 and fusogenic co-receptor CXCR4 or CCR5 on the membrane of producer cells. From these cells, LVs were generated that infect HIV-1 envelope gp expressing cells next to cells infected with HIV-1, enabling the development of novel antiviral therapy approaches (Somia, Miyoshi et al. 2000). Since the transduction efficiency was relatively low, LV co-enveloped with the HIV-1 cellular receptor CD4 and the E2 protein from sindbis virus were created. These turned out to have a higher infectivity level than in the former strategy (Lee, Dang et al. 2011). In another study the binding defective but fusogenic E1/E2 heterodimer was used to be co-displayed with a separate membrane bound anti-CD20 antibody in order to transduce

B cells (Lei, Joo et al. 2009). Today, numerous examples are found that ap‐

HSCs (Ziegler, Yang et al. 2008; Froelich, Ziegler et al. 2009; Yang, Joo et al.

ply this principle to target the following: immunoglobulin-expressing B cells, CD3+ T cells

2009). However, clinical applications with LVs displaying scFvs are hampered by lack of sta‐ bility, size and immunogenicity leading to the development of neutralizing antibodies. To solve these problems, we developed the Nanobody (Nb) display technology (Goyvaerts, De Groeve et al. 2012). In this strategy, a fusogenic but binding-defective form of VSV.G (VSV.GS) (Zhang, Kutner et al. 2010) is co-displayed with a surface bound form of a cell-spe‐ cific Nb to confer target binding (Figure 4). Some twenty years ago, Hamers-Casterman et al. discovered that part of the humoral response of Camelids is based on a unique repertoire of antibodies, which only consisted of two heavy chains (Hamers-Casterman, Atarhouch et al. 1993). The antigen binding part of these antibodies is composed of only one single variable region, termed VHH or Nb. These Nbs have unique characteristics and offer many advan‐ tages over scFvs to target LVs to specific cell types. These include (1) they are highly soluble, (2) they can refold after denaturation whilst retaining their binding capacity, (3) cloning and selection of antigen-specific Nbs obviate the need for construction and screening of large li‐ braries, (4) as Nbs can be fused to other proteins, it is possible to present them on the cell membrane of a producer cell line, thus generating LVs that incorporate a cell-specific Nb in their envelope during budding. Using the Nb display technology, we demonstrated produc‐ tion of stable Nb pseudotyped LV stocks at high titers with a DC subtype specific transduc‐ tion profile both *in vitro* as well as *in vivo* (Goyvaerts, De Groeve et al. 2012). As ligand specific Nbs can be generated to potentially every cell surface molecule, this technology will be applicable to target LVs to every cell type for which cell specific surface molecules are

characterized (Gainkam, Huang et al. 2008; Vaneycken, Devoogdt et al. 2011).

and Russell 2008)

exclusively CD20+

and CD117+

tentially hazardous therapeutic protein into targeted cell populations with virtual absence of background transduction in non-target cells (Funke, Maisner et al. 2008). Meanwhile, a broad variety of surface antigens has been successfully targeted using this strategy (Blechacz and Russell 2008)

cule recognizes both the viral gp as well as the molecular determinant on the target cell. This concept is based on a bridging system that was introduced more than 20 years ago and where three different linker molecules were involved: two biotinylated antibodies that bound the MLV gp and MHC class I or II proteins on the target cells respectively, and a bridging streptavidin molecule linking both antibodies. This led to the generation of a MLV that was capable of transducing MHC class I and II expressing cells (Roux, Jeanteur et al. 1989). Subsequently, two-protein molecular bridges have been exploited based on the avi‐ din-biotin system. A recent example is provided by O'Leary et al. who used a detoxified re‐ combinant form of the full-length botulinum neurotoxin, fused to core streptavidin that for its part was coupled to a biotinylated LV. This envelope gp construct endowed the LV parti‐ cle with considerable neuron selectivity *in vitro* as well as *in vivo* after injection into the tra‐ chea (O'Leary, Ovsepian et al. 2011). Nowadays, alternative linkers such as ligand-receptor, chemical conjugations and monoclonal antibodies have been exploited to retarget LVs as well (Roux, Jeanteur et al. 1989; Boerger, Snitkovsky et al. 1999). For the latter, the E2 protein of the sindbis gp has been modified to contain the Fc-binding domain (ZZ domain) of pro‐ tein A, making it possible to bind to a monoclonal antibody specific for a target molecule *via* its Fab antigen recognition end (Morizono, Xie et al. 2005). However, doubts are raised about the affinity of the adaptor-virus complex, as this may not be sufficient to prevent dis‐ sociation within the patient's blood. Moreover, complexity ascends as both the virion as the adaptor must be produced, purified and fully characterized for clinical approval. Another alternative possibility is to co-display a chimeric envelope gp together with a wild type gp such as VSV.G to enhance the transduction efficiency (Maurice, Verhoeyen et al. 2002; Ver‐ hoeyen, Dardalhon et al. 2003; Verhoeyen, Wiznerowicz et al. 2005). However, this had also limited success due to partial loss of targeting specificity. Therefore, a final alternative is the usage of a mutated fusogenic but binding-defective envelope gp to mediate fusion upon binding by the chimeric gp. The group of Lin et al. co-expressed the MLV gp fused to solu‐ ble Fms-like tyrosine kinase 3 (Flt3)-ligand together with a binding-defective influenza he‐ magglutinin protein from the fowl plague virus rostock 34 (HAmu). When LVs were pseudotyped with both of these gps, Flt3-targeted transduction was enhanced when com‐ pared to LVs without HAmu and could be competed away by the addition of soluble Flt3 ligand (Lin, Kasahara et al. 2001). Another more straightforward strategy is the use of the E1/E2 heterodimer gp of sindbis virus as the fusion and binding functions are already sepa‐ rated over two different monomers. By mutating the binding E2 monomer, its binding prop‐ erty can be completely abolished. Therefore, this binding defective E2 protein forms an ideal scaffold for cell-specific antibody conjugation to confer specific tropism to an endless list of cell types such as P-gp-expressing melanoma progenitor cells and endothelial cells (Morizo‐ no, Xie et al. 2005; Pariente, Mao et al. 2008). A drawback is that they only induce fusion upon low pH. Therefore alternatives were explored such as the H and F protein of the mea‐ sles virus, which induce fusion without the need for endocytosis (Earp, Delos et al. 2005; Funke, Schneider et al. 2009). This is exemplified by a study were a binding defective form of the H protein was fused to a CD20 specific scFv to pseudotype LVs. When these LVs were

360 Gene Therapy - Tools and Potential Applications

used to kill cells in culture, they selectively killed the CD20+

ture with CD20- cells. This demonstrated the ability of these LVs to exclusively transfer a po‐

human lymphocytes in co-cul‐

A fourth strategy to target LVs is based on two concepts: (1) the separation of binding and fusion functions over two distinct envelope molecules and (2) the ability of LVs to incorpo‐ rate host cell proteins into their envelope as they bud from the plasmamembrane of their producer cells (Chandrashekran, Gordon et al. 2004; Kueng, Leb et al. 2007). Chandrashek‐ ran et al. reported on efficient and specific targeting to human cells expressing stem cell fac‐ tor (SCF) receptor (c-kit) by an ecotropic gp pseudotyped LV which also displayed surface SCF. Another example is the overexpression of the HIV-1 derived primary receptor CD4 and fusogenic co-receptor CXCR4 or CCR5 on the membrane of producer cells. From these cells, LVs were generated that infect HIV-1 envelope gp expressing cells next to cells infected with HIV-1, enabling the development of novel antiviral therapy approaches (Somia, Miyoshi et al. 2000). Since the transduction efficiency was relatively low, LV co-enveloped with the HIV-1 cellular receptor CD4 and the E2 protein from sindbis virus were created. These turned out to have a higher infectivity level than in the former strategy (Lee, Dang et al. 2011). In another study the binding defective but fusogenic E1/E2 heterodimer was used to be co-displayed with a separate membrane bound anti-CD20 antibody in order to transduce exclusively CD20+ B cells (Lei, Joo et al. 2009). Today, numerous examples are found that ap‐ ply this principle to target the following: immunoglobulin-expressing B cells, CD3+ T cells and CD117+ HSCs (Ziegler, Yang et al. 2008; Froelich, Ziegler et al. 2009; Yang, Joo et al. 2009). However, clinical applications with LVs displaying scFvs are hampered by lack of sta‐ bility, size and immunogenicity leading to the development of neutralizing antibodies. To solve these problems, we developed the Nanobody (Nb) display technology (Goyvaerts, De Groeve et al. 2012). In this strategy, a fusogenic but binding-defective form of VSV.G (VSV.GS) (Zhang, Kutner et al. 2010) is co-displayed with a surface bound form of a cell-spe‐ cific Nb to confer target binding (Figure 4). Some twenty years ago, Hamers-Casterman et al. discovered that part of the humoral response of Camelids is based on a unique repertoire of antibodies, which only consisted of two heavy chains (Hamers-Casterman, Atarhouch et al. 1993). The antigen binding part of these antibodies is composed of only one single variable region, termed VHH or Nb. These Nbs have unique characteristics and offer many advan‐ tages over scFvs to target LVs to specific cell types. These include (1) they are highly soluble, (2) they can refold after denaturation whilst retaining their binding capacity, (3) cloning and selection of antigen-specific Nbs obviate the need for construction and screening of large li‐ braries, (4) as Nbs can be fused to other proteins, it is possible to present them on the cell membrane of a producer cell line, thus generating LVs that incorporate a cell-specific Nb in their envelope during budding. Using the Nb display technology, we demonstrated produc‐ tion of stable Nb pseudotyped LV stocks at high titers with a DC subtype specific transduc‐ tion profile both *in vitro* as well as *in vivo* (Goyvaerts, De Groeve et al. 2012). As ligand specific Nbs can be generated to potentially every cell surface molecule, this technology will be applicable to target LVs to every cell type for which cell specific surface molecules are characterized (Gainkam, Huang et al. 2008; Vaneycken, Devoogdt et al. 2011).

The downside of the use of the above-described strategies is that they rely on the fusogenic capacity of a gp that is derived from viruses infectious to humans such as VSV, measles vi‐ rus, sindbis virus and MLV. Their exposure to the complement or immune system, leading to anti-gp antibodies, might limit their clinical applicability. To surmount these obstacles, Frecha et al. pseudotyped LVs with a mutant fusogenic gp derived from an endogenous fe‐ line virus, named RD114. The mutant RD114 gp is an attractive candidate for in *vivo* use as it is resistant to degradation by the human complement. By co-displaying the early-acting-cy‐ tokine SCF together with mutant RD114 gp, human CD34+ HSCs could be targeted *in vivo* (Frecha, Fusil et al. 2011; Frecha, Costa et al. 2012). SCF was responsible for a slight and tran‐ sient stimulation of the HSCs while preserving the 'stemness' of the targeted HSCs. In that way, the need for CD3/CD28 or cytokine pretreatment was obviated. Springfeld et al. recent‐ ly pseudotyped LVs with the H and F gps of the *Tupaia paramyxovirus* (TPMV), an animal virus without close human pathogenic relatives. Moreover, as this virus does not infect hu‐ man cells, detargeting the H protein from its natural receptors is unnecessary. When LVs were pseudotyped with the TPMV envelope protein linked to an anti-CD20 single chain an‐ tibody, selective transduction of CD20+ cells, including quiescent primary human B cells, was reported (Enkirch, Kneissl et al. 2012).

cles, which were in turn coupled to lipid microbubbles. LVs coupled to magnetic nanoparticles to target them to specific cell types *in vitro* using an external magnetic field has been described before. However, when these LV-nanoparticle constructs are considered for *in vivo* use, a sufficient magnetic moment is needed as the particles are subject to flow velocity within the blood vessels. As the magnetic moment is proportional to particle size, Mannell et al. coated the LV-nanoparticle constructs with magnetic microbubbles for en‐ largement. Upon intravenous delivery, the LV magnetic microbubbles were first trapped at the site of interest. Next ultrasound mediated destruction of the microbubbles resulted in fast release of the LVs at the site of interest with high transduction efficiency without the

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

363

In conclusion, there seem to be some general prerequisites for successful transductional tar‐ geting of LVs: (1) use envelope gps with defined receptor binding sites, (2) abolish the natu‐ ral recognition sites of the attachment gp, (3) separate fusion and attachment functions over two different molecules, (4) avoid the construction of large fusion constructs since their fu‐ sogenic capacities can be severely compromised and (5) avoid the use of immunogenic gps

Nowadays, LVs have become valuable tools for the treatment of several monogenic disor‐ ders such as hemophilia B, β-thalassemia and X-linked adrenoleukodystrophy (Cartier, Ha‐ cein-Bey-Abina et al. 2012; Payen, Colomb et al. 2012). However, the use of viral vectors that integrate their cargo into the genome of the host cell can trigger oncogenesis by insertional mutagenesis. This is exemplified by the incident where two out of 11 patients treated with a γ-retroviral vector to correct X-linked SCID, developed leukemia. This was caused by the γretroviral construct's tendency to insert into active genes, in this case the LMO-2 oncogene (Marshall 2002). Later on, using the same vector type to treat chronic granulomatous dis‐ ease, genomic instability and myelodysplasia was observed (Stein, Ott et al. 2010). These in‐ cidents prompted substantial research into design, safety testing and optimization of integrating vectors. Thus far several measures have been taken to pose a reduced risk on in‐ sertional mutagenesis such as the development of SIN LVs containing a moderate cellular promoter (Modlich and Baum 2009; Montini, Cesana et al. 2009). Furthermore LVs are in‐ trinsically less genotoxic than their retroviral counterparts (Montini, Cesana et al. 2006). Nevertheless, LVs have a higher transduction efficiency, which could counterbalance the re‐ duced risk of mutagenic vector integration into the patient's genome. In addition, accumu‐ lating studies report the concept of LV-induced clonal dominance related to growth and/or survival advantage *e.g.* induced by vector integration and subsequent formation of aberrant‐ ly spliced mRNA forms (Fehse and Roeder 2008; Cavazzana-Calvo, Payen et al. 2010; Cesa‐ na, Sgualdino et al. 2012; Moiani, Paleari et al. 2012). In an extensive analysis to explore the effect of promoter-enhancer selection on efficacy and safety of LVs, no clear underlying mechanism could be provided for the observed. They concluded that other ill-defined risk factors must be involved for oncogenesis, including replicative stress (Ginn, Liao et al. 2010).

cost of higher cytotoxicity (Mannell, Pircher et al. 2012).

(Buchholz, Muhlebach et al. 2009).

**5. Genomic targeting**

**Figure 4. Principle of the Nb display technology.** The Nb display technology is based on the fact that LVs need to bind and fuse with the membrane of a target cell for proper infection. While VSV.G accounts for both of these func‐ tions, we propose to separate these functions over two different molecules: (1) binding via a membrane bound Nb against the target cell of choice and (2) fusion via VSV.GS, which is a binding defective truncated version of VSV.G.

Recently an innovative alternative strategy has been described by Mannell et al. for site spe‐ cific vascular gene delivery. In this case, the LVs were first coupled to magnetic nanoparti‐ cles, which were in turn coupled to lipid microbubbles. LVs coupled to magnetic nanoparticles to target them to specific cell types *in vitro* using an external magnetic field has been described before. However, when these LV-nanoparticle constructs are considered for *in vivo* use, a sufficient magnetic moment is needed as the particles are subject to flow velocity within the blood vessels. As the magnetic moment is proportional to particle size, Mannell et al. coated the LV-nanoparticle constructs with magnetic microbubbles for en‐ largement. Upon intravenous delivery, the LV magnetic microbubbles were first trapped at the site of interest. Next ultrasound mediated destruction of the microbubbles resulted in fast release of the LVs at the site of interest with high transduction efficiency without the cost of higher cytotoxicity (Mannell, Pircher et al. 2012).

In conclusion, there seem to be some general prerequisites for successful transductional tar‐ geting of LVs: (1) use envelope gps with defined receptor binding sites, (2) abolish the natu‐ ral recognition sites of the attachment gp, (3) separate fusion and attachment functions over two different molecules, (4) avoid the construction of large fusion constructs since their fu‐ sogenic capacities can be severely compromised and (5) avoid the use of immunogenic gps (Buchholz, Muhlebach et al. 2009).

#### **5. Genomic targeting**

The downside of the use of the above-described strategies is that they rely on the fusogenic capacity of a gp that is derived from viruses infectious to humans such as VSV, measles vi‐ rus, sindbis virus and MLV. Their exposure to the complement or immune system, leading to anti-gp antibodies, might limit their clinical applicability. To surmount these obstacles, Frecha et al. pseudotyped LVs with a mutant fusogenic gp derived from an endogenous fe‐ line virus, named RD114. The mutant RD114 gp is an attractive candidate for in *vivo* use as it is resistant to degradation by the human complement. By co-displaying the early-acting-cy‐

(Frecha, Fusil et al. 2011; Frecha, Costa et al. 2012). SCF was responsible for a slight and tran‐ sient stimulation of the HSCs while preserving the 'stemness' of the targeted HSCs. In that way, the need for CD3/CD28 or cytokine pretreatment was obviated. Springfeld et al. recent‐ ly pseudotyped LVs with the H and F gps of the *Tupaia paramyxovirus* (TPMV), an animal virus without close human pathogenic relatives. Moreover, as this virus does not infect hu‐ man cells, detargeting the H protein from its natural receptors is unnecessary. When LVs were pseudotyped with the TPMV envelope protein linked to an anti-CD20 single chain an‐

**Figure 4. Principle of the Nb display technology.** The Nb display technology is based on the fact that LVs need to bind and fuse with the membrane of a target cell for proper infection. While VSV.G accounts for both of these func‐ tions, we propose to separate these functions over two different molecules: (1) binding via a membrane bound Nb against the target cell of choice and (2) fusion via VSV.GS, which is a binding defective truncated version of VSV.G.

Recently an innovative alternative strategy has been described by Mannell et al. for site spe‐ cific vascular gene delivery. In this case, the LVs were first coupled to magnetic nanoparti‐

HSCs could be targeted *in vivo*

cells, including quiescent primary human B cells,

tokine SCF together with mutant RD114 gp, human CD34+

tibody, selective transduction of CD20+

362 Gene Therapy - Tools and Potential Applications

was reported (Enkirch, Kneissl et al. 2012).

Nowadays, LVs have become valuable tools for the treatment of several monogenic disor‐ ders such as hemophilia B, β-thalassemia and X-linked adrenoleukodystrophy (Cartier, Ha‐ cein-Bey-Abina et al. 2012; Payen, Colomb et al. 2012). However, the use of viral vectors that integrate their cargo into the genome of the host cell can trigger oncogenesis by insertional mutagenesis. This is exemplified by the incident where two out of 11 patients treated with a γ-retroviral vector to correct X-linked SCID, developed leukemia. This was caused by the γretroviral construct's tendency to insert into active genes, in this case the LMO-2 oncogene (Marshall 2002). Later on, using the same vector type to treat chronic granulomatous dis‐ ease, genomic instability and myelodysplasia was observed (Stein, Ott et al. 2010). These in‐ cidents prompted substantial research into design, safety testing and optimization of integrating vectors. Thus far several measures have been taken to pose a reduced risk on in‐ sertional mutagenesis such as the development of SIN LVs containing a moderate cellular promoter (Modlich and Baum 2009; Montini, Cesana et al. 2009). Furthermore LVs are in‐ trinsically less genotoxic than their retroviral counterparts (Montini, Cesana et al. 2006). Nevertheless, LVs have a higher transduction efficiency, which could counterbalance the re‐ duced risk of mutagenic vector integration into the patient's genome. In addition, accumu‐ lating studies report the concept of LV-induced clonal dominance related to growth and/or survival advantage *e.g.* induced by vector integration and subsequent formation of aberrant‐ ly spliced mRNA forms (Fehse and Roeder 2008; Cavazzana-Calvo, Payen et al. 2010; Cesa‐ na, Sgualdino et al. 2012; Moiani, Paleari et al. 2012). In an extensive analysis to explore the effect of promoter-enhancer selection on efficacy and safety of LVs, no clear underlying mechanism could be provided for the observed. They concluded that other ill-defined risk factors must be involved for oncogenesis, including replicative stress (Ginn, Liao et al. 2010). Finally, next to transcriptional activation of neighboring genes, also transcriptional shut off of the tg has been reported. This was due to chromatin remodeling at the site of insertion and cessation of the therapeutic effect (Stein, Ott et al. 2010).

ciogullari et al. 2011). Site-specific proviral integration can also be mediated by the use of site-specific recombinases. The best known are derived from the lambda integrase family of enzymes and include the bacteriophage P1 Cre recombinase, bacteriophage lambda inte‐ grase, the yeast Flp recombinase and bacterial XerCD recombinase. They catalyze site specif‐ ic recombination by a transient DNA-protein covalent linkage that brings two specific DNA repeats together (Van Duyne 2001). Depending on the orientation of the DNA repeats, the DNA segment will either be excised or inverted when in the same or opposite orientation respectively (Figure 5A, adapted from http://www.ruf.rice.edu/~rur/issue1\_files/ norman.html). The Cre-loxP system has been developed for gene studies to conditionally knock out a target gene in a cell- or tissue specific manner to overcome embryonic lethality due to permanent inactivation of the target gene in an early developmental stage (Ray, Fa‐ gan et al. 2000). This system is based on two palindromic loxP sites of 34 bp that flank the gene of interest. Although these loxP sites are prevalent in the genomes of bacteriophages, they are absent in the mouse genome where they have to be introduced by targeted muta‐ genesis (Kos 2004). Throughout the human genome, however, loxP-like sequences or pseu‐ do-loxP sites are present that can be recognized by either wild-type Cre or Cre variants. This last feature enables site-specific insertion of a gene in a defined loxP site in the human ge‐ nome if a Cre recombinase is provided in *cis* or *trans.* Michel et al. evaluated the feasibility of combining the Cre-loxP system for gene targeting with the versatile gene delivery system of LVs for site-specific gene insertion in human cell lines. They transduced a loxP site contain‐ ing cell line with a LV containing Cre recombinase in *trans* as a fusion protein to the HIV accessory protein Vpr. Moreover the LV contained a cassette containing a loxP site followed by the neomycin resistant gene, inserted in the U3 region of the 3'LTR. Upon reverse tran‐ scription, the loxP-neo sequence would appear in both LTRs, thereby providing a substrate for recombination that could be catalyzed by the virion-associated Vpr-Cre. Upon this re‐ combination step, a circular product was produced that was on his turn inserted into the loxP site of the cell line, again catalyzed by virion-associated Vpr-Cre. Another example is provided by the group of Jiang et al. who demonstrated a selective inhibitory effect on the lens epithelial cells and not the retinal pigment epithelial cells (Jiang, Lu et al. 2011). There‐ fore they used an enhanced Cre/loxP system with a LV expressing Cre under the control of the lens-specific promoter LEP503 in combination with another LV that contained a stiffer sequence encoding eGFP with a functional polyadenylation signal between two loxP sites, followed by the HSV-TK gene, both under the control of the human phosphoglycerate kin‐ ase promoter. Expression of the downstream HSV-TK was activated by co-expression of Cre under the control of the lens-specific promoter LEP503. Although this technology allows site-specific tg insertion, there are only a limited amount of pseudo-loxP sites in the human genome and even none in the mouse genome, which makes this technique unusable for fun‐ damental research in laboratory animals. Furthermore, two recombination events are re‐

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

365

quired which has a major impact on its efficiency.

A recent strategy makes use of site-specific endonucleases to target the tg to neutral 'safe harbor' genome regions or stimulate the process of homologous recombination for gene re‐ pair (Fischer, Hacein-Bey-Abina et al. 2011). Endonucleases induce site-specific ds breaks that can be repaired by homology-directed repair, a form of homologous recombination that

Therefore additional strategies have been considered to reduce the side effects related to random insertion. The most straightforward strategy is to prevent integration of the proviral cargo by the use of IDLVs. These IDLVs are produced with a mutated integrase, which re‐ sults in prevention of proviral integration and generation of increased levels of circular vec‐ tor episomes within the infected cells. They appear to be safer with only a 0,1 to 2,3% chance that the episomal transcript gets integrated without a marked loss in effectiveness in terms of immune stimulatory potential of the IDLV-based vaccines (Vargas, Gusella et al. 2004; Philippe, Sarkis et al. 2006; Karwacz, Mukherjee et al. 2009; Wanisch and Yanez-Munoz 2009). However, as the lentiviral episomes lack replication signals, they are gradually lost by dilution in dividing cells and only stable in quiescent cells, which is undesirable for perma‐ nent correction of any genetic disorder. Furthermore also lower tg expression levels have been reported compared to integrative vectors (Bayer, Kantor et al. 2008). Therefore several alternative strategies have been brought forward to target the integrative process to a specif‐ ic 'safe' genomic site.

In a first attempt, several groups tried to fuse a heterologous DNA binding domain directly to the integrase. Bushman et al. were the first to evaluate the activity of a hybrid, composed of the HIV-1 integrase and the lambda repressor. They reported on integration primarily near the lambda operator sites on the same face of the β-DNA helix (Bushman 1994). Later a model system was used were the integrase, derived from the avian sarcoma virus or HIV-1 respectively was fused to the *Escherichia coli* LexA repressor protein DNA binding domain (Katz, Merkel et al. 1996; Holmes-Son and Chow 2000). When this construct was packaged into the virion in *trans* either by replacing the original integrase gene or by cloning it adja‐ cent to the HIV-1 accessory protein Vpr, they observed that this enhanced the use of integra‐ tion sites adjacent to the *lex*A operators. In another study, the HIV-1 derived integrase was fused to a synthetic polydactyl zinc finger protein E2C, which binds specifically to a contig‐ uous 18 bp E2C recognition site (Tan, Dong et al. 2006). Although in all studies clearly a higher preference for integration near the target sequence of choice was observed, this also implicated reduced DNA-binding specificity of the fusion protein with associated decrease of integration frequency of about 80 percent compared to viruses containing wild type inte‐ grase. Furthermore this strategy is also limited by the difficulty to incorporate the fusion protein into infectious virions (Michel, Yu et al. 2010).

Another strategy is targeting the integration away from genes using tethering domains linked to the host cell-encoded transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75), a cellular integrase binding protein. For example the LEDGF/p75 chromatin interaction-binding domain has been replaced with CBX1, which binds histone H3 di- or trimethylated on K9. Subsequently proviral integration was directed to pericentric heterochromatin and intergenic regions (Llano, Vanegas et al. 2006; Ferris, Wu et al. 2010; Gijsbers, Ronen et al. 2010; Silvers, Smith et al. 2010). As this requires engineering of a host cell protein, it is not feasible for clinical applications at the present stage (Izmiryan, Basma‐ ciogullari et al. 2011). Site-specific proviral integration can also be mediated by the use of site-specific recombinases. The best known are derived from the lambda integrase family of enzymes and include the bacteriophage P1 Cre recombinase, bacteriophage lambda inte‐ grase, the yeast Flp recombinase and bacterial XerCD recombinase. They catalyze site specif‐ ic recombination by a transient DNA-protein covalent linkage that brings two specific DNA repeats together (Van Duyne 2001). Depending on the orientation of the DNA repeats, the DNA segment will either be excised or inverted when in the same or opposite orientation respectively (Figure 5A, adapted from http://www.ruf.rice.edu/~rur/issue1\_files/ norman.html). The Cre-loxP system has been developed for gene studies to conditionally knock out a target gene in a cell- or tissue specific manner to overcome embryonic lethality due to permanent inactivation of the target gene in an early developmental stage (Ray, Fa‐ gan et al. 2000). This system is based on two palindromic loxP sites of 34 bp that flank the gene of interest. Although these loxP sites are prevalent in the genomes of bacteriophages, they are absent in the mouse genome where they have to be introduced by targeted muta‐ genesis (Kos 2004). Throughout the human genome, however, loxP-like sequences or pseu‐ do-loxP sites are present that can be recognized by either wild-type Cre or Cre variants. This last feature enables site-specific insertion of a gene in a defined loxP site in the human ge‐ nome if a Cre recombinase is provided in *cis* or *trans.* Michel et al. evaluated the feasibility of combining the Cre-loxP system for gene targeting with the versatile gene delivery system of LVs for site-specific gene insertion in human cell lines. They transduced a loxP site contain‐ ing cell line with a LV containing Cre recombinase in *trans* as a fusion protein to the HIV accessory protein Vpr. Moreover the LV contained a cassette containing a loxP site followed by the neomycin resistant gene, inserted in the U3 region of the 3'LTR. Upon reverse tran‐ scription, the loxP-neo sequence would appear in both LTRs, thereby providing a substrate for recombination that could be catalyzed by the virion-associated Vpr-Cre. Upon this re‐ combination step, a circular product was produced that was on his turn inserted into the loxP site of the cell line, again catalyzed by virion-associated Vpr-Cre. Another example is provided by the group of Jiang et al. who demonstrated a selective inhibitory effect on the lens epithelial cells and not the retinal pigment epithelial cells (Jiang, Lu et al. 2011). There‐ fore they used an enhanced Cre/loxP system with a LV expressing Cre under the control of the lens-specific promoter LEP503 in combination with another LV that contained a stiffer sequence encoding eGFP with a functional polyadenylation signal between two loxP sites, followed by the HSV-TK gene, both under the control of the human phosphoglycerate kin‐ ase promoter. Expression of the downstream HSV-TK was activated by co-expression of Cre under the control of the lens-specific promoter LEP503. Although this technology allows site-specific tg insertion, there are only a limited amount of pseudo-loxP sites in the human genome and even none in the mouse genome, which makes this technique unusable for fun‐ damental research in laboratory animals. Furthermore, two recombination events are re‐ quired which has a major impact on its efficiency.

Finally, next to transcriptional activation of neighboring genes, also transcriptional shut off of the tg has been reported. This was due to chromatin remodeling at the site of insertion

Therefore additional strategies have been considered to reduce the side effects related to random insertion. The most straightforward strategy is to prevent integration of the proviral cargo by the use of IDLVs. These IDLVs are produced with a mutated integrase, which re‐ sults in prevention of proviral integration and generation of increased levels of circular vec‐ tor episomes within the infected cells. They appear to be safer with only a 0,1 to 2,3% chance that the episomal transcript gets integrated without a marked loss in effectiveness in terms of immune stimulatory potential of the IDLV-based vaccines (Vargas, Gusella et al. 2004; Philippe, Sarkis et al. 2006; Karwacz, Mukherjee et al. 2009; Wanisch and Yanez-Munoz 2009). However, as the lentiviral episomes lack replication signals, they are gradually lost by dilution in dividing cells and only stable in quiescent cells, which is undesirable for perma‐ nent correction of any genetic disorder. Furthermore also lower tg expression levels have been reported compared to integrative vectors (Bayer, Kantor et al. 2008). Therefore several alternative strategies have been brought forward to target the integrative process to a specif‐

In a first attempt, several groups tried to fuse a heterologous DNA binding domain directly to the integrase. Bushman et al. were the first to evaluate the activity of a hybrid, composed of the HIV-1 integrase and the lambda repressor. They reported on integration primarily near the lambda operator sites on the same face of the β-DNA helix (Bushman 1994). Later a model system was used were the integrase, derived from the avian sarcoma virus or HIV-1 respectively was fused to the *Escherichia coli* LexA repressor protein DNA binding domain (Katz, Merkel et al. 1996; Holmes-Son and Chow 2000). When this construct was packaged into the virion in *trans* either by replacing the original integrase gene or by cloning it adja‐ cent to the HIV-1 accessory protein Vpr, they observed that this enhanced the use of integra‐ tion sites adjacent to the *lex*A operators. In another study, the HIV-1 derived integrase was fused to a synthetic polydactyl zinc finger protein E2C, which binds specifically to a contig‐ uous 18 bp E2C recognition site (Tan, Dong et al. 2006). Although in all studies clearly a higher preference for integration near the target sequence of choice was observed, this also implicated reduced DNA-binding specificity of the fusion protein with associated decrease of integration frequency of about 80 percent compared to viruses containing wild type inte‐ grase. Furthermore this strategy is also limited by the difficulty to incorporate the fusion

Another strategy is targeting the integration away from genes using tethering domains linked to the host cell-encoded transcriptional co-activator lens epithelium-derived growth factor/p75 (LEDGF/p75), a cellular integrase binding protein. For example the LEDGF/p75 chromatin interaction-binding domain has been replaced with CBX1, which binds histone H3 di- or trimethylated on K9. Subsequently proviral integration was directed to pericentric heterochromatin and intergenic regions (Llano, Vanegas et al. 2006; Ferris, Wu et al. 2010; Gijsbers, Ronen et al. 2010; Silvers, Smith et al. 2010). As this requires engineering of a host cell protein, it is not feasible for clinical applications at the present stage (Izmiryan, Basma‐

and cessation of the therapeutic effect (Stein, Ott et al. 2010).

364 Gene Therapy - Tools and Potential Applications

protein into infectious virions (Michel, Yu et al. 2010).

ic 'safe' genomic site.

A recent strategy makes use of site-specific endonucleases to target the tg to neutral 'safe harbor' genome regions or stimulate the process of homologous recombination for gene re‐ pair (Fischer, Hacein-Bey-Abina et al. 2011). Endonucleases induce site-specific ds breaks that can be repaired by homology-directed repair, a form of homologous recombination that uses a copy of the genetic information from the broken DNA molecule. When the latter is provided by the same or another LV, this copy will be used to repair the ds break (Urnov, Miller et al. 2005; O'Driscoll and Jeggo 2006). The advantage of gene repair/correction is that both function and expression of the affected gene are restored while the risk associated with random vector integration is avoided. Besides the advantage of the reduced risk for inser‐ tional mutagenesis, this strategy is also used to target genes in order to knock them down or replace them with another gene by homologous recombination. The disadvantage is that the nuclease coding sequences are expressed for several days, which is not optimal for transla‐ tion to the clinic due to the background off-target generation of dsDNA breaks.

nomic targeting is relatively new for LV-based gene therapy, it opens a tremendous amount

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

367

**Figure 5.** Schematic representation of targeted genome modification by the Cre-loxP system (A) or by the zinc-finger nuclease complex (B). Cre recombinases recognize specific loxP sites in the genome, bind them en bring them togeth‐ er. Depending on their orientation this leads to excision (same orientation) or inversion (opposite orientation) of the sequence flanked by the two loxP sites(A). Two individual zinc finger molecules each recognize a 9 to 18 bp DNA se‐ quence using between three and six individual zinc finger repeats that bind the major groove of DNA. The DNA se‐ quences are non-palindromic DNA sites located respectively up -and downstream of the intended cleavage site, which is mostly about 5-7 bp long. If the zinc finger domains are perfectly specific for their intended target site then even a pair of three-finger ZFNs that recognize a total of 18 bp can theoretically target a single locus in a mammalian ge‐ nome. Next, the associated Fok1 nucleases dimerize and induce a double stranded break which can be restored by either non-homologous end-joining or homology-directed repair, which faithfully restores the original sequence by

copying it from the sister chromatid or using the homologous sequence provided by a LV(B).

of new possibilities.

One possibility is the use of the zinc finger nuclease strategy. For this, the Cys2His2 class of zinc finger DNA binding domains is engineered to recognize a DNA sequence of interest, fused to the nuclease domain of the FokI type II restriction endonuclease to yield a highly specific zinc finger (Figure 5 B, adapted from http://biol1020-2011-2.blogspot.be/2011/09/ zinc-finger-nucleases-zfn-emerging.html) (Kim, Cha et al. 1996; Pabo, Peisach et al. 2001). When two different zinc fingers are designed to bind the same sequence of interest in the opposite orientation, this will allow dimerization of the FokI domains which leads to a zinc finger induced dsDNA break (Bitinaite, Wah et al. 1998). Various strategies have been devel‐ oped to engineer the Cys2His2 zinc fingers in order to bind a specific sequence either by modular assembly or by selection strategies using phage display or a cellular selection sys‐ tem. Naldini et al. evaluated the use of zinc finger nucleases in combination with an IDLV for gene editing. Therefore they co-transduced several cell lines with three different IDLVs, one encoding the donor sequence and two encoding the two zinc fingers (Lombardo, Geno‐ vese et al. 2007). A few years later they also used this strategy to assess zinc finger specificity genome-wide by comprehensively mapping the locations of the IDLV integration sites in cells co-transduced with GFP and zinc finger encoding LVs (Gabriel, Lombardo et al. 2011). They observed a very high efficiency and specificity, yet a measurable rate of vector integra‐ tion at unidentified sites occurred with this approach, which is the sum of zinc finger medi‐ ated and background levels of IDLV integration. Moreover co-transduction with three different LVs may be a rate-limiting step in this system. Therefore the use of a single con‐ struct to express the zinc fingers and deliver the donor tg must be evaluated, especially for less permissive cells such as hematopoietic progenitors.

Another way to target the proviral genome is by the provision of a vector-associated mega‐ nuclease encoded by a separate vector or supplied as a protein within the viral particle. (Izmiryan, Basmaciogullari et al. 2011). For the latter, Ismiryan et al. fused the prototypic meganuclease I-SceI from yeast to Vpr. This avoided the potentially toxic sustained expres‐ sion of the introduced endonuclease. IDLVs encoding the donor sequence and containing the meganuclease-SceI fusion construct were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. They reported a two-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector and therefore improved both the safety and efficacy of this LV-based gene targeting system. In conclusion, although the field of ge‐ nomic targeting is relatively new for LV-based gene therapy, it opens a tremendous amount of new possibilities.

uses a copy of the genetic information from the broken DNA molecule. When the latter is provided by the same or another LV, this copy will be used to repair the ds break (Urnov, Miller et al. 2005; O'Driscoll and Jeggo 2006). The advantage of gene repair/correction is that both function and expression of the affected gene are restored while the risk associated with random vector integration is avoided. Besides the advantage of the reduced risk for inser‐ tional mutagenesis, this strategy is also used to target genes in order to knock them down or replace them with another gene by homologous recombination. The disadvantage is that the nuclease coding sequences are expressed for several days, which is not optimal for transla‐

One possibility is the use of the zinc finger nuclease strategy. For this, the Cys2His2 class of zinc finger DNA binding domains is engineered to recognize a DNA sequence of interest, fused to the nuclease domain of the FokI type II restriction endonuclease to yield a highly specific zinc finger (Figure 5 B, adapted from http://biol1020-2011-2.blogspot.be/2011/09/ zinc-finger-nucleases-zfn-emerging.html) (Kim, Cha et al. 1996; Pabo, Peisach et al. 2001). When two different zinc fingers are designed to bind the same sequence of interest in the opposite orientation, this will allow dimerization of the FokI domains which leads to a zinc finger induced dsDNA break (Bitinaite, Wah et al. 1998). Various strategies have been devel‐ oped to engineer the Cys2His2 zinc fingers in order to bind a specific sequence either by modular assembly or by selection strategies using phage display or a cellular selection sys‐ tem. Naldini et al. evaluated the use of zinc finger nucleases in combination with an IDLV for gene editing. Therefore they co-transduced several cell lines with three different IDLVs, one encoding the donor sequence and two encoding the two zinc fingers (Lombardo, Geno‐ vese et al. 2007). A few years later they also used this strategy to assess zinc finger specificity genome-wide by comprehensively mapping the locations of the IDLV integration sites in cells co-transduced with GFP and zinc finger encoding LVs (Gabriel, Lombardo et al. 2011). They observed a very high efficiency and specificity, yet a measurable rate of vector integra‐ tion at unidentified sites occurred with this approach, which is the sum of zinc finger medi‐ ated and background levels of IDLV integration. Moreover co-transduction with three different LVs may be a rate-limiting step in this system. Therefore the use of a single con‐ struct to express the zinc fingers and deliver the donor tg must be evaluated, especially for

Another way to target the proviral genome is by the provision of a vector-associated mega‐ nuclease encoded by a separate vector or supplied as a protein within the viral particle. (Izmiryan, Basmaciogullari et al. 2011). For the latter, Ismiryan et al. fused the prototypic meganuclease I-SceI from yeast to Vpr. This avoided the potentially toxic sustained expres‐ sion of the introduced endonuclease. IDLVs encoding the donor sequence and containing the meganuclease-SceI fusion construct were tested in reporter cells in which targeting events were scored by the repair of a puromycin resistance gene. They reported a two-fold higher frequency of the expected recombination event when the nuclease was delivered as a protein rather than encoded by a separate vector and therefore improved both the safety and efficacy of this LV-based gene targeting system. In conclusion, although the field of ge‐

tion to the clinic due to the background off-target generation of dsDNA breaks.

366 Gene Therapy - Tools and Potential Applications

less permissive cells such as hematopoietic progenitors.

**Figure 5.** Schematic representation of targeted genome modification by the Cre-loxP system (A) or by the zinc-finger nuclease complex (B). Cre recombinases recognize specific loxP sites in the genome, bind them en bring them togeth‐ er. Depending on their orientation this leads to excision (same orientation) or inversion (opposite orientation) of the sequence flanked by the two loxP sites(A). Two individual zinc finger molecules each recognize a 9 to 18 bp DNA se‐ quence using between three and six individual zinc finger repeats that bind the major groove of DNA. The DNA se‐ quences are non-palindromic DNA sites located respectively up -and downstream of the intended cleavage site, which is mostly about 5-7 bp long. If the zinc finger domains are perfectly specific for their intended target site then even a pair of three-finger ZFNs that recognize a total of 18 bp can theoretically target a single locus in a mammalian ge‐ nome. Next, the associated Fok1 nucleases dimerize and induce a double stranded break which can be restored by either non-homologous end-joining or homology-directed repair, which faithfully restores the original sequence by copying it from the sister chromatid or using the homologous sequence provided by a LV(B).

#### **6. Concluding remarks**

LVs have proven to be efficient vehicles to deliver one or more tgs to any cell type of choice, which has led to a promising list of therapeutic applications. As the demand for experimen‐ tation in gene delivery to specific cell types increases, technologies that precisely target LVbased gene expression will become more important for research and clinical applications. Four main groups of strategies with their own possibilities as well as difficulties have been developed so far. Self-evidently, further optimization and fine-tuning of these strategies is a necessity to fulfill the expectations for targeted LV delivery *in vivo*. In addition to extra opti‐ mization steps, combinations of two or more of these strategies can also lead to an overall more selective, efficient and most importantly, safe LV system. Several attempts to combine the different strategies have been reported (Brown, Cantore et al. 2007; Pariente, Morizono et al. 2007; Escors and Breckpot 2011). Pariente et al. for example, reported on a LV that was transductionally targeted to prostate cancer bone metastases by a modified sindbis virus en‐ velope that interacts with PSCA and transcriptionally targeted with a prostate cell specific promoter. This dual-targeted LV enhanced specificity to prostate cancer bone metastases af‐ ter systemic delivery with respect to individual transcriptional or transductional targeting. As the developed targeting strategies already resulted in a major step forward for LV-based gene therapy, their potential will most likely be more exploited in the future, paving the way towards an all-embracing LV-based tg vehicle for the gene therapeutic field.

[3] Annoni, A., M. Battaglia, et al. (2007). "The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

369

[4] Annoni, A., B. D. Brown, et al. (2009). "In vivo delivery of a microRNA-regulated transgene induces antigen-specific regulatory T cells and promotes immunologic tol‐

[5] Azzouz, M., G. S. Ralph, et al. (2004). "VEGF delivery with retrogradely transported lentivector prolongs survival in a mouse ALS model." Nature 429(6990): 413-7.

[6] Bahi, A., F. Boyer, et al. (2004). "CD81-induced behavioural changes during chronic cocaine administration: in vivo gene delivery with regulatable lentivirus." Eur J Neu‐

[7] Barde, I., M. A. Zanta-Boussif, et al. (2006). "Efficient control of gene expression in the hematopoietic system using a single Tet-on inducible lentiviral vector." Mol Ther

[8] Barrilleaux, B. and P. Knoepfler (2011). "Transduction of human cells with polymercomplexed ecotropic lentivirus for enhanced biosafety." J Vis Exp 2011(53).

[9] Bartel, D. P. (2004). "MicroRNAs: genomics, biogenesis, mechanism, and function."

[10] Bauer, G., M. A. Dao, et al. (2008). "In vivo biosafety model to assess the risk of ad‐ verse events from retroviral and lentiviral vectors." Mol Ther 16(7): 1308-15.

[11] Baup, D., L. Fraga, et al. (2010). "Variegation and silencing in a lentiviral-based mur‐

[12] Bayer, M., B. Kantor, et al. (2008). "A large U3 deletion causes increased in vivo ex‐ pression from a nonintegrating lentiviral vector." Mol Ther 16(12): 1968-76.

[13] Benabdellah, K., M. Cobo, et al. (2011). "Development of an all-in-one lentiviral vec‐ tor system based on the original TetR for the easy generation of Tet-ON cell lines."

[14] Benzekhroufa, K., B. H. Liu, et al. (2009). "Targeting central serotonergic neurons with lentiviral vectors based on a transcriptional amplification strategy." Gene Ther

[15] Beyer, W. R., M. Westphal, et al. (2002). "Oncoretrovirus and lentivirus vectors pseu‐ dotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concen‐

[16] Bitinaite, J., D. A. Wah, et al. (1998). "FokI dimerization is required for DNA cleav‐

[17] Blechacz, B. and S. J. Russell (2008). "Measles virus as an oncolytic vector platform."

ine transgenic model." Transgenic Res 19(3): 399-414.

tration, and broad host range." J Virol 76(3): 1488-95.

age." Proc Natl Acad Sci U S A 95(18): 10570-5.

not by CD4+CD25+ regulatory T cells." Blood 110(6): 1788-96.

erance." Blood 114(25): 5152-61.

rosci 19(6): 1621-33.

Cell 116(2): 281-97.

PLoS One 6(8): e23734.

Curr Gene Ther 8(3): 162-75.

16(5): 681-8.

13(2): 382-90.

#### **Author details**

Cleo Goyvaerts1 , Therese Liechtenstein2 , Christopher Bricogne2 , David Escors2 and Karine Breckpot1

1 Laboratory of Molecular and Cellular Therapy, Department of Immunology-Physiology, Vrije Universiteit Brussel, Jette, Belgium

2 Division of Infection and Immunity, Rayne Institute, University College London, London, UK

#### **References**


[3] Annoni, A., M. Battaglia, et al. (2007). "The immune response to lentiviral-delivered transgene is modulated in vivo by transgene-expressing antigen-presenting cells but not by CD4+CD25+ regulatory T cells." Blood 110(6): 1788-96.

**6. Concluding remarks**

368 Gene Therapy - Tools and Potential Applications

**Author details**

Cleo Goyvaerts1

Karine Breckpot1

**References**

UK

LVs have proven to be efficient vehicles to deliver one or more tgs to any cell type of choice, which has led to a promising list of therapeutic applications. As the demand for experimen‐ tation in gene delivery to specific cell types increases, technologies that precisely target LVbased gene expression will become more important for research and clinical applications. Four main groups of strategies with their own possibilities as well as difficulties have been developed so far. Self-evidently, further optimization and fine-tuning of these strategies is a necessity to fulfill the expectations for targeted LV delivery *in vivo*. In addition to extra opti‐ mization steps, combinations of two or more of these strategies can also lead to an overall more selective, efficient and most importantly, safe LV system. Several attempts to combine the different strategies have been reported (Brown, Cantore et al. 2007; Pariente, Morizono et al. 2007; Escors and Breckpot 2011). Pariente et al. for example, reported on a LV that was transductionally targeted to prostate cancer bone metastases by a modified sindbis virus en‐ velope that interacts with PSCA and transcriptionally targeted with a prostate cell specific promoter. This dual-targeted LV enhanced specificity to prostate cancer bone metastases af‐ ter systemic delivery with respect to individual transcriptional or transductional targeting. As the developed targeting strategies already resulted in a major step forward for LV-based gene therapy, their potential will most likely be more exploited in the future, paving the

way towards an all-embracing LV-based tg vehicle for the gene therapeutic field.

, Christopher Bricogne2

1 Laboratory of Molecular and Cellular Therapy, Department of Immunology-Physiology,

2 Division of Infection and Immunity, Rayne Institute, University College London, London,

[1] Abruzzese, R. V., D. Godin, et al. (2000). "Ligand-dependent regulation of vascular endothelial growth factor and erythropoietin expression by a plasmid-based autoin‐

[2] Adriani, W., F. Boyer, et al. (2010). "Social withdrawal and gambling-like profile after lentiviral manipulation of DAT expression in the rat accumbens." Int J Neuropsycho‐

, David Escors2

and

, Therese Liechtenstein2

ducible GeneSwitch system." Mol Ther 2(3): 276-87.

Vrije Universiteit Brussel, Jette, Belgium

pharmacol 13(10): 1329-42.


[18] Blesch, A., J. Conner, et al. (2005). "Regulated lentiviral NGF gene transfer controls rescue of medial septal cholinergic neurons." Mol Ther 11(6): 916-25.

[33] Bushman, F. D. (1994). "Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences." Proc Natl Acad Sci U S A 91(20):

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

371

[34] Calame, M., M. Cachafeiro, et al. (2011). "Retinal degeneration progression changes

[35] Canivet, M., A. D. Hoffman, et al. (1990). "Replication of HIV-1 in a wide variety of animal cells following phenotypic mixing with murine retroviruses." Virology 178(2):

[36] Cao, J., K. Sodhi, et al. (2011). "Lentiviral-human heme oxygenase targeting endothe‐ lium improved vascular function in angiotensin II animal model of hypertension."

[37] Cao, O., C. Furlan-Freguia, et al. (2007). "Emerging role of regulatory T cells in gene

[38] Cartier, N., S. Hacein-Bey-Abina, et al. (2012). "Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy." Methods Enzymol 507: 187-98.

[39] Cartier, N., S. Hacein-Bey-Abina, et al. (2009). "Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy." Science 326(5954):

[40] Cattoglio, C., G. Facchini, et al. (2007). "Hot spots of retroviral integration in human

[41] Cavazzana-Calvo, M., S. Hacein-Bey, et al. (2000). "Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease." Science 288(5466): 669-72.

[42] Cavazzana-Calvo, M., E. Payen, et al. (2010). "Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassaemia." Nature

[43] Cesana, D., J. Sgualdino, et al. (2012). "Whole transcriptome characterization of aber‐ rant splicing events induced by lentiviral vector integrations." J Clin Invest 122(5):

[44] Chadwick, M. P., F. J. Morling, et al. (1999). "Modification of retroviral tropism by

[45] Chandrashekran, A., M. Y. Gordon, et al. (2004). "Targeted retroviral transduction of c-kit+ hematopoietic cells using novel ligand display technology." Blood 104(9):

[46] Chowdhury, S., K. A. Chester, et al. (2004). "Efficient retroviral vector targeting of

[47] Colin, A., M. Faideau, et al. (2009). "Engineered lentiviral vector targeting astrocytes

carcinoembryonic antigen-positive tumors." Mol Ther 9(1): 85-92.

lentiviral vector cell targeting in the retina." PLoS One 6(8): e23782.

9233-7.

543-51.

818-23.

467(7313): 318-22.

1667-76.

2697-703.

in vivo." Glia 57(6): 667-79.

Hum Gene Ther 22(3): 271-82.

transfer." Curr Gene Ther 7(5): 381-90.

CD34+ hematopoietic cells." Blood 110(6): 1770-8.

display of IGF-I." J Mol Biol 285(2): 485-94.


[33] Bushman, F. D. (1994). "Tethering human immunodeficiency virus 1 integrase to a DNA site directs integration to nearby sequences." Proc Natl Acad Sci U S A 91(20): 9233-7.

[18] Blesch, A., J. Conner, et al. (2005). "Regulated lentiviral NGF gene transfer controls

[19] Blomer, U., L. Naldini, et al. (1997). "Highly efficient and sustained gene transfer in

[20] Boerger, A. L., S. Snitkovsky, et al. (1999). "Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types." Proc Natl Acad Sci

[21] Bonci, D., V. Coppola, et al. (2008). "The miR-15a-miR-16-1 cluster controls prostate cancer by targeting multiple oncogenic activities." Nat Med 14(11): 1271-7.

[22] Breckpot, K., P. Emeagi, et al. (2007). "Activation of immature monocyte-derived dendritic cells after transduction with high doses of lentiviral vectors." Hum Gene

[23] Breckpot, K., P. U. Emeagi, et al. (2008). "Lentiviral vectors for anti-tumor immuno‐

[24] Breckpot, K., D. Escors, et al. (2010). "HIV-1 lentiviral vector immunogenicity is mediated by Toll-like receptor 3 (TLR3) and TLR7." J Virol 84(11): 5627-36.

[25] Brown, B. D., A. Cantore, et al. (2007). "A microRNA-regulated lentiviral vector me‐

[26] Brown, B. D., B. Gentner, et al. (2007). "Endogenous microRNA can be broadly ex‐ ploited to regulate transgene expression according to tissue, lineage and differentia‐

[27] Brown, B. D. and D. Lillicrap (2002). "Dangerous liaisons: the role of "danger" signals

[28] Brown, B. D. and L. Naldini (2009). "Exploiting and antagonizing microRNA regula‐ tion for therapeutic and experimental applications." Nat Rev Genet 10(8): 578-85.

[29] Brown, B. D., M. A. Venneri, et al. (2006). "Endogenous microRNA regulation sup‐ presses transgene expression in hematopoietic lineages and enables stable gene

[30] Buchholz, C. J., L. J. Duerner, et al. (2008). "Retroviral display and high throughput

[31] Buchholz, C. J., M. D. Muhlebach, et al. (2009). "Lentiviral vectors with measles virus glycoproteins - dream team for gene transfer?" Trends Biotechnol 27(5): 259-65.

[32] Burns, J. C., T. Friedmann, et al. (1993). "Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells." Proc Natl Acad Sci U S A 90(17):

diates stable correction of hemophilia B mice." Blood 110(13): 4144-52.

in the immune response to gene therapy." Blood 100(4): 1133-40.

screening." Comb Chem High Throughput Screen 11(2): 99-110.

rescue of medial septal cholinergic neurons." Mol Ther 11(6): 916-25.

adult neurons with a lentivirus vector." J Virol 71(9): 6641-9.

U S A 96(17): 9867-72.

370 Gene Therapy - Tools and Potential Applications

Ther 18(6): 536-46.

therapy." Curr Gene Ther 8(6): 438-48.

tion state." Nat Biotechnol 25(12): 1457-67.

transfer." Nat Med 12(5): 585-91.

8033-7.


[48] Cronin, J., X. Y. Zhang, et al. (2005). "Altering the tropism of lentiviral vectors through pseudotyping." Curr Gene Ther 5(4): 387-98.

[62] Dylla, D. E., L. Xie, et al. (2011). "Altering alpha-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism." Genet Vaccines

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

373

[63] Earp, L. J., S. E. Delos, et al. (2005). "The many mechanisms of viral membrane fusion

[64] Ebert, M. S., J. R. Neilson, et al. (2007). "MicroRNA sponges: competitive inhibitors of

[65] Efrat, S., D. Fusco-DeMane, et al. (1995). "Conditional transformation of a pancreatic beta-cell line derived from transgenic mice expressing a tetracycline-regulated onco‐

[66] Emi, N., T. Friedmann, et al. (1991). "Pseudotype formation of murine leukemia virus

[67] Enkirch, T., S. Kneissl, et al. (2012). "Targeted lentiviral vectors pseudotyped with the

[68] Escors, D. and K. Breckpot (2010). "Lentiviral vectors in gene therapy: their current status and future potential." Arch Immunol Ther Exp (Warsz) 58(2): 107-19.

[69] Escors, D. and K. Breckpot (2011). "Lentiviral vectors in gene therapy: their current status and future potential." Arch Immunol Ther Exp (Warsz) 58(2): 107-19.

[70] Fehse, B. and I. Roeder (2008). "Insertional mutagenesis and clonal dominance: bio‐

[71] Ferris, A. L., X. Wu, et al. (2010). "Lens epithelium-derived growth factor fusion pro‐ teins redirect HIV-1 DNA integration." Proc Natl Acad Sci U S A 107(7): 3135-40. [72] Fielding, A. K., M. Maurice, et al. (1998). "Inverse targeting of retroviral vectors: se‐ lective gene transfer in a mixed population of hematopoietic and nonhematopoietic

[73] Fischer, A., S. Hacein-Bey-Abina, et al. (2011). "Gene therapy for primary adaptive

[74] Frecha, C., C. Costa, et al. (2012). "A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure

[75] Frecha, C., F. Fusil, et al. (2011). "In vivo gene delivery into hCD34+ cells in a human‐

[76] Frecha, C., C. Levy, et al. (2010). "Advances in the field of lentivector-based transduc‐

[77] Frecha, C., C. Levy, et al. (2011). "Measles virus glycoprotein-pseudotyped lentiviral vector-mediated gene transfer into quiescent lymphocytes requires binding to both

tion of T and B lymphocytes for gene therapy." Mol Ther 18(10): 1748-57.

with the G protein of vesicular stomatitis virus." J Virol 65(3): 1202-7.

logical and statistical considerations." Gene Ther 15(2): 143-53.

immune deficiencies." J Allergy Clin Immunol 127(6): 1356-9.

ized mouse model." Methods Mol Biol 737: 367-90.

SLAM and CD46 entry receptors." J Virol 85(12): 5975-85.

syndrome and in vivo in humanized mice." Blood 119(5): 1139-50.

proteins." Curr Top Microbiol Immunol 285: 25-66.

gene." Proc Natl Acad Sci U S A 92(8): 3576-80.

Tupaia paramyxovirus glycoproteins." Gene Ther.

cells." Blood 91(5): 1802-9.

small RNAs in mammalian cells." Nat Methods 4(9): 721-6.

Ther 9: 8.


[62] Dylla, D. E., L. Xie, et al. (2011). "Altering alpha-dystroglycan receptor affinity of LCMV pseudotyped lentivirus yields unique cell and tissue tropism." Genet Vaccines Ther 9: 8.

[48] Cronin, J., X. Y. Zhang, et al. (2005). "Altering the tropism of lentiviral vectors

[49] Croyle, M. A., S. M. Callahan, et al. (2004). "PEGylation of a vesicular stomatitis virus G pseudotyped lentivirus vector prevents inactivation in serum." J Virol 78(2):

[50] Cui, Y., J. Golob, et al. (2002). "Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/

[51] De Palma, M., E. Montini, et al. (2005). "Promoter trapping reveals significant differ‐ ences in integration site selection between MLV and HIV vectors in primary hemato‐

[52] De Palma, M., M. A. Venneri, et al. (2003). "In vivo targeting of tumor endothelial cells by systemic delivery of lentiviral vectors." Hum Gene Ther 14(12): 1193-206.

[53] Delenda, C. (2004). "Lentiviral vectors: optimization of packaging, transduction and

[54] DePolo, N. J., J. D. Reed, et al. (2000). "VSV-G pseudotyped lentiviral vector particles produced in human cells are inactivated by human serum." Mol Ther 2(3): 218-22.

[55] Deuschle, U., R. A. Hipskind, et al. (1990). "RNA polymerase II transcription blocked

[56] Di Domenico, C., D. Di Napoli, et al. (2006). "Limited transgene immune response and long-term expression of human alpha-L-iduronidase in young adult mice with mucopolysaccharidosis type I by liver-directed gene therapy." Hum Gene Ther

[57] Di Nunzio, F., G. Maruggi, et al. (2008). "Correction of laminin-5 deficiency in human epidermal stem cells by transcriptionally targeted lentiviral vectors." Mol Ther

[58] Dreja, H. and M. Piechaczyk (2006). "The effects of N-terminal insertion into VSV-G

[59] Dresch, C., S. L. Edelmann, et al. (2008). "Lentiviral-mediated transcriptional target‐ ing of dendritic cells for induction of T cell tolerance in vivo." J Immunol 181(7):

[60] Dropulic, B. (2011). "Lentiviral vectors: their molecular design, safety, and use in lab‐

[61] Dull, T., R. Zufferey, et al. (1998). "A third-generation lentivirus vector with a condi‐

oratory and preclinical research." Hum Gene Ther 22(6): 649-57.

tional packaging system." J Virol 72(11): 8463-71.

through pseudotyping." Curr Gene Ther 5(4): 387-98.

progenitor cells." Blood 99(2): 399-408.

poietic cells." Blood 105(6): 2307-15.

gene expression." J Gene Med 6 Suppl 1: S125-38.

by Escherichia coli lac repressor." Science 248(4954): 480-3.

912-21.

372 Gene Therapy - Tools and Potential Applications

17(11): 1112-21.

16(12): 1977-85.

4495-506.

of an scFv peptide." Virol J 3: 69.


[78] Friedrich, R. I., K. Nopora, et al. (2012). "Transcriptional targeting of B cells with viral vectors." European Journal of Cell Biology 91(1): 86-96.

[93] Gilham, D. E., A. L. M. Lie, et al. (2010). "Cytokine stimulation and the choice of pro‐ moter are critical factors for the efficient transduction of mouse T cells with HIV-1

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

375

[94] Ginn, S. L., S. H. Liao, et al. (2010). "Lymphomagenesis in SCID-X1 mice following lentivirus-mediated phenotype correction independent of insertional mutagenesis

[95] Gossen, M. and H. Bujard (1992). "Tight control of gene expression in mammalian cells by tetracycline-responsive promoters." Proc Natl Acad Sci U S A 89(12): 5547-51.

[96] Goyvaerts, C., K. De Groeve, et al. (2012). "Development of the Nanobody display technology to target lentiviral vectors to antigen-presenting cells." Gene Ther. [97] Guibinga, G. H., F. L. Hall, et al. (2004). "Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus as‐

[98] Hamers-Casterman, C., T. Atarhouch, et al. (1993). "Naturally occurring antibodies

[99] Hanawa, H., D. A. Persons, et al. (2002). "High-level erythroid lineage-directed gene expression using globin gene regulatory elements after lentiviral vector-mediated gene transfer into primitive human and murine hematopoietic cells." Hum Gene

[100] Hawley, T. S., A. Z. Fong, et al. (1998). "Leukemic predisposition of mice transplant‐ ed with gene-modified hematopoietic precursors expressing flt3 ligand." Blood 92(6):

[101] Heckl, D., D. C. Wicke, et al. (2011). "Lentiviral gene transfer regenerates hemato‐ poietic stem cells in a mouse model for Mpl-deficient aplastic anemia." Blood 117(14):

[102] Herold, M. J., J. van den Brandt, et al. (2008). "Inducible and reversible gene silencing by stable integration of an shRNA-encoding lentivirus in transgenic rats." Proc Natl

[103] Hioki, H., H. Kameda, et al. (2007). "Efficient gene transduction of neurons by lentivi‐ rus with enhanced neuron-specific promoters." Gene Ther 14(11): 872-82.

[104] Hioki, H., E. Kuramoto, et al. (2009). "High-level transgene expression in neurons by

[105] Hofmann, H., K. Hattermann, et al. (2004). "S protein of severe acute respiratory syn‐ drome-associated coronavirus mediates entry into hepatoma cell lines and is targeted

[106] Holmes-Son, M. L. and S. A. Chow (2000). "Integrase-lexA fusion proteins incorpo‐ rated into human immunodeficiency virus type 1 that contains a catalytically inactive

integrase gene are functional to mediate integration." J Virol 74(24): 11548-56.

by neutralizing antibodies in infected patients." J Virol 78(12): 6134-42.

lentivirus with Tet-Off system." Neurosci Res 63(2): 149-54.

vectors." J Gene Med 12(2): 129-36.

sembly phenotype." Mol Ther 9(1): 76-84.

Ther 13(17): 2007-16.

Acad Sci U S A 105(47): 18507-12.

2003-11.

3737-47.

devoid of light chains." Nature 363(6428): 446-8.

and gammac overexpression." Mol Ther 18(5): 965-76.


[93] Gilham, D. E., A. L. M. Lie, et al. (2010). "Cytokine stimulation and the choice of pro‐ moter are critical factors for the efficient transduction of mouse T cells with HIV-1 vectors." J Gene Med 12(2): 129-36.

[78] Friedrich, R. I., K. Nopora, et al. (2012). "Transcriptional targeting of B cells with viral

[79] Froelich, S., L. Ziegler, et al. (2009). "Targeted gene delivery to CD117-expressing cells in vivo with lentiviral vectors co-displaying stem cell factor and a fusogenic

[80] Funke, S., A. Maisner, et al. (2008). "Targeted cell entry of lentiviral vectors." Mol

[81] Funke, S., I. C. Schneider, et al. (2009). "Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity." Gene Ther

[82] Furth, P. A., L. St Onge, et al. (1994). "Temporal control of gene expression in trans‐ genic mice by a tetracycline-responsive promoter." Proc Natl Acad Sci U S A 91(20):

[83] Fussenegger, M., R. P. Morris, et al. (2000). "Streptogramin-based gene regulation

[84] Gabriel, R., A. Lombardo, et al. (2011). "An unbiased genome-wide analysis of zinc-

[85] Gainkam, L. O., L. Huang, et al. (2008). "Comparison of the biodistribution and tu‐ mor targeting of two 99mTc-labeled anti-EGFR nanobodies in mice, using pinhole

[86] Galimi, F., E. Saez, et al. (2005). "Development of ecdysone-regulated lentiviral vec‐

[87] Gascon, S., J. A. Paez-Gomez, et al. (2008). "Dual-promoter lentiviral vectors for con‐ stitutive and regulated gene expression in neurons." J Neurosci Methods 168(1):

[88] Gaspar, H. B., K. L. Parsley, et al. (2004). "Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector." Lancet

[89] Gennari, F., L. Lopes, et al. (2009). "Single-chain antibodies that target lentiviral vec‐ tors to MHC class II on antigen-presenting cells." Hum Gene Ther 20(6): 554-62. [90] Gentner, B., G. Schira, et al. (2009). "Stable knockdown of microRNA in vivo by lenti‐

[91] Gentner, B., I. Visigalli, et al. (2010). "Identification of hematopoietic stem cell-specific miRNAs enables gene therapy of globoid cell leukodystrophy." Sci Transl Med 2(58):

[92] Gijsbers, R., K. Ronen, et al. (2010). "LEDGF hybrids efficiently retarget lentiviral in‐

systems for mammalian cells." Nat Biotechnol 18(11): 1203-8.

finger nuclease specificity." Nat Biotechnol 29(9): 816-23.

SPECT/micro-CT." J Nucl Med 49(5): 788-95.

viral vectors." Nat Methods 6(1): 63-6.

tegration into heterochromatin." Mol Ther 18(3): 552-60.

tors." Mol Ther 11(1): 142-8.

vectors." European Journal of Cell Biology 91(1): 86-96.

molecule." Biotechnol Bioeng 104(1): 206-15.

Ther 16(8): 1427-36.

374 Gene Therapy - Tools and Potential Applications

16(5): 700-5.

9302-6.

104-12.

58ra84.

364(9452): 2181-7.


[107] Hsieh, Y. J., F. D. Chen, et al. (2011). "The EIIAPA Chimeric Promoter for Tumor Spe‐ cific Gene Therapy of Hepatoma." Mol Imaging Biol.

[121] Kimura, T., R. C. Koya, et al. (2007). "Lentiviral vectors with CMV or MHCII promot‐ ers administered in vivo: immune reactivity versus persistence of expression." Mol

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

377

[122] Kobinger, G. P., S. Deng, et al. (2004). "Transduction of human islets with pseudotyp‐

[123] Kobinger, G. P., D. J. Weiner, et al. (2001). "Filovirus-pseudotyped lentiviral vector can efficiently and stably transduce airway epithelia in vivo." Nat Biotechnol 19(3):

[124] Kos, C. H. (2004). "Cre/loxP system for generating tissue-specific knockout mouse

[125] Kueng, H. J., V. M. Leb, et al. (2007). "General strategy for decoration of enveloped viruses with functionally active lipid-modified cytokines." J Virol 81(16): 8666-76.

[126] Kumar, M. S., S. J. Erkeland, et al. (2008). "Suppression of non-small cell lung tumor development by the let-7 microRNA family." Proc Natl Acad Sci U S A 105(10):

[127] Kuroda, H., R. H. Kutner, et al. (2008). "A comparative analysis of constitutive and cell-specific promoters in the adult mouse hippocampus using lentivirus vector-

[128] Lachmann, N., J. Jagielska, et al. (2011). "MicroRNA-150-regulated vectors allow lym‐ phocyte-sparing transgene expression in hematopoietic gene therapy." Gene Ther.

[129] Lai, E. C. (2002). "Micro RNAs are complementary to 3' UTR sequence motifs that mediate negative post-transcriptional regulation." Nat Genet 30(4): 363-4.

[130] Lakso, M., B. Sauer, et al. (1992). "Targeted oncogene activation by site-specific re‐ combination in transgenic mice." Proc Natl Acad Sci U S A 89(14): 6232-6.

[131] Latta-Mahieu, M., M. Rolland, et al. (2002). "Gene transfer of a chimeric trans-activa‐ tor is immunogenic and results in short-lived transgene expression." Hum Gene Ther

[132] Lavillette, D., S. J. Russell, et al. (2001). "Retargeting gene delivery using surface-engi‐

[133] Lee, C. J., X. Fan, et al. (2011). "Promoter-specific lentivectors for long-term, cardiac-

[134] Lee, C. L., J. Dang, et al. (2011). "Engineered lentiviral vectors pseudotyped with a CD4 receptor and a fusogenic protein can target cells expressing HIV-1 envelope pro‐

[135] Lei, Y., K. I. Joo, et al. (2009). "Engineering fusogenic molecules to achieve targeted

neered retroviral vector particles." Curr Opin Biotechnol 12(5): 461-6.

directed therapy of Fabry disease." J Cardiol 57(1): 115-22.

transduction of enveloped lentiviral vectors." J Biol Eng 3: 8.

ed lentiviral vectors." Hum Gene Ther 15(2): 211-9.

mediated gene transfer." J Gene Med 10(11): 1163-75.

models." Nutr Rev 62(6 Pt 1): 243-6.

Ther 15(7): 1390-9.

225-30.

3903-8.

13(13): 1611-20.

teins." Virus Res 160(1-2): 340-50.


[121] Kimura, T., R. C. Koya, et al. (2007). "Lentiviral vectors with CMV or MHCII promot‐ ers administered in vivo: immune reactivity versus persistence of expression." Mol Ther 15(7): 1390-9.

[107] Hsieh, Y. J., F. D. Chen, et al. (2011). "The EIIAPA Chimeric Promoter for Tumor Spe‐

[108] Hu, B., B. Dai, et al. (2010). "Vaccines delivered by integration-deficient lentiviral vec‐ tors targeting dendritic cells induces strong antigen-specific immunity." Vaccine

[109] Izmiryan, A., S. Basmaciogullari, et al. (2011). "Efficient gene targeting mediated by a lentiviral vector-associated meganuclease." Nucleic Acids Res 39(17): 7610-9.

[110] Jakobsson, J., C. Ericson, et al. (2003). "Targeted transgene expression in rat brain us‐

[111] Jiang, Y. X., Y. Lu, et al. (2011). "Using HSV-TK/GCV suicide gene therapy to inhibit lens epithelial cell proliferation for treatment of posterior capsular opacification."

[112] Kang, Y., C. S. Stein, et al. (2002). "In vivo gene transfer using a nonprimate lentiviral vector pseudotyped with Ross River Virus glycoproteins." J Virol 76(18): 9378-88.

[113] Karavanas, G., M. Marin, et al. (2002). "The insertion of an anti-MHC I ScFv into the N-terminus of an ecotropic MLV glycoprotein does not alter its fusiogenic potential

[114] Karwacz, K., S. Mukherjee, et al. (2009). "Nonintegrating lentivector vaccines stimu‐ late prolonged T-cell and antibody responses and are effective in tumor therapy." J

[115] Katane, M., E. Takao, et al. (2002). "Factors affecting the direct targeting of murine leukemia virus vectors containing peptide ligands in the envelope protein." EMBO

[116] Katz, R. A., G. Merkel, et al. (1996). "Targeting of retroviral integrase by fusion to a heterologous DNA binding domain: in vitro activities and incorporation of a fusion

[117] Kelly, E. J., E. M. Hadac, et al. (2008). "Engineering microRNA responsiveness to de‐

[118] Kerns, H. M., B. Y. Ryu, et al. (2010). "B cell-specific lentiviral gene therapy leads to sustained B-cell functional recovery in a murine model of X-linked agammaglobuli‐

[119] Kim, S., G. J. Kim, et al. (2007). "Efficiency of the elongation factor-1alpha promoter in mammalian embryonic stem cells using lentiviral gene delivery systems." Stem

[120] Kim, Y. G., J. Cha, et al. (1996). "Hybrid restriction enzymes: zinc finger fusions to

Fok I cleavage domain." Proc Natl Acad Sci U S A 93(3): 1156-60.

cific Gene Therapy of Hepatoma." Mol Imaging Biol.

ing lentiviral vectors." J Neurosci Res 73(6): 876-85.

on murine cells." Virus Res 83(1-2): 57-69.

protein into viral particles." Virology 217(1): 178-90.

crease virus pathogenicity." Nat Med 14(11): 1278-83.

28(41): 6675-83.

376 Gene Therapy - Tools and Potential Applications

Mol Vis 17: 291-9.

Virol 83(7): 3094-103.

Rep 3(9): 899-904.

nemia." Blood 115(11): 2146-55.

Cells Dev 16(4): 537-45.


[136] Leuci, V., L. Gammaitoni, et al. (2009). "Efficient transcriptional targeting of human hematopoietic stem cells and blood cell lineages by lentiviral vectors containing the regulatory element of the Wiskott-Aldrich syndrome gene." Stem Cells 27(11): 2815-23.

[149] Mannell, H., J. Pircher, et al. (2012). "Targeted endothelial gene delivery by ultrasonic destruction of magnetic microbubbles carrying lentiviral vectors." Pharm Res 29(5):

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

379

[150] Marshall, E. (2002). "Gene therapy. What to do when clear success comes with an un‐

[151] Matrai, J., A. Cantore, et al. (2011). "Hepatocyte-targeted expression by integrase-de‐ fective lentiviral vectors induces antigen-specific tolerance in mice with low genotox‐

[152] Matsui, H., C. Hegadorn, et al. (2011). "A microRNA-regulated and GP64-pseudotyp‐ ed lentiviral vector mediates stable expression of FVIII in a murine model of Hemo‐

[153] Maurice, M., S. Mazur, et al. (1999). "Efficient gene delivery to quiescent interleukin-2 (IL-2)-dependent cells by murine leukemia virus-derived vectors harboring IL-2 chi‐

[154] Maurice, M., E. Verhoeyen, et al. (2002). "Efficient gene transfer into human primary blood lymphocytes by surface-engineered lentiviral vectors that display a T cell-acti‐

[155] Mazarakis, N. D., M. Azzouz, et al. (2001). "Rabies virus glycoprotein pseudotyping of lentiviral vectors enables retrograde axonal transport and access to the nervous

[156] McIver, S. R., C. S. Lee, et al. (2005). "Lentiviral transduction of murine oligodendro‐

[157] Michel, G., Y. Yu, et al. (2010). "Site-specific gene insertion mediated by a Cre-loxP-

[158] Miletic, H., Y. H. Fischer, et al. (2004). "Selective transduction of malignant glioma by lentiviral vectors pseudotyped with lymphocytic choriomeningitis virus glycopro‐

[159] Mitomo, K., U. Griesenbach, et al. (2010). "Toward gene therapy for cystic fibrosis us‐ ing a lentivirus pseudotyped with Sendai virus envelopes." Mol Ther 18(6): 1173-82.

[160] Modlich, U. and C. Baum (2009). "Preventing and exploiting the oncogenic potential

[161] Moiani, A., Y. Paleari, et al. (2012). "Lentiviral vector integration in the human ge‐ nome induces alternative splicing and generates aberrant transcripts." J Clin Invest

[162] Montini, E., D. Cesana, et al. (2009). "The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model

system after peripheral delivery." Hum Mol Genet 10(19): 2109-21.

1282-94.

clear risk?" Science 298(5593): 510-1.

ic risk." Hepatology 53(5): 1696-707.

philia A." Mol Ther 19(4): 723-30.

meric envelope glycoproteins." Blood 94(2): 401-10.

vating polypeptide." Blood 99(7): 2342-50.

cytes in vivo." J Neurosci Res 82(3): 397-403.

teins." Hum Gene Ther 15(11): 1091-100.

122(5): 1653-66.

carrying lentiviral vector." Mol Ther 18(10): 1814-21.

of integrating gene vectors." J Clin Invest 119(4): 755-8.

of HSC gene therapy." J Clin Invest 119(4): 964-75.


[149] Mannell, H., J. Pircher, et al. (2012). "Targeted endothelial gene delivery by ultrasonic destruction of magnetic microbubbles carrying lentiviral vectors." Pharm Res 29(5): 1282-94.

[136] Leuci, V., L. Gammaitoni, et al. (2009). "Efficient transcriptional targeting of human hematopoietic stem cells and blood cell lineages by lentiviral vectors containing the regulatory element of the Wiskott-Aldrich syndrome gene." Stem Cells 27(11):

[137] Li, M., N. Husic, et al. (2010). "Optimal promoter usage for lentiviral vector-mediated transduction of cultured central nervous system cells." J Neurosci Methods 189(1):

[138] Lin, A. H., N. Kasahara, et al. (2001). "Receptor-specific targeting mediated by the co‐ expression of a targeted murine leukemia virus envelope protein and a binding-de‐

[139] Liu, B., J. F. Paton, et al. (2008). "Viral vectors based on bidirectional cell-specific mammalian promoters and transcriptional amplification strategy for use in vitro and

[140] Liu, B., S. Wang, et al. (2008). "Enhancement of cell-specific transgene expression from a Tet-Off regulatory system using a transcriptional amplification strategy in the

[141] Liu, B. H., X. Wang, et al. (2004). "CMV enhancer/human PDGF-beta promoter for

[142] Llano, M., M. Vanegas, et al. (2006). "Identification and characterization of the chro‐ matin-binding domains of the HIV-1 integrase interactor LEDGF/p75." J Mol Biol

[143] Lodish, H. F. and R. A. Weiss (1979). "Selective isolation of mutants of vesicular sto‐ matitis virus defective in production of the viral glycoprotein." J Virol 30(1): 177-89.

[144] Lombardo, A., P. Genovese, et al. (2007). "Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery." Nat Biotech‐

[145] Lopes, L., M. Dewannieux, et al. (2008). "Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell re‐

[146] Lopes, L., M. Dewannieux, et al. (2011). "A lentiviral vector pseudotype suitable for

[147] Lopez-Ornelas, A., T. Mejia-Castillo, et al. (2011). "Lentiviral transfer of an inducible transgene expressing a soluble form of Gas1 causes glioma cell arrest, apoptosis and

[148] Manilla, P., T. Rebello, et al. (2005). "Regulatory considerations for novel gene thera‐ py products: a review of the process leading to the first clinical lentiviral vector."

neuron-specific transgene expression." Gene Ther 11(1): 52-60.

fective influenza hemagglutinin protein." Hum Gene Ther 12(4): 323-32.

2815-23.

378 Gene Therapy - Tools and Potential Applications

56-64.

in vivo." BMC Biotechnol 8: 49.

360(4): 760-73.

nol 25(11): 1298-306.

sponses." J Virol 82(1): 86-95.

Hum Gene Ther 16(1): 17-25.

vaccine development." J Gene Med 13(3): 181-7.

inhibits tumor growth." Cancer Gene Ther 18(2): 87-99.

rat brain." J Gene Med 10(5): 583-92.


[163] Montini, E., D. Cesana, et al. (2006). "Hematopoietic stem cell gene transfer in a tu‐ mor-prone mouse model uncovers low genotoxicity of lentiviral vector integration." Nat Biotechnol 24(6): 687-96.

[177] Page, K. A., N. R. Landau, et al. (1990). "Construction and use of a human immuno‐ deficiency virus vector for analysis of virus infectivity." J Virol 64(11): 5270-6.

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

381

[178] Palmowski, M. J., L. Lopes, et al. (2004). "Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response." J Immunol 172(3): 1582-7.

[179] Papapetrou, E. P., D. Kovalovsky, et al. (2009). "Harnessing endogenous miR-181a to segregate transgenic antigen receptor expression in developing versus post-thymic T

[180] Pariente, N., S. H. Mao, et al. (2008). "Efficient targeted transduction of primary hu‐ man endothelial cells with dual-targeted lentiviral vectors." J Gene Med 10(3): 242-8.

[181] Pariente, N., K. Morizono, et al. (2007). "A novel dual-targeted lentiviral vector leads to specific transduction of prostate cancer bone metastases in vivo after systemic ad‐

[182] Parker, D. G., H. M. Brereton, et al. (2009). "A steroid-inducible promoter for the cor‐

[183] Payen, E., C. Colomb, et al. (2012). "Lentivirus vectors in beta-thalassemia." Methods

[184] Petrigliano, F. A., M. S. Virk, et al. (2009). "Targeting of prostate cancer cells by a cy‐ totoxic lentiviral vector containing a prostate stem cell antigen (PSCA) promoter."

[185] Peviani, M., M. Kurosaki, et al. (2012). "Lentiviral vectors carrying enhancer elements of Hb9 promoter drive selective transgene expression in mouse spinal cord motor

[186] Philippe, S., C. Sarkis, et al. (2006). "Lentiviral vectors with a defective integrase al‐ low efficient and sustained transgene expression in vitro and in vivo." Proc Natl

[187] Pollock, R., R. Issner, et al. (2000). "Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector." Proc Natl Acad Sci U S A 97(24):

[188] Ramezani, A. and R. G. Hawley (2002). "Overview of the HIV-1 Lentiviral Vector

[189] Rasko, J. E., J. L. Battini, et al. (1999). "The RD114/simian type D retrovirus receptor is a neutral amino acid transporter." Proc Natl Acad Sci U S A 96(5): 2129-34.

[190] Ray, M. K., S. P. Fagan, et al. (2000). "The Cre-loxP system: a versatile tool for target‐ ing genes in a cell- and stage-specific manner." Cell Transplant 9(6): 805-15.

[191] Reiser, J., Z. Lai, et al. (2000). "Development of multigene and regulated lentivirus

cells in murine hematopoietic chimeras." J Clin Invest 119(1): 157-68.

ministration." Mol Ther 15(11): 1973-81.

neurons." J Neurosci Methods 205(1): 139-47.

System." Curr Protoc Mol Biol Chapter 16: Unit 16 21.

nea." Br J Ophthalmol 93(9): 1255-9.

Enzymol 507: 109-24.

Prostate 69(13): 1422-34.

Acad Sci U S A 103(47): 17684-9.

vectors." J Virol 74(22): 10589-99.

13221-6.


[177] Page, K. A., N. R. Landau, et al. (1990). "Construction and use of a human immuno‐ deficiency virus vector for analysis of virus infectivity." J Virol 64(11): 5270-6.

[163] Montini, E., D. Cesana, et al. (2006). "Hematopoietic stem cell gene transfer in a tu‐ mor-prone mouse model uncovers low genotoxicity of lentiviral vector integration."

[164] Morizono, K., A. Ku, et al. (2010). "Redirecting lentiviral vectors pseudotyped with Sindbis virus-derived envelope proteins to DC-SIGN by modification of N-linked

[165] Morizono, K., Y. Xie, et al. (2005). "Lentiviral vector retargeting to P-glycoprotein on metastatic melanoma through intravenous injection." Nat Med 11(3): 346-52.

[166] Munch, R. C., M. D. Muhlebach, et al. (2011). "DARPins: an efficient targeting do‐

[167] Naldini, L., U. Blomer, et al. (1996). "In vivo gene delivery and stable transduction of

[168] Neda, H., C. H. Wu, et al. (1991). "Chemical modification of an ecotropic murine leu‐ kemia virus results in redirection of its target cell specificity." J Biol Chem 266(22):

[169] Neddermann, P., C. Gargioli, et al. (2003). "A novel, inducible, eukaryotic gene ex‐ pression system based on the quorum-sensing transcription factor TraR." EMBO Rep

[170] Nefkens, I., J. M. Garcia, et al. (2007). "Hemagglutinin pseudotyped lentiviral parti‐ cles: characterization of a new method for avian H5N1 influenza sero-diagnosis." J

[171] Nelson, P., M. Kiriakidou, et al. (2003). "The microRNA world: small is mighty."

[172] Nguyen, T. H., J. C. Pages, et al. (1998). "Amphotropic retroviral vectors displaying hepatocyte growth factor-envelope fusion proteins improve transduction efficiency

[173] O'Driscoll, M. and P. A. Jeggo (2006). "The role of double-strand break repair - in‐

[174] O'Leary, V. B., S. V. Ovsepian, et al. (2011). "Innocuous full-length botulinum neuro‐ toxin targets and promotes the expression of lentiviral vectors in central and auto‐

[175] Ogueta, S. B., F. Yao, et al. (2001). "Design and in vitro characterization of a single regulatory module for efficient control of gene expression in both plasmid DNA and

[176] Pabo, C. O., E. Peisach, et al. (2001). "Design and selection of novel Cys2His2 zinc fin‐

of primary hepatocytes." Hum Gene Ther 9(17): 2469-79.

sights from human genetics." Nat Rev Genet 7(1): 45-54.

a self-inactivating lentiviral vector." Mol Med 7(8): 569-79.

nondividing cells by a lentiviral vector." Science 272(5259): 263-7.

glycans of envelope proteins." J Virol 84(14): 6923-34.

main for lentiviral vectors." Mol Ther 19(4): 686-93.

Nat Biotechnol 24(6): 687-96.

380 Gene Therapy - Tools and Potential Applications

14143-6.

4(2): 159-65.

Clin Virol 39(1): 27-33.

Trends Biochem Sci 28(10): 534-40.

nomic neurons." Gene Ther 18(7): 656-65.

ger proteins." Annu Rev Biochem 70: 313-40.


[192] Roberts, M. C. (2002). "Resistance to tetracycline, macrolide-lincosamide-streptogra‐ min, trimethoprim, and sulfonamide drug classes." Mol Biotechnol 20(3): 261-83.

[206] Shinoda, Y., K. Hieda, et al. (2009). "Efficient transduction of cytotoxic and anti-HIV-1 genes by a gene-regulatable lentiviral vector." Virus Genes 39(2): 165-75. [207] Silvers, R. M., J. A. Smith, et al. (2010). "Modification of integration site preferences of an HIV-1-based vector by expression of a novel synthetic protein." Hum Gene Ther

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

383

[208] Singhal, R., X. Deng, et al. (2011). "Long-distance effects of insertional mutagenesis."

[209] Sirin, O. and F. Park (2003). "Regulating gene expression using self-inactivating lenti‐ viral vectors containing the mifepristone-inducible system." Gene 323: 67-77. [210] Somia, N. V., H. Miyoshi, et al. (2000). "Retroviral vector targeting to human immu‐ nodeficiency virus type 1-infected cells by receptor pseudotyping." J Virol 74(9):

[211] Steffens, S., J. Tebbets, et al. (2004). "Transduction of human glial and neuronal tumor cells with different lentivirus vector pseudotypes." J Neurooncol 70(3): 281-8. [212] Steidl, S., J. Stitz, et al. (2002). "Coreceptor Switch of [MLV(SIVagm)] pseudotype vec‐

[213] Stein, C. S., I. Martins, et al. (2005). "The lymphocytic choriomeningitis virus enve‐ lope glycoprotein targets lentiviral gene transfer vector to neural progenitors in the

[214] Stein, S., M. G. Ott, et al. (2010). "Genomic instability and myelodysplasia with mon‐ osomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous

[215] Stitz, J., C. J. Buchholz, et al. (2000). "Lentiviral vectors pseudotyped with envelope glycoproteins derived from gibbon ape leukemia virus and murine leukemia virus

[216] Strang, B. L., Y. Ikeda, et al. (2004). "Characterization of HIV-1 vectors with gammar‐ etrovirus envelope glycoproteins produced from stable packaging cells." Gene Ther

[217] Strang, B. L., Y. Takeuchi, et al. (2005). "Human immunodeficiency virus type 1 vec‐ tors with alphavirus envelope glycoproteins produced from stable packaging cells." J

[218] Szulc, J., M. Wiznerowicz, et al. (2006). "A versatile tool for conditional gene expres‐

[219] Tai, A., S. Froelich, et al. (2011). "Production of lentiviral vectors with enhanced effi‐ ciency to target dendritic cells by attenuating mannosidase activity of mammalian

[220] Tan, W., Z. Dong, et al. (2006). "Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger

tors by V3-loop exchange." Virology 300(2): 205-16.

sion and knockdown." Nat Methods 3(2): 109-16.

murine brain." Mol Ther 11(3): 382-9.

disease." Nat Med 16(2): 198-204.

10A1." Virology 273(1): 16-20.

11(7): 591-8.

Virol 79(3): 1765-71.

cells." J Biol Eng 5(1): 1.

21(3): 337-49.

4420-4.

PLoS One 6(1): e15832.


[206] Shinoda, Y., K. Hieda, et al. (2009). "Efficient transduction of cytotoxic and anti-HIV-1 genes by a gene-regulatable lentiviral vector." Virus Genes 39(2): 165-75.

[192] Roberts, M. C. (2002). "Resistance to tetracycline, macrolide-lincosamide-streptogra‐ min, trimethoprim, and sulfonamide drug classes." Mol Biotechnol 20(3): 261-83. [193] Roet, K. C., R. Eggers, et al. (2012). "Non-invasive bioluminescence imaging of olfac‐ tory ensheathing glia and Schwann cells following transplantation into the lesioned

[194] Romano, G., P. P. Claudio, et al. (2003). "Human immunodeficiency virus type 1 (HIV-1) derived vectors: safety considerations and controversy over therapeutic ap‐

[195] Roux, P., P. Jeanteur, et al. (1989). "A versatile and potentially general approach to the targeting of specific cell types by retroviruses: application to the infection of hu‐ man cells by means of major histocompatibility complex class I and class II antigens by mouse ecotropic murine leukemia virus-derived viruses." Proc Natl Acad Sci U S

[196] Sachdeva, R., M. E. Jonsson, et al. (2010). "Tracking differentiating neural progenitors in pluripotent cultures using microRNA-regulated lentiviral vectors." Proc Natl Acad

[197] Sakuma, T., S. S. De Ravin, et al. (2010). "Characterization of retroviral and lentiviral vectors pseudotyped with xenotropic murine leukemia virus-related virus envelope

[198] Sanchez-Danes, A., A. Consiglio, et al. (2012). "Efficient generation of A9 midbrain dopaminergic neurons by lentiviral delivery of LMX1A in human embryonic stem

[199] Sarkar, N. N. (2002). "Mifepristone: bioavailability, pharmacokinetics and use-effec‐

[200] Sayed, D., S. Rane, et al. (2008). "MicroRNA-21 targets Sprouty2 and promotes cellu‐

[201] Schaffer, D. V., J. T. Koerber, et al. (2008). "Molecular engineering of viral gene deliv‐

[202] Scherr, M., L. Venturini, et al. (2007). "Lentivirus-mediated antagomir expression for

[203] Semple-Rowland, S. L., W. E. Coggin, et al. (2010). "Expression characteristics of du‐ al-promoter lentiviral vectors targeting retinal photoreceptors and Muller cells." Mol

[204] Semple-Rowland, S. L., K. S. Eccles, et al. (2007). "Targeted expression of two pro‐ teins in neural retina using self-inactivating, insulated lentiviral vectors carrying two

[205] Seo, E., S. Kim, et al. (2009). "Induction of cancer cell-specific death via MMP2 pro‐

specific inhibition of miRNA function." Nucleic Acids Res 35(22): e149.

cells and induced pluripotent stem cells." Hum Gene Ther 23(1): 56-69.

tiveness." Eur J Obstet Gynecol Reprod Biol 101(2): 113-20.

rat spinal cord." Cell Transplant.

A 86(23): 9079-83.

382 Gene Therapy - Tools and Potential Applications

Vis 16: 916-34.

Sci U S A 107(25): 11602-7.

plications." Eur J Dermatol 13(5): 424-9.

glycoprotein." Hum Gene Ther 21(12): 1665-73.

lar outgrowths." Mol Biol Cell 19(8): 3272-82.

ery vehicles." Annu Rev Biomed Eng 10: 169-94.

internal independent promoters." Mol Vis 13: 2001-11.

moterdependent Bax expression." BMB Rep 42(4): 217-22.


protein E2C can bias integration of viral DNA into a predetermined chromosomal re‐ gion in human cells." J Virol 80(4): 1939-48.

[235] Watson, D. J., G. P. Kobinger, et al. (2002). "Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or

Targeted Lentiviral Vectors: Current Applications and Future Potential

http://dx.doi.org/10.5772/52770

385

[236] Wiederschain, D., S. Wee, et al. (2009). "Single-vector inducible lentiviral RNAi sys‐

[237] Wong, L. F., M. Azzouz, et al. (2004). "Transduction patterns of pseudotyped lentivi‐

[238] Xu, Z. L., H. Mizuguchi, et al. (2003). "Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems." Gene 309(2): 145-51.

[239] Yang, H., K. I. Joo, et al. (2009). "Cell type-specific targeting with surface-engineered lentiviral vectors co-displaying OKT3 antibody and fusogenic molecule." Pharm Res

[240] Yang, L., H. Yang, et al. (2008). "Engineered lentivector targeting of dendritic cells for

[241] Yu, D., D. Chen, et al. (2001). "Prostate-specific targeting using PSA promoter-based

[242] Yu, D., C. Scott, et al. (2006). "Targeting and killing of prostate cancer cells using len‐ tiviral constructs containing a sequence recognized by translation factor eIF4E and a

[243] Yu, S. T., C. Li, et al. (2011). "Noninvasive and real-time monitoring of the therapeu‐ tic response of tumors in vivo with an optimized hTERT promoter." Cancer 118(7):

[244] Zeilfelder, U. and V. Bosch (2001). "Properties of wild-type, C-terminally truncated, and chimeric maedi-visna virus glycoprotein and putative pseudotyping of retroviral

[245] Zhang, J., L. Zou, et al. (2009). "Rapid generation of dendritic cell specific transgenic

[246] Zhang, X. Y., R. H. Kutner, et al. (2010). "Cell-specific targeting of lentiviral vectors mediated by fusion proteins derived from Sindbis virus, vesicular stomatitis virus, or

[247] Zheng, J. Y., D. Chen, et al. (2003). "Regression of prostate cancer xenografts by a len‐ tiviral vector specifically expressing diphtheria toxin A." Cancer Gene Ther 10(10):

[248] Zhu, Z. H., S. S. Chen, et al. (1990). "Phenotypic mixing between human immunodefi‐ ciency virus and vesicular stomatitis virus or herpes simplex virus." J Acquir Im‐

MuLV envelope proteins." Mol Ther 5(5 Pt 1): 528-37.

tem for oncology target validation." Cell Cycle 8(3): 498-504.

ral vectors in the nervous system." Mol Ther 9(1): 101-11.

in vivo immunization." Nat Biotechnol 26(3): 326-34.

lentiviral vectors." Cancer Gene Ther 8(9): 628-35.

vector particles." J Virol 75(1): 548-55.

mune Defic Syndr 3(3): 215-9.

prostate-specific promoter." Cancer Gene Ther 13(1): 32-43.

mice by lentiviral vectors." Transgenic Res 18(6): 921-31.

avian sarcoma/leukosis virus." Retrovirology 7: 3.

26(6): 1432-45.

1884-93.

764-70.


[235] Watson, D. J., G. P. Kobinger, et al. (2002). "Targeted transduction patterns in the mouse brain by lentivirus vectors pseudotyped with VSV, Ebola, Mokola, LCMV, or MuLV envelope proteins." Mol Ther 5(5 Pt 1): 528-37.

protein E2C can bias integration of viral DNA into a predetermined chromosomal re‐

[221] Tian, J., P. Lei, et al. (2008). "Regulated insulin delivery from human epidermal cells

[222] Toniatti, C., H. Bujard, et al. (2004). "Gene therapy progress and prospects: transcrip‐

[223] Uch, R., R. Gerolami, et al. (2003). "Hepatoma cell-specific ganciclovir-mediated tox‐ icity of a lentivirally transduced HSV-TkEGFP fusion protein gene placed under the control of rat alpha-fetoprotein gene regulatory sequences." Cancer Gene Ther 10(9):

[224] Urnov, F. D., J. C. Miller, et al. (2005). "Highly efficient endogenous human gene cor‐

[225] Valastyan, S., F. Reinhardt, et al. (2009). "A pleiotropically acting microRNA, miR-31,

[226] Van Duyne, G. D. (2001). "A structural view of cre-loxp site-specific recombination."

[227] Vandendriessche, T., L. Thorrez, et al. (2007). "Efficacy and safety of adeno-associat‐ ed viral vectors based on serotype 8 and 9 vs. lentiviral vectors for hemophilia B gene

[228] Vaneycken, I., N. Devoogdt, et al. (2011). "Preclinical screening of anti-HER2 nano‐

[229] Vargas, J., Jr., G. L. Gusella, et al. (2004). "Novel integrase-defective lentiviral episo‐

[230] Verhoeyen, E., V. Dardalhon, et al. (2003). "IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes."

[231] Verhoeyen, E., M. Wiznerowicz, et al. (2005). "Novel lentiviral vectors displaying "early-acting cytokines" selectively promote survival and transduction of NOD/SCID

[232] Waehler, R., S. J. Russell, et al. (2007). "Engineering targeted viral vectors for gene

[233] Wang, Y., H. H. Hu, et al. (2012). "Lentiviral transgenic microRNA-based shRNA suppressed mouse cytochromosome P450 3A (CYP3A) expression in a dose-depend‐

[234] Wanisch, K. and R. J. Yanez-Munoz (2009). "Integration-deficient lentiviral vectors: a

repopulating human hematopoietic stem cells." Blood 106(10): 3386-95.

bodies for molecular imaging of breast cancer." FASEB J 25(7): 2433-46.

mal vectors for gene transfer." Hum Gene Ther 15(4): 361-72.

rection using designed zinc-finger nucleases." Nature 435(7042): 646-51.

gion in human cells." J Virol 80(4): 1939-48.

689-95.

384 Gene Therapy - Tools and Potential Applications

reverses hyperglycemia." Mol Ther 16(6): 1146-53.

tion regulatory systems." Gene Ther 11(8): 649-57.

inhibits breast cancer metastasis." Cell 137(6): 1032-46.

Annu Rev Biophys Biomol Struct 30: 87-104.

therapy." J Thromb Haemost 5(1): 16-24.

therapy." Nat Rev Genet 8(8): 573-87.

ent and inheritable manner." PLoS One 7(1): e30560.

slow coming of age." Mol Ther 17(8): 1316-32.

Blood 101(6): 2167-74.


[249] Ziegler, L., L. Yang, et al. (2008). "Targeting lentiviral vectors to antigen-specific im‐ munoglobulins." Hum Gene Ther 19(9): 861-72.

**Chapter 15**

**Vectors for Highly Efficient and Neuron-Specific**

**Retrograde Gene Transfer for Gene Therapy of**

Viral vectors have been widely used to deliver several therapeutic genes in the clinical ap‐ proach of gene therapy. The lentiviral vector permits stable and efficient gene transfer into non-dividing cells in the central nervous system of neurological and neurodegenerative dis‐ eases (Deeks, et al., 2002; Mavilo, et al., 2006; Rossi et al., 2007; Ciceri, et al., 2009; Naldini, 2011). Moreover, long-term expression of delivered gene attributed to genome integration has an advantage not only for clinical application, but also for gene therapy trials in animal models (Naldini et al., 1996; Reiser et al., 1996; Mochizuki et al., 1998; Mitrophanous et al., 1999; Wong et al., 2006; Lundberg et al., 2008). Among many lentiviral vector systems, the most familiar is the human immunodeficiency virus type-1 (HIV-1)-based vector of which molecular biological property has been extensively studied (Rabson and Martin, 1985; Joshi

Axonal transport in the retrograde direction, as observed in the case of some viral vectors, has a considerable advantage for transferring genes into neuronal cell bodies situated in re‐ gions remote from the injection sites of the vectors (see Fig.1). These viral vectors, for exam‐ ple, injected into the striatum, transfer the genes via retrograde transport into nigrostriatal dopaminergic neurons, which are the major target for gene therapy of Parkinson's disease (Zheng et al., 2005; Barkats et al., 2006). Intramuscular injection of the vectors also delivers retrogradely the genes into motor neurons that are the target for gene therapy of motor neu‐ ron diseases (Baumgartner & Shine, 1998; Perrelet et al., 2000; Mazarakis et al., 2001; Saka‐

> © 2013 Kato et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

**Neurological Diseases**

http://dx.doi.org/10.5772/52611

**1. Introduction**

Shigeki Kato, Kenta Kobayashi, Ken-ichi Inoue,

Masahiko Takada and Kazuto Kobayashi

Additional information is available at the end of the chapter

and Joshi, 1996; Nielsen et al., 2005; Pluta and Kacprzak, 2009).

moto et al., 2003; Azzouz et al., 2004).

[250] Zufferey, R., D. Nagy, et al. (1997). "Multiply attenuated lentiviral vector achieves ef‐ ficient gene delivery in vivo." Nat Biotechnol 15(9): 871-5.

## **Vectors for Highly Efficient and Neuron-Specific Retrograde Gene Transfer for Gene Therapy of Neurological Diseases**

Shigeki Kato, Kenta Kobayashi, Ken-ichi Inoue, Masahiko Takada and Kazuto Kobayashi

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52611

#### **1. Introduction**

[249] Ziegler, L., L. Yang, et al. (2008). "Targeting lentiviral vectors to antigen-specific im‐

[250] Zufferey, R., D. Nagy, et al. (1997). "Multiply attenuated lentiviral vector achieves ef‐

munoglobulins." Hum Gene Ther 19(9): 861-72.

386 Gene Therapy - Tools and Potential Applications

ficient gene delivery in vivo." Nat Biotechnol 15(9): 871-5.

Viral vectors have been widely used to deliver several therapeutic genes in the clinical ap‐ proach of gene therapy. The lentiviral vector permits stable and efficient gene transfer into non-dividing cells in the central nervous system of neurological and neurodegenerative dis‐ eases (Deeks, et al., 2002; Mavilo, et al., 2006; Rossi et al., 2007; Ciceri, et al., 2009; Naldini, 2011). Moreover, long-term expression of delivered gene attributed to genome integration has an advantage not only for clinical application, but also for gene therapy trials in animal models (Naldini et al., 1996; Reiser et al., 1996; Mochizuki et al., 1998; Mitrophanous et al., 1999; Wong et al., 2006; Lundberg et al., 2008). Among many lentiviral vector systems, the most familiar is the human immunodeficiency virus type-1 (HIV-1)-based vector of which molecular biological property has been extensively studied (Rabson and Martin, 1985; Joshi and Joshi, 1996; Nielsen et al., 2005; Pluta and Kacprzak, 2009).

Axonal transport in the retrograde direction, as observed in the case of some viral vectors, has a considerable advantage for transferring genes into neuronal cell bodies situated in re‐ gions remote from the injection sites of the vectors (see Fig.1). These viral vectors, for exam‐ ple, injected into the striatum, transfer the genes via retrograde transport into nigrostriatal dopaminergic neurons, which are the major target for gene therapy of Parkinson's disease (Zheng et al., 2005; Barkats et al., 2006). Intramuscular injection of the vectors also delivers retrogradely the genes into motor neurons that are the target for gene therapy of motor neu‐ ron diseases (Baumgartner & Shine, 1998; Perrelet et al., 2000; Mazarakis et al., 2001; Saka‐ moto et al., 2003; Azzouz et al., 2004).

© 2013 Kato et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

In our previous study, we generated an HIV-1-based vector pseudotyped with a variant of rabies virus glycoprotein (RV-G) gene and tested gene transfer through retrograde axonal transport into several brain regions (Kato et al., 2007). Although this pseudotyped vector showed gene transfer through retrograde transport in the rodent and nonhuman primate brains, higher titer stocks of the vector was required for the application of gene therapy tri‐ als. To enhance the efficiency of retrograde gene transfer, we subsequently developed a nov‐ el type of lentiviral vector that shows highly efficient retrograde gene transfer (HiRet) by pseudotyping an HIV-1-based vector with fusion glycoprotein B type (FuG-B) composed of parts of RV-G and vesicular stomatitis virus glycoprotein (VSV-G) (Kato et al., 2011a,b).

The viral vectors enter nerve terminals and are retrogradely transported through axons into

Vectors for Highly Efficient and Neuron-Specific Retrograde Gene Transfer for Gene Therapy of Neurological Diseases

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In this chapter, we recapitulate gene transduction property of the HiRet and NeuRet vectors, and then describe the application of the NeuRet vector for retrograde gene transfer into the

The HiRet vector is a pseudotype of the HIV-1 lentiviral vector with FuG-B, which is com‐ posed of the extracellular and transmembrane domains of RV-G (challenged virus standard strain) and the cytoplasmic domain of VSV-G (Fig. 2A) (Kato et al., 2011a). When the HiRet vector encoding green fluorescent protein (GFP) was injected into the dorsal striatum of mice, we observed high efficiency of retrograde gene transfer into the brain regions inner‐ vating the striatum, including the primary motor cortex (M1), primary somatosensory cortex (S1), parafascicular nucleus (PF) in the thalamus, and substantia nigra pars compacta (SNc) in the ventral midbrain (Fig. 2B). The extent of gene transfer efficiency increased compared with that of the RV-G pseudotype, ranging from 8- to 14-folds dependent on the neural pathways. The high efficiency of gene transfer was also detected in the brain regions that project to the nucleus accumbens or medial prefrontal cortex in mice. In addition, we ob‐ served gene transduction of the HiRet vector into glial cells (~75%) and a small number of neuronal cells (~20%) in the striatum around the injection sites (Fig. 2C). Recently, we creat‐ ed a variant of FuG-B (termed FuG-B2), in which the extracellular and transmembrane do‐ mains of RV-G derived from the challenged virus standard strain was exchanged with the counterparts of Pasteur virus strain, and the vector pseudotyped with FuG-B2 exhibited a further increase in the retrograde gene transfer efficiency in the rodent brain (Kato et al., 2011b). More recently, Carpentier et al. (2012) reported the increased psudotyping efficiency of an HIV-1 vector by a chimeric envelope glycoprotein composed of RV-G and VSV-G do‐

The host range of lentiviral vectors is altered by pseudotyping with different envelope glycoproteins (Cronin et al., 2005). Therefore, the possibility arises that some mutations in RV-G shift the efficiency of gene transduction or host cell specificity of the pseudotyped vector. Indeed, substitution of the cytoplasmic domain of RV-G with the corresponding part of the VSV-G enhanced the efficiency of retrograde gene transfer. The cytoplasmic domain differs in length between RV-G (44 amino acids) and VSV-G (29 amino acids), but their amino acid sequences do not show any particular homology (Rose et al., 1982). It ap‐ pears that the cytoplasmic domain is involved in the mechanism underlying vector entry into synaptic terminals or the transduction level of the vector, resulting in enhanced retro‐

neuronal cell bodies, resulting in the induction of transgene expression.

**2. Gene transduction property of HiRet and NeuRet vectors**

nigrostriatal dopamine system in nonhuman primates.

mains, which corresponds to our FuG-B.

grade gene transfer.

**2.1. HiRet vector**

More recently, we developed another vector system for neuron-specific retrograde gene transfer (NeuRet) by pseudotyping the HIV-1-based vector with fusion glycoprotein C type (FuG-C) composed of a different set of parts of RV-G and VSV-G (Kato et al., 2011c). Inter‐ estingly, the NeuRet vector shows high efficiency of retrograde gene transfer into various neuronal populations, whereas it remarkably reduces gene transduction into dividing cells including glial and nerural stem/progenitor cells around the vector injection sites. One sig‐ nificant issue on the therapeutic use of lentiviral vectors is transgene integration into the host genome in dividing cells, which may lead to tumorigenesis by altering the expression of proto-oncogenes adjacent to the integration sites (De Palma et al., 2005; Themis et al., 2005; Montini et al., 2006). In this context, the NeuRet vector can reduce the risk of vector transduction into dividing cells in the brain and improve the safety of future gene therapy trials for neurological and neurodegenerative disorders.

**Figure 1.** Gene transfer process through retrograde axonal transport.

The viral vectors enter nerve terminals and are retrogradely transported through axons into neuronal cell bodies, resulting in the induction of transgene expression.

In this chapter, we recapitulate gene transduction property of the HiRet and NeuRet vectors, and then describe the application of the NeuRet vector for retrograde gene transfer into the nigrostriatal dopamine system in nonhuman primates.

#### **2. Gene transduction property of HiRet and NeuRet vectors**

#### **2.1. HiRet vector**

In our previous study, we generated an HIV-1-based vector pseudotyped with a variant of rabies virus glycoprotein (RV-G) gene and tested gene transfer through retrograde axonal transport into several brain regions (Kato et al., 2007). Although this pseudotyped vector showed gene transfer through retrograde transport in the rodent and nonhuman primate brains, higher titer stocks of the vector was required for the application of gene therapy tri‐ als. To enhance the efficiency of retrograde gene transfer, we subsequently developed a nov‐ el type of lentiviral vector that shows highly efficient retrograde gene transfer (HiRet) by pseudotyping an HIV-1-based vector with fusion glycoprotein B type (FuG-B) composed of parts of RV-G and vesicular stomatitis virus glycoprotein (VSV-G) (Kato et al., 2011a,b).

More recently, we developed another vector system for neuron-specific retrograde gene transfer (NeuRet) by pseudotyping the HIV-1-based vector with fusion glycoprotein C type (FuG-C) composed of a different set of parts of RV-G and VSV-G (Kato et al., 2011c). Inter‐ estingly, the NeuRet vector shows high efficiency of retrograde gene transfer into various neuronal populations, whereas it remarkably reduces gene transduction into dividing cells including glial and nerural stem/progenitor cells around the vector injection sites. One sig‐ nificant issue on the therapeutic use of lentiviral vectors is transgene integration into the host genome in dividing cells, which may lead to tumorigenesis by altering the expression of proto-oncogenes adjacent to the integration sites (De Palma et al., 2005; Themis et al., 2005; Montini et al., 2006). In this context, the NeuRet vector can reduce the risk of vector transduction into dividing cells in the brain and improve the safety of future gene therapy

trials for neurological and neurodegenerative disorders.

388 Gene Therapy - Tools and Potential Applications

**Figure 1.** Gene transfer process through retrograde axonal transport.

The HiRet vector is a pseudotype of the HIV-1 lentiviral vector with FuG-B, which is com‐ posed of the extracellular and transmembrane domains of RV-G (challenged virus standard strain) and the cytoplasmic domain of VSV-G (Fig. 2A) (Kato et al., 2011a). When the HiRet vector encoding green fluorescent protein (GFP) was injected into the dorsal striatum of mice, we observed high efficiency of retrograde gene transfer into the brain regions inner‐ vating the striatum, including the primary motor cortex (M1), primary somatosensory cortex (S1), parafascicular nucleus (PF) in the thalamus, and substantia nigra pars compacta (SNc) in the ventral midbrain (Fig. 2B). The extent of gene transfer efficiency increased compared with that of the RV-G pseudotype, ranging from 8- to 14-folds dependent on the neural pathways. The high efficiency of gene transfer was also detected in the brain regions that project to the nucleus accumbens or medial prefrontal cortex in mice. In addition, we ob‐ served gene transduction of the HiRet vector into glial cells (~75%) and a small number of neuronal cells (~20%) in the striatum around the injection sites (Fig. 2C). Recently, we creat‐ ed a variant of FuG-B (termed FuG-B2), in which the extracellular and transmembrane do‐ mains of RV-G derived from the challenged virus standard strain was exchanged with the counterparts of Pasteur virus strain, and the vector pseudotyped with FuG-B2 exhibited a further increase in the retrograde gene transfer efficiency in the rodent brain (Kato et al., 2011b). More recently, Carpentier et al. (2012) reported the increased psudotyping efficiency of an HIV-1 vector by a chimeric envelope glycoprotein composed of RV-G and VSV-G do‐ mains, which corresponds to our FuG-B.

The host range of lentiviral vectors is altered by pseudotyping with different envelope glycoproteins (Cronin et al., 2005). Therefore, the possibility arises that some mutations in RV-G shift the efficiency of gene transduction or host cell specificity of the pseudotyped vector. Indeed, substitution of the cytoplasmic domain of RV-G with the corresponding part of the VSV-G enhanced the efficiency of retrograde gene transfer. The cytoplasmic domain differs in length between RV-G (44 amino acids) and VSV-G (29 amino acids), but their amino acid sequences do not show any particular homology (Rose et al., 1982). It ap‐ pears that the cytoplasmic domain is involved in the mechanism underlying vector entry into synaptic terminals or the transduction level of the vector, resulting in enhanced retro‐ grade gene transfer.

ber of striatal neuronal cells (~6%), its transduction level into striatal glial cells was quite low (~0.3%) (Fig. 3C). The property of gene transduction of the NeuRet vector around the injec‐ tion sites was quite different from that of the HiRet vector, and in particular, the transduc‐ tion of glial cells was largely declined in the NeuRet vector. Furthermore, when the NeuRet vector was injected into the subventricular zone, gene transduction of the vector into neural

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FuG-C pseudotyping of the NeuRet vector enhanced the efficiency of retrograde gene trans‐ fer into various neuronal populations, whereas it caused less efficiency of gene transduction into glial and neural stem/progenitor cells. The N-terminal segment of the RV-G extracellu‐ lar domain of 439 amino acids appears to be involved in the retrograde gene transfer, proba‐ bly by promoting the interaction with synaptic terminals required for retrograde transport. Actually, amino acid residues essential for rabies virus virulence are reported to exist in the RV-G-derived extracellular domain used for FuG-C construction (Prehaud et al., 1988; Cou‐ lon et al., 1998). In contrast, pseudotyping with FuG-B (FuG-B2) and FuG-C generates a marked difference in gene transduction into glial and neural stem/progenitor cells around the injection areas. This difference suggests that the C-terminal part of 16 amino acids in the extracellular domain of envelope glycoproteins may be implicated in determining the host cell specificity of vector transduction, and that this C-terminal part may contribute to the in‐

For gene therapy trials with lentiviral vectors, there is a significant issue that vector inser‐ tion into the host genome may lead to tumorigenesis by altering the expression of cellular oncogenes surrounding the integration sites (De Palma et al., 2005; Themis et al., 2005; Mon‐ tini et al., 2006). One useful approach to protect this issue is to restrict vector transduction to neuronal cells. The NeuRet vector system provides a useful approach for gene therapy trials for neurological diseases through enhanced retrograde gene transfer and improves the safe‐ ty of gene therapy by profoundly suppressing the efficacy of gene transduction into divid‐

**3. Retrograde gene delivery into monkey nigrostriatal pathway by**

The nigrostriatal dopamine system is a major target for gene therapy of Parkinson's disease. The availability of the HiRet vector for gene transfer via retrograde transport into the nigros‐ triatal dopamine system in nonhuman primates was described in our previous review (Kato et al., 2011d). To verify the capability of the NeuRet vector for efficient retrograde gene transfer into the nigrostriatal pathway, we injected the NeuRetvector encoding the GFP transgene into the striatum (caudate nucleus and putamen) of crab-eating monkeys (Fig. 4A). Intrastriatal injection of the NeuRet vector produced a larger number of GFP-positive neurons in the SNc (Fig. 4B). These positive signals were in register with immunostaining for tyrosine hydroxylase, a marker of dopaminergic neurons (Fig. 4C), indicating the trans‐ gene expression in the nigrostriatal dopaminergic neurons. In addition, we assessed the

stem/progenitor cells was also inefficient.

teraction with glial and neural stem/progenitor cells.

ing cells in the brain.

**NeuRet vector**

**Figure 2.** Gene trasnfer by HiRet vector. (**A**)Fusion envelope glycoprotein. The structure of viral envelope glycoprotein is schematically illustrated in the left panel. FuG-B is composed of the extracellular and transmembrane (TM) domains of RV-G derived from the challenge virus standard (CVS) strain fused to the cytoplasmic domain of VSV-G. In FuG-B2, the RV-G domains are exchanged by the counterparts of RV-G derived from Pasteur virus (PV) strain. S, signal peptide. (**B**) Gene transfer through retrograde transport. The HiRet vector pseudotyped with FuG-B, encoding GFP transgene was injected into the mouse striatum. Four weeks later, sections were processed for GFP immunostaining (right pan‐ el). GFP expression can be seen in the brain regions innervating the striatum, including the M1, S1, PF, and SNc. (**C**) Gene transduction around the injection sites. Sections through the striatum were stained by double immunofluores‐ cence histochemistry for GFP/NeuN or for GFP/glial fibrillary acidic protein (GFAP). Scale bars: 50 μm. (Data from Kato et al., 2011a)

#### **2.2. NeuRet vector**

The NeuRet vector is another pseudotype of the HIV-1 lentiviral vector with FuG-C, which is composed of the N-terminal segment of the extracellular domain (439 amino acids) of RV-G and the C-terminal segment of the extracellular domain (16 amino acids) and transmem‐ brane/cytoplasmic domains of VSV-G (Fig. 3A) (Kato et al., 2011c). After injection of the NeuRet vector encoding GFP transgene into the mouse striatum, we found enhanced retro‐ grade gene transfer into the brain regions innervating the striatum, such as the M1, S1, PF, and SNc (Fig. 3B). The efficiency of gene transfer of the NeuRet vector was slightly different with that of the HiRet vector (FuG-B2 pseudo type), depending on the neural pathways (see a review by Kato et al. 2012). In addition, we tested gene transduction of the NeuRet vector surrounding the injection sites. Although the NeuRet vector transduced only a small num‐ ber of striatal neuronal cells (~6%), its transduction level into striatal glial cells was quite low (~0.3%) (Fig. 3C). The property of gene transduction of the NeuRet vector around the injec‐ tion sites was quite different from that of the HiRet vector, and in particular, the transduc‐ tion of glial cells was largely declined in the NeuRet vector. Furthermore, when the NeuRet vector was injected into the subventricular zone, gene transduction of the vector into neural stem/progenitor cells was also inefficient.

FuG-C pseudotyping of the NeuRet vector enhanced the efficiency of retrograde gene trans‐ fer into various neuronal populations, whereas it caused less efficiency of gene transduction into glial and neural stem/progenitor cells. The N-terminal segment of the RV-G extracellu‐ lar domain of 439 amino acids appears to be involved in the retrograde gene transfer, proba‐ bly by promoting the interaction with synaptic terminals required for retrograde transport. Actually, amino acid residues essential for rabies virus virulence are reported to exist in the RV-G-derived extracellular domain used for FuG-C construction (Prehaud et al., 1988; Cou‐ lon et al., 1998). In contrast, pseudotyping with FuG-B (FuG-B2) and FuG-C generates a marked difference in gene transduction into glial and neural stem/progenitor cells around the injection areas. This difference suggests that the C-terminal part of 16 amino acids in the extracellular domain of envelope glycoproteins may be implicated in determining the host cell specificity of vector transduction, and that this C-terminal part may contribute to the in‐ teraction with glial and neural stem/progenitor cells.

For gene therapy trials with lentiviral vectors, there is a significant issue that vector inser‐ tion into the host genome may lead to tumorigenesis by altering the expression of cellular oncogenes surrounding the integration sites (De Palma et al., 2005; Themis et al., 2005; Mon‐ tini et al., 2006). One useful approach to protect this issue is to restrict vector transduction to neuronal cells. The NeuRet vector system provides a useful approach for gene therapy trials for neurological diseases through enhanced retrograde gene transfer and improves the safe‐ ty of gene therapy by profoundly suppressing the efficacy of gene transduction into divid‐ ing cells in the brain.

### **3. Retrograde gene delivery into monkey nigrostriatal pathway by NeuRet vector**

**Figure 2.** Gene trasnfer by HiRet vector. (**A**)Fusion envelope glycoprotein. The structure of viral envelope glycoprotein is schematically illustrated in the left panel. FuG-B is composed of the extracellular and transmembrane (TM) domains of RV-G derived from the challenge virus standard (CVS) strain fused to the cytoplasmic domain of VSV-G. In FuG-B2, the RV-G domains are exchanged by the counterparts of RV-G derived from Pasteur virus (PV) strain. S, signal peptide. (**B**) Gene transfer through retrograde transport. The HiRet vector pseudotyped with FuG-B, encoding GFP transgene was injected into the mouse striatum. Four weeks later, sections were processed for GFP immunostaining (right pan‐ el). GFP expression can be seen in the brain regions innervating the striatum, including the M1, S1, PF, and SNc. (**C**) Gene transduction around the injection sites. Sections through the striatum were stained by double immunofluores‐ cence histochemistry for GFP/NeuN or for GFP/glial fibrillary acidic protein (GFAP). Scale bars: 50 μm. (Data from Kato

The NeuRet vector is another pseudotype of the HIV-1 lentiviral vector with FuG-C, which is composed of the N-terminal segment of the extracellular domain (439 amino acids) of RV-G and the C-terminal segment of the extracellular domain (16 amino acids) and transmem‐ brane/cytoplasmic domains of VSV-G (Fig. 3A) (Kato et al., 2011c). After injection of the NeuRet vector encoding GFP transgene into the mouse striatum, we found enhanced retro‐ grade gene transfer into the brain regions innervating the striatum, such as the M1, S1, PF, and SNc (Fig. 3B). The efficiency of gene transfer of the NeuRet vector was slightly different with that of the HiRet vector (FuG-B2 pseudo type), depending on the neural pathways (see a review by Kato et al. 2012). In addition, we tested gene transduction of the NeuRet vector surrounding the injection sites. Although the NeuRet vector transduced only a small num‐

et al., 2011a)

**2.2. NeuRet vector**

390 Gene Therapy - Tools and Potential Applications

The nigrostriatal dopamine system is a major target for gene therapy of Parkinson's disease. The availability of the HiRet vector for gene transfer via retrograde transport into the nigros‐ triatal dopamine system in nonhuman primates was described in our previous review (Kato et al., 2011d). To verify the capability of the NeuRet vector for efficient retrograde gene transfer into the nigrostriatal pathway, we injected the NeuRetvector encoding the GFP transgene into the striatum (caudate nucleus and putamen) of crab-eating monkeys (Fig. 4A). Intrastriatal injection of the NeuRet vector produced a larger number of GFP-positive neurons in the SNc (Fig. 4B). These positive signals were in register with immunostaining for tyrosine hydroxylase, a marker of dopaminergic neurons (Fig. 4C), indicating the trans‐ gene expression in the nigrostriatal dopaminergic neurons. In addition, we assessed the property of gene transduction with the NeuRet vector around the injection sites in the mon‐ key striatum. The vector displayed a low level of gene transfer into neuronal cell bodies (~13%), and the level of vector transduction into glial cells was also quite low in the monkey striatum (~0.6%) (Fig. 4D).The pattern of gene transduction around the injection sites was similar to that obtained from the analysis of the mouse brain sections. Therefore, the NeuRet vector mediates enhanced retrograde gene transfer, whereas it reduces the gene transfer into glial cells around the injection areas in both rodent and monkey brains.

**Figure 4. Transgene expression in the nigrostriatal dopamine system by NeuRet vector injection into the mon‐ key striatum.** (**A**) Gene transfer through retrograde transport after intrastriatal injection. The NeuRet vector encoding GFP transgene was stereotaxically injected into the caudate nucleus and the putamen, and histological analysis was performed on the brains fixed at the 4-week postinjection period. (**B**) GFP immunostaining in the SNc.Cp, cerebral pe‐ duncle; SNr, substantia nigra pars reticulata. (**C**) Double immunofluorescence staining for GFP and tyrosine hydroxy‐ lase (TH) in the SNc. (**D**) Double immunofluorescence staining for GFP/NeuN or GFP/glial fibrillary acidic protein

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In this chapter, we mentioned the gene transduction property of the HiRet and NeuRet vec‐ tors pseudotyped with different fusion envelope glycoproteins. These two vectors showed the enhancement in gene transfer through retrograde axonal transport into various neuronal populations in both rodent and nonhuman primate brains. The HiRet vector transduced prominently glial cells around the injection sites, whereas gene transduction of the NeuRet vector into glial cells was much less efficient. The transduction level of the NeuRet vector into neural stem/progenitor cells was also low. The variation in the structure of envelope glycoproteins shifted the efficiency of retrograde gene transfer and the preference of host range. In addition, we described the application of the NeuRet vector for retrograde gene transfer into the nigrostriatal dopamine system of monkeys. The NeuRet vector, together with the HiRet vector, will offer a promising technology for gene therapy of neurological diseases through enhanced retrograde gene transfer. In particular, the NeuRet vector system will improve the safety of gene therapy by greatly suppressing the risk of gene transduction

(GFAP) in the striatum. Scale bars: 500 μm (**B**), and 50 μm (**C, D**). (Data from Kato et al., 2011c)

into dividing cells in the central nervous system.

**4. Conclusion**

**Figure 3.** Gene delivery by NeuRet vector. (**A**) Structure of fusion envelope glycoprotein. FuG-C is composed of the Nterminal segment of the extracellular domain of RV-G and the C-terminal segment of the extracellular domain and the transmembrane(TM)/cytoplasmic domains of VSV-G. Amino acid sequences around the junction between the RV-G and VSV-G segments are shown. S, signal peptide. (**B**) Gene transfer through retrograde transport. The NeuRet vector encoding GFP transgene was injected into the mouse striatum, and four weeks later sections were processed for GFP immunostaining. GFP expression can be visualized in the M1, S1, PF, and SNc. (**C**) Gene transduction around the injec‐ tion sites. Sections through the striatum were stained by double immunofluorescence histochemistry for GFP/NeuN or for GFP/glial fibrillary acidic protein (GFAP). Scale bars: 50 μm. (Data from Kato et al., 2011c)

The NeuRet vector system successfully achieved efficient gene transfer through retrograde transport into the nigrostriatal dopaminergic neurons in nonhuman primates. Our vector system will provide a powerful strategy for gene therapy of Parkinson's disease with en‐ hanced retrograde gene transfer in the near future. This system will improve the safety of gene therapy by reducing the risk of gene transduction into proliferating cells (glial and neural stem/progenitor cells) in the brain.

Vectors for Highly Efficient and Neuron-Specific Retrograde Gene Transfer for Gene Therapy of Neurological Diseases http://dx.doi.org/10.5772/52611 393

**Figure 4. Transgene expression in the nigrostriatal dopamine system by NeuRet vector injection into the mon‐ key striatum.** (**A**) Gene transfer through retrograde transport after intrastriatal injection. The NeuRet vector encoding GFP transgene was stereotaxically injected into the caudate nucleus and the putamen, and histological analysis was performed on the brains fixed at the 4-week postinjection period. (**B**) GFP immunostaining in the SNc.Cp, cerebral pe‐ duncle; SNr, substantia nigra pars reticulata. (**C**) Double immunofluorescence staining for GFP and tyrosine hydroxy‐ lase (TH) in the SNc. (**D**) Double immunofluorescence staining for GFP/NeuN or GFP/glial fibrillary acidic protein (GFAP) in the striatum. Scale bars: 500 μm (**B**), and 50 μm (**C, D**). (Data from Kato et al., 2011c)

#### **4. Conclusion**

property of gene transduction with the NeuRet vector around the injection sites in the mon‐ key striatum. The vector displayed a low level of gene transfer into neuronal cell bodies (~13%), and the level of vector transduction into glial cells was also quite low in the monkey striatum (~0.6%) (Fig. 4D).The pattern of gene transduction around the injection sites was similar to that obtained from the analysis of the mouse brain sections. Therefore, the NeuRet vector mediates enhanced retrograde gene transfer, whereas it reduces the gene transfer into

**Figure 3.** Gene delivery by NeuRet vector. (**A**) Structure of fusion envelope glycoprotein. FuG-C is composed of the Nterminal segment of the extracellular domain of RV-G and the C-terminal segment of the extracellular domain and the transmembrane(TM)/cytoplasmic domains of VSV-G. Amino acid sequences around the junction between the RV-G and VSV-G segments are shown. S, signal peptide. (**B**) Gene transfer through retrograde transport. The NeuRet vector encoding GFP transgene was injected into the mouse striatum, and four weeks later sections were processed for GFP immunostaining. GFP expression can be visualized in the M1, S1, PF, and SNc. (**C**) Gene transduction around the injec‐ tion sites. Sections through the striatum were stained by double immunofluorescence histochemistry for GFP/NeuN or

The NeuRet vector system successfully achieved efficient gene transfer through retrograde transport into the nigrostriatal dopaminergic neurons in nonhuman primates. Our vector system will provide a powerful strategy for gene therapy of Parkinson's disease with en‐ hanced retrograde gene transfer in the near future. This system will improve the safety of gene therapy by reducing the risk of gene transduction into proliferating cells (glial and

for GFP/glial fibrillary acidic protein (GFAP). Scale bars: 50 μm. (Data from Kato et al., 2011c)

neural stem/progenitor cells) in the brain.

glial cells around the injection areas in both rodent and monkey brains.

392 Gene Therapy - Tools and Potential Applications

In this chapter, we mentioned the gene transduction property of the HiRet and NeuRet vec‐ tors pseudotyped with different fusion envelope glycoproteins. These two vectors showed the enhancement in gene transfer through retrograde axonal transport into various neuronal populations in both rodent and nonhuman primate brains. The HiRet vector transduced prominently glial cells around the injection sites, whereas gene transduction of the NeuRet vector into glial cells was much less efficient. The transduction level of the NeuRet vector into neural stem/progenitor cells was also low. The variation in the structure of envelope glycoproteins shifted the efficiency of retrograde gene transfer and the preference of host range. In addition, we described the application of the NeuRet vector for retrograde gene transfer into the nigrostriatal dopamine system of monkeys. The NeuRet vector, together with the HiRet vector, will offer a promising technology for gene therapy of neurological diseases through enhanced retrograde gene transfer. In particular, the NeuRet vector system will improve the safety of gene therapy by greatly suppressing the risk of gene transduction into dividing cells in the central nervous system.

#### **Acknowledgements**

This work was supported by grants-in aid from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST). A part of this work was supported by "Highly Creative Animal Model Development for Brain Sciences" carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank St. Jude Children's Research Hospital (Dr. A. Nienhuis) and the George Washington University for providing the HIV-1 based vector system. We also grateful to M. Kikuchi, N. Sato, M. Watanabe, and T. Kobaya‐ shi for their technical support in the animal experiments.

Vectors by a Rabies/Vesicular Stomatitis Virus Chimeric Envelope Glycoprotein.

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[5] Ciceri F., Bonini C., Stanghellini M. T., Bondanza A., Traversari C., SalomoniM., Turchetto L., Colombi S., Bernardi M., Peccatori J., Pescarollo A., Servida P., Magnani Z., Perna S. K., Valtolina V., Crippa F., Callegaro L., Spoldi E., Crocchiolo R., Fleisch‐ hauer K., Ponzoni M., Vago L., Rossini S., Santoro A., Todisco E., Apperley J., Olavar‐ ria E., Slavin S., Weissinger E. M., Ganser A., Stadler M., Yannaki E., Fassas A., Anagnostopoulos A., Bregni M., Stampino C. G., Bruzzi P. & Bordignon C. (2009). In‐ fusion of Suicide-gene-engineered Donor Lymphocytes after Family Haploidentical Haemopoietic Stem-cell Transplantation for Leukaemia (the TK007 Trial): a Nonrandomised Phase I-II Study. *The Lancet Oncology,* Vol. 10, No. 5, (May), pp. 489-500. [6] Coulon P., Ternaux J. P., Flamand A. & Tuffereau C. (1998). An Avirulent Mutant of Rabies Virus Is Unable to Infect Motoneurons *In Vivo* and *In Vitro*. *Journal of Virology*,

Vectors for Highly Efficient and Neuron-Specific Retrograde Gene Transfer for Gene Therapy of Neurological Diseases

[7] Cronin, J., Zhang, X. Y., & Reiser, J. (2005). Altering the Tropism of Lentiviral Vectors

[8] Deeks, S. G., Wagner, B., Anton, P. A., Mitsuyasu, R. T., Scadden, D. T., Huang, C., Macken, C., Richman, D. D., Christopherson, C., June, C. H., Lazar, R., Broad, D. F., Jalali, S., & Hege, K. M. (2002). A Phase II Randomized Study of HIV-specific T-cell Gene Therapy in Subjects with Undetectable Plasma Viremia on Combination Anti‐

[9] De Palma M., Montini E., Santoni de Sio F. R. S., Benedicenti F., Gentile A., Medico E. & Naldini, L. (2005). Promoter Trapping Reveals Significant Differences in Integra‐ tion Site Selection between MLV and HIV Vectors in Primary Hematopoietic Cells.

[10] Joshi S. & Joshi R. L. (1996). Molecular Biology of Human Immunodeficiency Virus

[11] Kato S., Inoue K., Kobayashi K., Yasoshima Y., Miyachi S., Inoue S., Hanawa H., Shi‐ mada T., Takada M. & Kobayashi K. (2007). Efficient Gene Transfer via Retrograde Transport in Rodent and Primate Brains Using a Human Immunodeficiency Virus Type 1-Based Vector Pseudotyped with Rabies Virus Glycoprotein. *Human Gene*

[12] Kato S., Kobayashi K., Inoue K., Kuramochi M., OkadaT., Yaginuma H., Morimoto K., Shimada T., Takada M. & Kobayashi K. (2011a). A Lentiviral Strategy for Highly Efficient Retrograde Gene Transfer by Pseudotyping with Fusion Envelope Glyco‐

[13] Kato S., Kuramochi M., Kobayashi K., Fukabori R., Okada K., Uchigashima M., Wata‐ nabe M., Tsutsui Y. & Kobayashi K. (2011b). Selective Neural Pathway Targeting Re‐ veals Key Roles of Thalamostriatal Projection in the Control of Visual Discrimination.

Type-1. *Transfusion Science,* Vol. 17, No. 3, (September), pp. 351-378.

protein. *Human Gene Therapy*, Vol. 22, No. 2, (February), pp. 197-206.

*Journal of Neuroscience,* Vol. 31, No. 47, (November), pp. 17169-17179.

through Pseudotyping. *Current Gene Therapy*, (August), 5(4), 387-398.

retroviral Therapy. *Molecular Therapy*, (June), 5(6), 788-797.

*Therapy*, Vol. 18, No. 11, (November), pp. 1141-1151.

*Gene Therapy*, Vol. 19, No. 7, (September), pp. 761-774.

(January), 72(1), pp. 273-278.

*Blood,* (March), 105(6), pp. 2307-2315.

#### **Author details**

Shigeki Kato1 , Kenta Kobayashi2 , Ken-ichi Inoue3 , Masahiko Takada3 and Kazuto Kobayashi1

1 Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical University School of Medicine, Fukushima, Japan

2 Section of Viral Vector Development, National Institute of Physiological Sciences, Okazaki, Japan

3 Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Ja‐ pan

#### **References**


Vectors by a Rabies/Vesicular Stomatitis Virus Chimeric Envelope Glycoprotein. *Gene Therapy*, Vol. 19, No. 7, (September), pp. 761-774.

**Acknowledgements**

394 Gene Therapy - Tools and Potential Applications

**Author details**

Shigeki Kato1

Japan

pan

**References**

shi for their technical support in the animal experiments.

, Kenta Kobayashi2

No. 6900, (May), pp. 413-417.

(February), 83(2), pp. 233-242.

54, No. 6, (December), pp. 766-777.

University School of Medicine, Fukushima, Japan

This work was supported by grants-in aid from Core Research for Evolutional Science and Technology (CREST) of Japan Science and Technology Agency (JST). A part of this work was supported by "Highly Creative Animal Model Development for Brain Sciences" carried out under the Strategic Research Program for Brain Sciences by the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank St. Jude Children's Research Hospital (Dr. A. Nienhuis) and the George Washington University for providing the HIV-1 based vector system. We also grateful to M. Kikuchi, N. Sato, M. Watanabe, and T. Kobaya‐

, Ken-ichi Inoue3

1 Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical

2 Section of Viral Vector Development, National Institute of Physiological Sciences, Okazaki,

3 Systems Neuroscience Section, Primate Research Institute, Kyoto University, Inuyama, Ja‐

[1] Azzouz M., Ralph G. S., Storkebaum E., Walmsley L. E., Mitrophanous K. A., Kings‐ man S. M., Carmeliet P. & Mazarakis N. D. (2004). VEGF Delivery with Retrogradely Transported Lentivector Prolongs Survival in a Mouse ALS Model. *Nature*, Vol. 429,

[2] Barkats M, Horellou P, Colin P, Millecamps S, Faucon-Biguet N, Mallet J. (2006). 1- Methyl-4-phenylpyridinium Neurotoxicity Is Attenuated by Adenoviral Gene Trans‐ fer of Human Cu/Zn Superoxide Dismutase. *Journal of Neuroscience Research,*

[3] Baumgartner B. J. & Shine H. D. (1998). Permanent Rescue of Lesioned Neonatal Mo‐ toneurons and Enhanced Axonal Regeneration by Adenovirus-Mediated Expression of Glial Cell Line-Derived Neurotrophic Factor. *Journal of Neuroscience Research*, Vol.

[4] Carpentier D. C. J., Vevis K., Trabalza A., Georgiadis C., Ellison S. M., Asfahani R. L. & Mazarakis N. D. (2012). Enhanced Pseudotyping Efficiency of HIV-1 Lentiviral

, Masahiko Takada3

and Kazuto Kobayashi1


[14] Kato S., Kuramochi M., Takasumi K., Kobayashi K., Inoue K., Takahara D., Hitoshi S., Ikenaka K., Shimada T., Takada M. & Kobayashi K. (2011c). Neuron-Specific Gene Transfer through Retrograde Transport of Lentiviral Vector Pseudotyped with a Novel Type of Fusion Envelope Glycoprotein. *Human Gene Therapy*, Vol. 22, No. 12, (December), pp. 1511-1523.

[24] Naldini L., Blömer U., Gage F. H., Trono D. & Verma, I. M. (1996). Efficient Transfer, Integration, and Sustained Long-Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector. *Proceedings of the National Academy of Sciences*

Vectors for Highly Efficient and Neuron-Specific Retrograde Gene Transfer for Gene Therapy of Neurological Diseases

http://dx.doi.org/10.5772/52611

397

[25] Nielsen, M.H., Pedersen, F.S. & Kjems, J. (2005). Molecular Strategy to Inhibit HIV-1

[26] Perrelet D., Ferri A., MacKenzie A. E., Smith G. M., Korneluk R. G., Liston P., Sagot Y., Terrado J., Monnier D. & Kato A. C. (2000). IAP Family Proteins Delay Motoneur‐ on Cell Death *In Vivo*. *European Journal of Neuroscience*, Vol. 12, No. 6, (June), pp.

[27] Pluta, K. & Kacprzak, M. M. (2009). Use of HIV as a Gene Transfer Vector. *Acta Bio‐*

[28] Prehaud C., Coulon P., Lafay F., Thiers C. & Flamand A. (1988). Antigenic Site II of the Rabies Virus Glycoprotein: Structure and Role in Viral Virulence. *Journal of Virolo‐*

[29] Rabson, A.B. & Martin, M.A. (1985). Molecular Organization of the AIDS Retrovirus.

[30] Reiser J., Harmison G., Kluepfel-Stahl S., Brady R. O., Karlsson S. & Schubert M. (1996). Transduction of Nondividing Cells Using Pseudotyped Defective High-Titer HIV Type 1 Particles. *Proceedings of the National Academy of Sciences of the United States*

[31] Rose J. K., Doolittle R. F., Anilionis A., Curtis P. J. & Wunner W. H. (1982). Homology between the Glycoproteins of Vesicular Stomatitis Virus and Rabies Virus. *Journal of*

[32] Rossi, J.J., June, C.H., & Kohn, D.B. (2007). Genetic Therapies against HIV. *Nature Bio‐*

[33] Sakamoto T., Kawagoe Y., Shen J. S., Takeda Y., Arakawa Y., Ogawa J., Oyanagi K., Ohashi T., Watanabe K., Inoue K., Eto Y. & Watabe K. (2003). Adenoviral Gene Transfer of GDNF, BDNF and TGF, but not CNTF, Cardiotrophin-1 or IGF1, Pro‐ tects Injured Adult Motoneurons after Facial Nerve Avulsion. *Journal of Neuroscience*

[34] Themis M., Waddington S. N., Schmidt M., von Kalle C., Wang Y., Al-Allaf F., Grego‐ ry L. G., Nivsarkar M., Themis M., Holder M. V., Buckley S. M., Dighe N., Ruthe A. T., Mistry A., Bigger B., Rahim A., Nguyen T. H., Trono D., Thrasher A. J. & Coutelle C. (2005). Oncogenesis Following Delivery of a Nonprimate Lentiviral Gene Therapy Vector to Fetal and Neonatal Mice. *Molecular Therapy*, (October), 12(4), pp. 763-771.

[35] Wong L. F., Goodhead L., Prat C., Mitrophanous K. A., Kingsman S. M. & Mazarakis N. D. (2006). Lentivirus-Mediated Gene Transfer to the Central Nervous System:

*of the United States of America*, (October), 93(21), pp. 11382-11388.

Replication. *Retrovirology*, (February), 2(10), 1-20.

*chimica Polonica*, (November), 56(4), 531-595.

*of America*, (December), 93(26), pp. 15266-15271.

*Virology,* Vol. 43, No. 1, (July), pp. 361-364.

*technology*, (December), 25(12), 1444-1454.

*Research*, Vol. 72, No. 1, (April), pp. 54-64.

*gy,* Vol. 62, No. 1, (January), pp. 1-7.

*Cell*, (March), 40(3), 477-480.

2059-2067.


[24] Naldini L., Blömer U., Gage F. H., Trono D. & Verma, I. M. (1996). Efficient Transfer, Integration, and Sustained Long-Term Expression of the Transgene in Adult Rat Brains Injected with a Lentiviral Vector. *Proceedings of the National Academy of Sciences of the United States of America*, (October), 93(21), pp. 11382-11388.

[14] Kato S., Kuramochi M., Takasumi K., Kobayashi K., Inoue K., Takahara D., Hitoshi S., Ikenaka K., Shimada T., Takada M. & Kobayashi K. (2011c). Neuron-Specific Gene Transfer through Retrograde Transport of Lentiviral Vector Pseudotyped with a Novel Type of Fusion Envelope Glycoprotein. *Human Gene Therapy*, Vol. 22, No. 12,

[15] Kato S., Kobayashi K., Kuramochi M., Inoue K., Takada M. & Kobayashi K. (2011d) Highly efficient retrograde gene transfer for genetic treatment of neurological diseas‐ es. *Viral Gene Therapy* (ed. KeXu) Chapter 17, InTech, Rijeka (Croatia), pp. 371-380.

[16] Kato, S., Kobayashi, K. & Kobayashi, K. (2012). Dissecting Circuit Mechanisms by Genetic Manipulation of Specific Neural Pathways. *Reviews in Neurosciences*, in press.

[17] Lundberg C., Björklund T., Carlsson T., Jakobsson J., Hantraye P., Déglon N. & Kirik D. (2008). Applications of Lentiviral Vectors for Biology and Gene Therapy of Neuro‐

logical Disorders. *Current Gene Therapy*, Vol. 8, No. 6, (December), pp. 461-473.

[18] Mavilio F., Pellegrini G., Ferrari S., Di Nunzio F., Di Iorio E., Recchia A., Maruggi G., Ferrari G., Provasi E., Bonini C., Capurro S., Conti A., Magnoni C., Giannetti A. & De Luca M. (2006). Correction of Junctional Epidermolysis Bullosa by Transplantation of Genetically Modified Epidermal Stem Cells. *Nature Medicine,* Vol. 12, No. 12, (Decem‐

[19] Mazarakis N. D., Azzouz M., Rohll J. B., Ellard F. M., Wilkes F. J., Olsen A. L., Carter E. E., Barber R. D., Baban D. F., Kingsman S. M., Kingsman A. J., O'Malley K. & Mi‐ trophanous K. A. (2001). RabiesVirus Glycoprotein Pseudotyping of Lentiviral Vec‐ tors Enables Retrograde Axonal Transport and Access to the Nervous System after Peripheral Delivery. *Human Molecular Genetics*, Vol. 10, No. 19, (September), pp.

[20] Mitrophanous K., Yoon S., Rohll J., Patil D., Wilkes F., Kim V., Kingsman S. Kings‐ man A. & Mazarakis N. (1999). Stable Gene Transfer to the Nervous System Using a Non-Primate Lentiviral Vector. *Gene Therapy*, Vol. 6, No. 11, (November), pp.

[21] Mochizuki H., Schwartz J. P., Tanaka K., Brady R. O. & Reiser J. (1998). High-Titer Human Immunodeficiency Virus Type 1-Based Vector Systems for Gene Delivery in‐ to Nondividing Cells. *Journalof Virology*, Vol. 72, No. 11, (November), pp. 8873-8883.

[22] Montini E., Cesana D., Schmidt M., Sanvito F., Ponzoni M., Bartholomae C., Sergi L. S., Benedicenti F., Ambrosi A., Di Serio C., Doglioni C., von Kalle C. & Naldini L. (2006). Hematopoietic Stem Cell Gene Transfer in a Tumor-Prone Mouse Model Un‐ covers Low Genotoxicity of LentiviralVector Integration. *Nature Biotechnology,* Vol.

[23] Naldini, L. (2011). *Ex Vivo* Gene Transfer and Correction for Cell-Based Therapies.

(December), pp. 1511-1523.

396 Gene Therapy - Tools and Potential Applications

ber), pp. 1397-1402.

2109-2121.

1808-1818.

24, No. 6, (June), pp. 687-696.

*Nature Reviews Genetics*, (May), 12 (5), 301-315.


Therapeutic and Research Applications. *Human Gene Therapy*, Vol. 17, No. 1, (Janu‐ ary), pp. 1-9.

**Chapter 16**

**Retroviral Genotoxicity**

http://dx.doi.org/10.5772/52530

**1. Introduction**

Dustin T. Rae and Grant D. Trobridge

Additional information is available at the end of the chapter

Gene therapies have enormous potential to cure human disease. In recent years, hematopoiet‐ ic stem cell (HSC) gene therapy has advanced tremendously, due in part to years of intense re‐ search to develop effective vectors and efficient ex vivo transduction protocols. In early clinical trials, inefficient gene transfer resulted in either a lack of therapeutic benefit or shortlived therapeutic benefit [1-3]. Advances in preclinical animal models, led to improved gene transfer in human clinical trials, where long-term efficacy has now been achieved. HSC gene therapy has been used to correct several monoallelic genetic diseases [4], such as X-linked se‐ vere combined immune-deficiency (SCID X-1) [5], chronic granulomatous disease (CGD) [6-8], adenine deaminase deficiency (ADA-SCID) [9-12], Wiskott-Aldrich syndrome [13-14], and X-linked adrenoleukodysrophy [15,16]. Recently HSC gene therapy has also been used to treat glioblastoma [17], X-linked hyper-immunoglobulin M syndrome (HIGM), and familial haemophagocyticlymphohistiocytosis syndrome (HLH) [18]. These successes are in large part due to advances in ex vivo transduction protocols and improvements with recombinant vec‐ tor technologies. The French SCID-X1 HSC gene therapy trial marked a major turning point in the field when nine of the ten patients treated exhibited therapeutic benefit. However, follow‐ ing this exciting achievement the field was dealt a major setback when it was initially report‐ ed, that two patients from the study had developed vector-mediated leukemia resulting from the treatment [19]. This was the first vector-mediated malignancy reported in a HSC gene therapy clinical trial. Four boys ultimately developed leukemia as a side effect of the gene therapy procedure [5]. Three of the four boys were successfully treated with chemotherapy, but one patient died due to vector-mediated T cell leukemia. In these patients, vector-mediat‐ ed dysregulation of host genes led to leukemia, and this unwanted adverse side effect is cur‐ rently a major challenge for HSC gene therapy. The effect of the integrated viral vector on host

gene expression resulting in an altered phenotype is known as genotoxicity.

© 2013 Rae and Trobridge; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

[36] Zheng J. S., Tang L. L., Zheng S. S., Zhan R. Y., Zhou Y. Q., Goudreau J., Kaufman D. & Chen A. F. (2005). Delayed Gene Therapy of Glial Cell Line-Derived Neurotrophic Factor is Efficacious in a Rat Model of Parkinson's Disease. *Molecular Brain Research*, Vol. 134, No. 1, (March), pp. 155-161.

**Chapter 16**

### **Retroviral Genotoxicity**

Dustin T. Rae and Grant D. Trobridge

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52530

#### **1. Introduction**

Therapeutic and Research Applications. *Human Gene Therapy*, Vol. 17, No. 1, (Janu‐

[36] Zheng J. S., Tang L. L., Zheng S. S., Zhan R. Y., Zhou Y. Q., Goudreau J., Kaufman D. & Chen A. F. (2005). Delayed Gene Therapy of Glial Cell Line-Derived Neurotrophic Factor is Efficacious in a Rat Model of Parkinson's Disease. *Molecular Brain Research*,

ary), pp. 1-9.

398 Gene Therapy - Tools and Potential Applications

Vol. 134, No. 1, (March), pp. 155-161.

Gene therapies have enormous potential to cure human disease. In recent years, hematopoiet‐ ic stem cell (HSC) gene therapy has advanced tremendously, due in part to years of intense re‐ search to develop effective vectors and efficient ex vivo transduction protocols. In early clinical trials, inefficient gene transfer resulted in either a lack of therapeutic benefit or shortlived therapeutic benefit [1-3]. Advances in preclinical animal models, led to improved gene transfer in human clinical trials, where long-term efficacy has now been achieved. HSC gene therapy has been used to correct several monoallelic genetic diseases [4], such as X-linked se‐ vere combined immune-deficiency (SCID X-1) [5], chronic granulomatous disease (CGD) [6-8], adenine deaminase deficiency (ADA-SCID) [9-12], Wiskott-Aldrich syndrome [13-14], and X-linked adrenoleukodysrophy [15,16]. Recently HSC gene therapy has also been used to treat glioblastoma [17], X-linked hyper-immunoglobulin M syndrome (HIGM), and familial haemophagocyticlymphohistiocytosis syndrome (HLH) [18]. These successes are in large part due to advances in ex vivo transduction protocols and improvements with recombinant vec‐ tor technologies. The French SCID-X1 HSC gene therapy trial marked a major turning point in the field when nine of the ten patients treated exhibited therapeutic benefit. However, follow‐ ing this exciting achievement the field was dealt a major setback when it was initially report‐ ed, that two patients from the study had developed vector-mediated leukemia resulting from the treatment [19]. This was the first vector-mediated malignancy reported in a HSC gene therapy clinical trial. Four boys ultimately developed leukemia as a side effect of the gene therapy procedure [5]. Three of the four boys were successfully treated with chemotherapy, but one patient died due to vector-mediated T cell leukemia. In these patients, vector-mediat‐ ed dysregulation of host genes led to leukemia, and this unwanted adverse side effect is cur‐ rently a major challenge for HSC gene therapy. The effect of the integrated viral vector on host gene expression resulting in an altered phenotype is known as genotoxicity.

© 2013 Rae and Trobridge; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Genotoxicity is a result of retroviral mediated delivery of the integrated form of the retrovi‐ ral vector genome known as the vector provirus into the host genome. Integration of the vector provirus into a host chromosome, by definition, alters the host DNA. In cases where a retrovirus or retroviral vector provirus has dysregulated host gene expression, insertional mutagenesis is said to have occurred. However, it is important to remember that provirus integration always results in mutation of the host genome, regardless of whether the vector provirus exerts an effect on host gene expression. The oncogenic properties of replicationcompetent retroviruses were well known prior to the development of retroviral vectors for gene therapy. However, vectors that are used in gene therapy have been engineered so that they do not have the ability to replicate, only to insert their genome into a target cell. These vectors are thus referred to as replication-incompetent. In numerous preclinical and clinical studies conducted prior to the SCID-X1 trial, malignancies were not observed when using replication-incompetent vector systems [20]. It was therefore assumed that the potential for malignant transformation from a replication-incompetent vector was very low. Unfortunate‐ ly, it has now been clearly shown in the French SCID-X1 trial and in subsequent HSC gene therapy trials, that genotoxicity is indeed a problem for replication-incompetent vectors. Here we review the mechanisms of vector-mediated genotoxicity in HSC gene therapy and describe efforts in the field to reduce genotoxicity which is currently a major challenge in the field [21, 22].

> **Figure 1.** Human Hematopoiesis. Long-term-hematopoietic stem cells (LT-HSCs) are a self-renewing population of stem cells that reconstitute the blood system throughout the entirety of our life span. Short-term-HSCs, reconstitute our blood system for only limited periods. The short-term-HSCs differentiate into multipotent progenitors (MPPs), which have the ability to differentiate into several transit amplifying cell lineages. Common lymphoid progenitors (CLPs) differentiate into (Pro- Dendritic Cell, Pro-T, Pro-NK, and Pro-B) lymphoid progenitor cells. Finally, these progeni‐ tors give rise to the mature lymphoid class cells of the blood system (T- lymphocytes, B-lymphocytes, and natural killer (NK) cells). Common myeloid progenitors (CMPs), give rise to granulocyte-macrophage progenitors (GMPs) and mega‐ karyocyte-erythroid progenitors (MEPs) that differentiate into :( macrophages, granulocytes, megakaryocytes, and er‐ ythroid) myeloid class progenitors. Finally, these progenitors give rise to the mature myeloid class cells of the blood

Retroviral Genotoxicity

401

http://dx.doi.org/10.5772/52530

We now know that the use of retroviral vectors for HSC gene therapy, though highly effi‐ cient, can dysregulate host genes near the vector provirus and ultimately lead to malignant transformation. The ability of replicating retroviruses to cause tumorigenesis is well estab‐ lished. In 1911, Peyton Rous showed that a sarcoma growing on a domestic chicken could be transferred to another chicken by exposing the healthy bird to a cell-free filtrate [23]. This filterable agent is now known to be the Rous sarcoma retrovirus. Since this report, many ret‐ roviruses have been discovered that cause diverse malignancies. There are several mecha‐ nisms whereby retroviruses can cause malignancy. Varmus et al. showed that acutely transforming onco-retroviruses capture and deliver cellular oncogenes, which allow these viruses to efficiently, convert target cells into a malignant phenotype [24]. It is important to

system.

**3. Retroviruses as insertional mutagens**

#### **2. Why integrating vectors are used for HSC gene therapy**

Why use integrating vectors for HSC gene therapy if we know that retroviral vectors muta‐ genize the genome and therefore carry a risk to induce genotoxicity? The answer is that pro‐ virus integration to HSCs is currently the only way to efficiently and stably deliver transgenes to the billions of mature blood cells produced every day in the body. Our mature blood cells are generated from a relatively small pool of self-renewing long term-repopulat‐ ing HSCs in the bone marrow through a process known as hematopoiesis (Figure 1). During hematopoiesis, long-term repopulating stem cells provide lifelong supplies of the mature cells of each blood cell lineage via massive expansion of transit-amplifying cells that include multi-potent and lineage restricted progenitors. By permanently modifying a long term re‐ populating HSC via proviral integration into the HSC genome we can ensure that all proge‐ ny produced from these gene-modified cells will inherit the transgene during mitosis. Thus, the mature blood cells that arise during hematopoiesis from gene-modified HSCs and their daughter transit amplifying cells all inherit the transgene. Using retroviral vectors to effi‐ ciently deliver a therapeutic transgene via integration of the vector provirus into a HSC is currently the only effective approach for HSC gene therapy. While there have been reports of some success with adenovirus and other non-integrating approaches in small animal models, to date only integrating vectors have been used successfully for HSC gene therapy in large animal models and in clinical trials.

**Figure 1.** Human Hematopoiesis. Long-term-hematopoietic stem cells (LT-HSCs) are a self-renewing population of stem cells that reconstitute the blood system throughout the entirety of our life span. Short-term-HSCs, reconstitute our blood system for only limited periods. The short-term-HSCs differentiate into multipotent progenitors (MPPs), which have the ability to differentiate into several transit amplifying cell lineages. Common lymphoid progenitors (CLPs) differentiate into (Pro- Dendritic Cell, Pro-T, Pro-NK, and Pro-B) lymphoid progenitor cells. Finally, these progeni‐ tors give rise to the mature lymphoid class cells of the blood system (T- lymphocytes, B-lymphocytes, and natural killer (NK) cells). Common myeloid progenitors (CMPs), give rise to granulocyte-macrophage progenitors (GMPs) and mega‐ karyocyte-erythroid progenitors (MEPs) that differentiate into :( macrophages, granulocytes, megakaryocytes, and er‐ ythroid) myeloid class progenitors. Finally, these progenitors give rise to the mature myeloid class cells of the blood system.

#### **3. Retroviruses as insertional mutagens**

Genotoxicity is a result of retroviral mediated delivery of the integrated form of the retrovi‐ ral vector genome known as the vector provirus into the host genome. Integration of the vector provirus into a host chromosome, by definition, alters the host DNA. In cases where a retrovirus or retroviral vector provirus has dysregulated host gene expression, insertional mutagenesis is said to have occurred. However, it is important to remember that provirus integration always results in mutation of the host genome, regardless of whether the vector provirus exerts an effect on host gene expression. The oncogenic properties of replicationcompetent retroviruses were well known prior to the development of retroviral vectors for gene therapy. However, vectors that are used in gene therapy have been engineered so that they do not have the ability to replicate, only to insert their genome into a target cell. These vectors are thus referred to as replication-incompetent. In numerous preclinical and clinical studies conducted prior to the SCID-X1 trial, malignancies were not observed when using replication-incompetent vector systems [20]. It was therefore assumed that the potential for malignant transformation from a replication-incompetent vector was very low. Unfortunate‐ ly, it has now been clearly shown in the French SCID-X1 trial and in subsequent HSC gene therapy trials, that genotoxicity is indeed a problem for replication-incompetent vectors. Here we review the mechanisms of vector-mediated genotoxicity in HSC gene therapy and describe efforts in the field to reduce genotoxicity which is currently a major challenge in the

**2. Why integrating vectors are used for HSC gene therapy**

in large animal models and in clinical trials.

Why use integrating vectors for HSC gene therapy if we know that retroviral vectors muta‐ genize the genome and therefore carry a risk to induce genotoxicity? The answer is that pro‐ virus integration to HSCs is currently the only way to efficiently and stably deliver transgenes to the billions of mature blood cells produced every day in the body. Our mature blood cells are generated from a relatively small pool of self-renewing long term-repopulat‐ ing HSCs in the bone marrow through a process known as hematopoiesis (Figure 1). During hematopoiesis, long-term repopulating stem cells provide lifelong supplies of the mature cells of each blood cell lineage via massive expansion of transit-amplifying cells that include multi-potent and lineage restricted progenitors. By permanently modifying a long term re‐ populating HSC via proviral integration into the HSC genome we can ensure that all proge‐ ny produced from these gene-modified cells will inherit the transgene during mitosis. Thus, the mature blood cells that arise during hematopoiesis from gene-modified HSCs and their daughter transit amplifying cells all inherit the transgene. Using retroviral vectors to effi‐ ciently deliver a therapeutic transgene via integration of the vector provirus into a HSC is currently the only effective approach for HSC gene therapy. While there have been reports of some success with adenovirus and other non-integrating approaches in small animal models, to date only integrating vectors have been used successfully for HSC gene therapy

field [21, 22].

400 Gene Therapy - Tools and Potential Applications

We now know that the use of retroviral vectors for HSC gene therapy, though highly effi‐ cient, can dysregulate host genes near the vector provirus and ultimately lead to malignant transformation. The ability of replicating retroviruses to cause tumorigenesis is well estab‐ lished. In 1911, Peyton Rous showed that a sarcoma growing on a domestic chicken could be transferred to another chicken by exposing the healthy bird to a cell-free filtrate [23]. This filterable agent is now known to be the Rous sarcoma retrovirus. Since this report, many ret‐ roviruses have been discovered that cause diverse malignancies. There are several mecha‐ nisms whereby retroviruses can cause malignancy. Varmus et al. showed that acutely transforming onco-retroviruses capture and deliver cellular oncogenes, which allow these viruses to efficiently, convert target cells into a malignant phenotype [24]. It is important to note that oncogene capture does not occur at a detectable frequency with current replica‐ tion-incompetent vectors used in gene therapy. Yet several mechanisms remain for cellular transformation from replication-incompetent retroviral vector proviruses (Figure 2). Despite the risks associated with malignant transformation from retroviral vectors via insertional mutagenesis, for several severe hematopoietic diseases the therapeutic benefit of HSC gene therapy outweighs the risks. Currently, major efforts are underway to further our under‐ standing of genotoxic events and to improve vector safety by reducing their genotoxic po‐ tential [18, 25, 26].

marker allows for rapid enrichment of HSCs, typically via column enrichment using CD34 antibody-conjugated magnetic beads. CD34-enriched cells that include repopulating HSCs are then exposed to the vector containing the therapeutic gene in an ex vivo transduction process. Following ex vivo transduction, gene modified cells must be infused into the pa‐ tient. Correction of the disease phenotype will occur if enough gene-modified repopulating cells engraft. Engraftment requires that gene modified cells survive, home to the bone mar‐

Retroviral Genotoxicity

403

http://dx.doi.org/10.5772/52530

Early preclinical HSC gene therapy studies in mice demonstrated high gene transfer rates. However, early clinical trials using similar approaches and culture conditions had inefficient gene transfer [28, 29]. Large animal models such as the dog and non-human primates more accurately model human HSC gene therapy and have since been used to establish the condi‐ tions for efficient ex vivo transduction [30]. More effective ex vivo gene protocols were de‐ veloped, that resulted in higher gene transfer efficiencies while maintaining efficient engraftment. These improvements included defining cytokine support and the extracellular matrix CH-296 fibronectin fragment [5, 31, 32]. Together these advances contributed to the success of HSC gene therapy clinical trials such as the French SCID-X1 trial, and now effi‐ cient transduction of human CD34+ cells can be routinely achieved using retroviral vectors. These improved gene transfer efficiencies also factored into the observed genotoxicity. For example, two patients in the French SCID-X1 trials were estimated to have a total of 4.3 × 106 and 11.3 × 106 CD34+γC + cells/kg body weight gene modified cells respectively [5]. Thus in each of these patients many proviral integrants exist with the potential to dysregulate many

**5. The SCID-X1 trials as an example of genotoxicity in HSC gene therapy**

SCID-X1 is a fatal X-linked inherited mutation of the IL2RG locus harboring the gamma c (γC) cytokine receptor common subunit [5]. Inactivating mutations in this gene prevent proper cellular communication and maturation of lymphoid progenitor cells. The loss of cellular signaling in lymphoid progenitors prevents the development of mature T, NK, and B cells. Many SCID-X1 patients fail to thrive or suffer morbidity and mortality in ear‐ ly life because of impaired immune function, which leaves them susceptible to life-threat‐ ening infection. Allogeneic HSC transplantation has been used to treat SCID-X1, but many patients do not have suitable donors. In addition, graft versus host disease is a major source of mortality for patients treated by this approach. Graft versus host disease occurs when transplanted (allogeneic) immune cells from a donor recognize the host recipient tis‐ sue as foreign and attack these cells. SCID-X1 HSC gene therapy using the γC transgene has a lower mortality rate and a higher treatment efficacy compared to conventional allo‐ geneic bone marrow transplants [25], and is currently the only therapeutic choice for pa‐ tients without a suitable donor. Prior to the French study, preclinical studies conducted in both murine and canine models corrected SCID-X1 deficiency was with no reported ad‐

row, and proliferate sufficiently to repopulate the blood system.

nearby genes, including proto-oncogenes.

verse events [20, 31, 33, and 34].

**Figure 2.** Mechanisms of Retroviral Mutagenesis. The black boxes represent promoters, and grey squares represent exons. A) The proviral 3' LTR can drive transcription of nearby cellular gene at an increased rate. B) Proviral LTR en‐ hancers can activate a nearby promoter, increasing transcription of cellular genes. C) Transcription from 5' LTR in con‐ junction with proviral cryptic splice sights creates novel isoforms and fusion transcripts of both cellular and viral genes. D) Proviral LTR methylation, induces epigenetic changes, silencing proviral genes and nearby cellular genes. E) Proviral integration can disrupt cellular gene expression by causing premature polyadenylation (pA) signaling.

#### **4. Overview of ex vivo HSC gene therapy**

It is important when studying genotoxicity to consider how the target cells are manipulated during the gene transfer process. HSC gene therapy is conceptually straightforward but re‐ quires culture of stem cells under the appropriate ex vivo conditions. A patient's cells are collected and enriched for repopulating stem cells using the CD34 marker. The CD34 pro‐ tein is a member of the sialomucin family, and is expressed in early HSCs [27]. The CD34 marker allows for rapid enrichment of HSCs, typically via column enrichment using CD34 antibody-conjugated magnetic beads. CD34-enriched cells that include repopulating HSCs are then exposed to the vector containing the therapeutic gene in an ex vivo transduction process. Following ex vivo transduction, gene modified cells must be infused into the pa‐ tient. Correction of the disease phenotype will occur if enough gene-modified repopulating cells engraft. Engraftment requires that gene modified cells survive, home to the bone mar‐ row, and proliferate sufficiently to repopulate the blood system.

note that oncogene capture does not occur at a detectable frequency with current replica‐ tion-incompetent vectors used in gene therapy. Yet several mechanisms remain for cellular transformation from replication-incompetent retroviral vector proviruses (Figure 2). Despite the risks associated with malignant transformation from retroviral vectors via insertional mutagenesis, for several severe hematopoietic diseases the therapeutic benefit of HSC gene therapy outweighs the risks. Currently, major efforts are underway to further our under‐ standing of genotoxic events and to improve vector safety by reducing their genotoxic po‐

**Figure 2.** Mechanisms of Retroviral Mutagenesis. The black boxes represent promoters, and grey squares represent exons. A) The proviral 3' LTR can drive transcription of nearby cellular gene at an increased rate. B) Proviral LTR en‐ hancers can activate a nearby promoter, increasing transcription of cellular genes. C) Transcription from 5' LTR in con‐ junction with proviral cryptic splice sights creates novel isoforms and fusion transcripts of both cellular and viral genes. D) Proviral LTR methylation, induces epigenetic changes, silencing proviral genes and nearby cellular genes. E) Proviral

It is important when studying genotoxicity to consider how the target cells are manipulated during the gene transfer process. HSC gene therapy is conceptually straightforward but re‐ quires culture of stem cells under the appropriate ex vivo conditions. A patient's cells are collected and enriched for repopulating stem cells using the CD34 marker. The CD34 pro‐ tein is a member of the sialomucin family, and is expressed in early HSCs [27]. The CD34

integration can disrupt cellular gene expression by causing premature polyadenylation (pA) signaling.

**4. Overview of ex vivo HSC gene therapy**

tential [18, 25, 26].

402 Gene Therapy - Tools and Potential Applications

Early preclinical HSC gene therapy studies in mice demonstrated high gene transfer rates. However, early clinical trials using similar approaches and culture conditions had inefficient gene transfer [28, 29]. Large animal models such as the dog and non-human primates more accurately model human HSC gene therapy and have since been used to establish the condi‐ tions for efficient ex vivo transduction [30]. More effective ex vivo gene protocols were de‐ veloped, that resulted in higher gene transfer efficiencies while maintaining efficient engraftment. These improvements included defining cytokine support and the extracellular matrix CH-296 fibronectin fragment [5, 31, 32]. Together these advances contributed to the success of HSC gene therapy clinical trials such as the French SCID-X1 trial, and now effi‐ cient transduction of human CD34+ cells can be routinely achieved using retroviral vectors. These improved gene transfer efficiencies also factored into the observed genotoxicity. For example, two patients in the French SCID-X1 trials were estimated to have a total of 4.3 × 106 and 11.3 × 106 CD34+γC + cells/kg body weight gene modified cells respectively [5]. Thus in each of these patients many proviral integrants exist with the potential to dysregulate many nearby genes, including proto-oncogenes.

#### **5. The SCID-X1 trials as an example of genotoxicity in HSC gene therapy**

SCID-X1 is a fatal X-linked inherited mutation of the IL2RG locus harboring the gamma c (γC) cytokine receptor common subunit [5]. Inactivating mutations in this gene prevent proper cellular communication and maturation of lymphoid progenitor cells. The loss of cellular signaling in lymphoid progenitors prevents the development of mature T, NK, and B cells. Many SCID-X1 patients fail to thrive or suffer morbidity and mortality in ear‐ ly life because of impaired immune function, which leaves them susceptible to life-threat‐ ening infection. Allogeneic HSC transplantation has been used to treat SCID-X1, but many patients do not have suitable donors. In addition, graft versus host disease is a major source of mortality for patients treated by this approach. Graft versus host disease occurs when transplanted (allogeneic) immune cells from a donor recognize the host recipient tis‐ sue as foreign and attack these cells. SCID-X1 HSC gene therapy using the γC transgene has a lower mortality rate and a higher treatment efficacy compared to conventional allo‐ geneic bone marrow transplants [25], and is currently the only therapeutic choice for pa‐ tients without a suitable donor. Prior to the French study, preclinical studies conducted in both murine and canine models corrected SCID-X1 deficiency was with no reported ad‐ verse events [20, 31, 33, and 34].

In the French SCID-X1 trial, four of the ten patients developed T-cell leukemia from inser‐ tional mutagenesis from the murine moloney leukemia virus (MLV)-based vector [5, 25]. Careful molecular analysis of leukemic cells showed that the MLV provirus integrated near the proto-oncogene, LMO2, and suggested that viral enhancer elements in the provirus con‐ tributed to leukemia (Figure 2B) [25]. The SCID-X1 trials were the first HSC gene therapy clinical trial where vector-mediated insertional mutagenesis led to cancer. In this trial, MLV vector LTR enhancers activated LMO2 expression, resulting in T-cell LMO2 dependent pro‐ liferation (Figure 2B). LMO2 is normally silenced in mature T-cells, and when viral enhanc‐ ers turn on expression, LMO2 drives T-cell proliferation by dysregulating transcription networks that affect the cell cycle. This promotes cell cycle escape and can result in higher proliferation rates compared to normal cells [35]. Leukemic transformation was a result of provirus integration near LMO2 with additional proviral integrants near other proto-onco‐ genes that resulted in expansion of cells with these mutations [5, 19].

pressed [39]. However, after proviral integration events, resulting in activation of LMO2, these gene-modified T-cells begin to expand from dysregulation of transcriptional regulato‐ ry pathways. In 2010, Oram et al. demonstrated that LMO2 expression in T cells activates FLI1 and ERG enhancers, known to be involved in blood stem/progenitor cells. These gene products of FLI1 and ERG in turn activate the enhancer of the HHEX/PRH gene locus, which has been shown to act in early progenitor cell expansion and formation of T-lineage acute lymphoblastic leukemia (T-ALL) [35]. LMO2 overexpression has also been demon‐ strated to reduce or eliminate cell cyclin dependent kinase (CDK) inhibitors promoting es‐ cape of the G1 cell cycle checkpoints during cellular division [40]. Once aberrantly expressed within a cell, LMO2 promotes cell cycle progression via multiple mechanisms, giving the

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Additional proto-oncogenes were activated in the French SCID-X1 patients. The BMI1 protooncogene normally functions in self-renewal and maintenance of hematopoietic primitive stem cells [40, 41]. Like LMO2, dysregulation of BMI-1 via enhancer activation results in clo‐ nal expansion. Additional mechanisms mediate clonal expansion, such as the high mobility group AT-hook2 (HMGA2) which can provide a proliferative advantage when the endoge‐ nous gene is truncated via insertional mutagenesis. Truncation results in the loss of regula‐ tory target sequences within the protein mRNA preventing degradation by the endogenous miRNA let7. These miRNA elements normally function to regulate HMGA2 via RNA inter‐ ference using the RNA induced silencing complex (RISC) machinery [42]. Truncated HMGA2 mRNA is not degraded thus continuing activation of gene networks involved with cell proliferation, and cell-cycle progression [42]. Another mechanism that can contribute to clonal expansion is the aberrant expression of septin proteins. SEPT9 functions as a microtu‐ bule regulator and plays an important function in cytokinesis and chromosome segregation, thus affecting genomic stability [43, 44]. When aberrantly expressed, SEPT9 causes dysregu‐ lated cytokinesis or cell division resulting in missegregation of chromosomes [43, 44]. Ge‐ nomic instability then results from SEPT9 dysregulation, leading to the accumulation of chromosomal deletions or amplifications from missegregation of chromosomes. These events in conjunction with additional mutations can enhance cell proliferation and survival.

Two additional genes identified near vector proviruses in the SCID-X1 trial were RUNX family members RUNX2 and RUNX3. RUNX proteins (RUNX 1, 2, 3) are a family of RUNT homology domain containing α-subunits that form heterodimeric transcription factors that mediate hematopoietic differentiation and expansion in conjunction with β subunit core binding factor (CBF). Aberrant expression of the RUNX proteins in mouse models hinders myeloid class progenitor differentiation capacity and represses expression of several target genes including Csf1R, Mpo, Cebpd, and the cell cycle inhibitor Cdkn1a [45]. Repression of these genes blocks hematopoietic stem cell differentiation leading to an accumulation of un‐ differentiated cells. These cells cannot pass the differentiation block to repopulate the de‐ pleted blood cell niche. The lack of differentiated mature cells continues to generate proliferative signaling pathways that further stimulate mutant HSC expansion. The expand‐ ed undifferentiated blast cells accumulate in the bone marrow, disrupting normal blood cell production, and can eventually give rise to various cytopenias and leukemic blast crisis.

cell a proliferative advantage.

#### **6. Clonal expansion in the SCID-X1 trials**

As evidenced in the French SCID-X1 trial, retroviral vector integration can dysregulate near‐ by host genes, thus affecting cell growth and survival. Once integrated, elements in the pro‐ virus, particularly enhancers in the LTR, can dysregulate nearby gene expression through several mechanisms (Figure 2). Aberrant gene expression can dysregulate host cell genes and regulatory networks involved with cell growth, including proto-oncogenes. A survival advantage can occur through the activation of proto-oncogenes giving cells a proliferative advantage or "go signal". Alternatively, tumor suppressors can be inactivated, causing a proliferative advantage with uncontrolled cellular division from the lack of a "stop signal". To date the genotoxicity described in HSC gene therapy trials has been through activation of growth promoting genes and proto-oncogenes, rather than inactivation of tumor suppres‐ sors. This is because activation requires a single integration into only one allele whereas in‐ activation of a tumor suppressor requires a second event, either a second integration or a loss of heterozygosity at that allele, to inactivate tumor suppressor activity. Tumor suppres‐ sors have however been identified in preclinical mouse studies [36-37]. These vector mediat‐ ed mutations along with additional accumulating mutagenic events in expanding genemodified repopulating cells can ultimately result in tumorigenesis. The proviral promoter and enhancer elements have been shown to act up to a distance of 500 Kb upstream and downstream of the site of proviral integration [38]. In the SCID-X1 trials where patients de‐ veloped T-cell leukemia, the integration sites of dominant repopulating clones near the genes LMO2, BMI1, HMGA2, SEPT9, RUNX2, and RUNX3 gave rise to cells with a prolifera‐ tive advantage or survival advantage over competitor repopulating cells. These advantages eventually led to an over-representation of these clones (Figure 3) [5].

We now know how vector-mediated dysregulation of these different genes may have con‐ tributed to clonal expansion and frank leukemia. LMO2 or Lim only 2 is a proto-oncogene that regulates early progenitor expansion during hematopoiesis [39]. LMO2 oncogenic prop‐ erties were first observed in mature gene-modified T-cells, where it is not normally ex‐ pressed [39]. However, after proviral integration events, resulting in activation of LMO2, these gene-modified T-cells begin to expand from dysregulation of transcriptional regulato‐ ry pathways. In 2010, Oram et al. demonstrated that LMO2 expression in T cells activates FLI1 and ERG enhancers, known to be involved in blood stem/progenitor cells. These gene products of FLI1 and ERG in turn activate the enhancer of the HHEX/PRH gene locus, which has been shown to act in early progenitor cell expansion and formation of T-lineage acute lymphoblastic leukemia (T-ALL) [35]. LMO2 overexpression has also been demon‐ strated to reduce or eliminate cell cyclin dependent kinase (CDK) inhibitors promoting es‐ cape of the G1 cell cycle checkpoints during cellular division [40]. Once aberrantly expressed within a cell, LMO2 promotes cell cycle progression via multiple mechanisms, giving the cell a proliferative advantage.

In the French SCID-X1 trial, four of the ten patients developed T-cell leukemia from inser‐ tional mutagenesis from the murine moloney leukemia virus (MLV)-based vector [5, 25]. Careful molecular analysis of leukemic cells showed that the MLV provirus integrated near the proto-oncogene, LMO2, and suggested that viral enhancer elements in the provirus con‐ tributed to leukemia (Figure 2B) [25]. The SCID-X1 trials were the first HSC gene therapy clinical trial where vector-mediated insertional mutagenesis led to cancer. In this trial, MLV vector LTR enhancers activated LMO2 expression, resulting in T-cell LMO2 dependent pro‐ liferation (Figure 2B). LMO2 is normally silenced in mature T-cells, and when viral enhanc‐ ers turn on expression, LMO2 drives T-cell proliferation by dysregulating transcription networks that affect the cell cycle. This promotes cell cycle escape and can result in higher proliferation rates compared to normal cells [35]. Leukemic transformation was a result of provirus integration near LMO2 with additional proviral integrants near other proto-onco‐

As evidenced in the French SCID-X1 trial, retroviral vector integration can dysregulate near‐ by host genes, thus affecting cell growth and survival. Once integrated, elements in the pro‐ virus, particularly enhancers in the LTR, can dysregulate nearby gene expression through several mechanisms (Figure 2). Aberrant gene expression can dysregulate host cell genes and regulatory networks involved with cell growth, including proto-oncogenes. A survival advantage can occur through the activation of proto-oncogenes giving cells a proliferative advantage or "go signal". Alternatively, tumor suppressors can be inactivated, causing a proliferative advantage with uncontrolled cellular division from the lack of a "stop signal". To date the genotoxicity described in HSC gene therapy trials has been through activation of growth promoting genes and proto-oncogenes, rather than inactivation of tumor suppres‐ sors. This is because activation requires a single integration into only one allele whereas in‐ activation of a tumor suppressor requires a second event, either a second integration or a loss of heterozygosity at that allele, to inactivate tumor suppressor activity. Tumor suppres‐ sors have however been identified in preclinical mouse studies [36-37]. These vector mediat‐ ed mutations along with additional accumulating mutagenic events in expanding genemodified repopulating cells can ultimately result in tumorigenesis. The proviral promoter and enhancer elements have been shown to act up to a distance of 500 Kb upstream and downstream of the site of proviral integration [38]. In the SCID-X1 trials where patients de‐ veloped T-cell leukemia, the integration sites of dominant repopulating clones near the genes LMO2, BMI1, HMGA2, SEPT9, RUNX2, and RUNX3 gave rise to cells with a prolifera‐ tive advantage or survival advantage over competitor repopulating cells. These advantages

genes that resulted in expansion of cells with these mutations [5, 19].

eventually led to an over-representation of these clones (Figure 3) [5].

We now know how vector-mediated dysregulation of these different genes may have con‐ tributed to clonal expansion and frank leukemia. LMO2 or Lim only 2 is a proto-oncogene that regulates early progenitor expansion during hematopoiesis [39]. LMO2 oncogenic prop‐ erties were first observed in mature gene-modified T-cells, where it is not normally ex‐

**6. Clonal expansion in the SCID-X1 trials**

404 Gene Therapy - Tools and Potential Applications

Additional proto-oncogenes were activated in the French SCID-X1 patients. The BMI1 protooncogene normally functions in self-renewal and maintenance of hematopoietic primitive stem cells [40, 41]. Like LMO2, dysregulation of BMI-1 via enhancer activation results in clo‐ nal expansion. Additional mechanisms mediate clonal expansion, such as the high mobility group AT-hook2 (HMGA2) which can provide a proliferative advantage when the endoge‐ nous gene is truncated via insertional mutagenesis. Truncation results in the loss of regula‐ tory target sequences within the protein mRNA preventing degradation by the endogenous miRNA let7. These miRNA elements normally function to regulate HMGA2 via RNA inter‐ ference using the RNA induced silencing complex (RISC) machinery [42]. Truncated HMGA2 mRNA is not degraded thus continuing activation of gene networks involved with cell proliferation, and cell-cycle progression [42]. Another mechanism that can contribute to clonal expansion is the aberrant expression of septin proteins. SEPT9 functions as a microtu‐ bule regulator and plays an important function in cytokinesis and chromosome segregation, thus affecting genomic stability [43, 44]. When aberrantly expressed, SEPT9 causes dysregu‐ lated cytokinesis or cell division resulting in missegregation of chromosomes [43, 44]. Ge‐ nomic instability then results from SEPT9 dysregulation, leading to the accumulation of chromosomal deletions or amplifications from missegregation of chromosomes. These events in conjunction with additional mutations can enhance cell proliferation and survival.

Two additional genes identified near vector proviruses in the SCID-X1 trial were RUNX family members RUNX2 and RUNX3. RUNX proteins (RUNX 1, 2, 3) are a family of RUNT homology domain containing α-subunits that form heterodimeric transcription factors that mediate hematopoietic differentiation and expansion in conjunction with β subunit core binding factor (CBF). Aberrant expression of the RUNX proteins in mouse models hinders myeloid class progenitor differentiation capacity and represses expression of several target genes including Csf1R, Mpo, Cebpd, and the cell cycle inhibitor Cdkn1a [45]. Repression of these genes blocks hematopoietic stem cell differentiation leading to an accumulation of un‐ differentiated cells. These cells cannot pass the differentiation block to repopulate the de‐ pleted blood cell niche. The lack of differentiated mature cells continues to generate proliferative signaling pathways that further stimulate mutant HSC expansion. The expand‐ ed undifferentiated blast cells accumulate in the bone marrow, disrupting normal blood cell production, and can eventually give rise to various cytopenias and leukemic blast crisis.

selective advantage to cells through its expression. This was true in the SCID-X1 trials, where expression of the corrective γC transgene gave cells a proliferative advantage allow‐ ing reconstitution of the lymphoid cell population from a modest number of gene modified HSCs [5]. To determine the mechanisms behind these expansion events investigators must first characterize the integration sites of the vectors being used to deliver the therapeutic transgene. This allows identification of nearby genes that may have been dysregulated lead‐

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**8. Vector integration sites in expanded repopulating clones allow clonal**

To understand the risk of genotoxicity we need to identify where different vectors tend to integrate. Conveniently, integrated vector proviruses serve as molecular tags to identify in‐ tegration sites and to track specific clones in order to study clonal expansion. Sequencing the unique vector-chromosome junctions can identify where in the genome the virus has inte‐ grated. Analysis of vector insertion sites has allowed researchers to compile comprehensive integration profiles for specific virus types and assess the safety of viral vectors based on the

Long term tracking of gene-modified cells is necessary to monitor potential adverse events that may occur over time resulting in clonal expansion. By identifying the spectrum of vec‐ tor integration sites in repopulating cells, the clonality of repopulating cells can be estimated and the expansion of specific clones can be monitored. Long-term tracking may also provide insight into specific mechanism of clonal expansion, such as emergent LMO2 expansion in SCID-X1 trials, and will direct novel approaches to reduce genotoxic effects [46]. It has be‐ come an important area of study to understand where retroviruses integrate in the human

**9. The integration profile of retroviruses and its relation to genotoxicity**

Identifying the integration profiles of different vector types has provided important data on the relative genotoxic risk associated with different vectors. Following the leukemias ob‐ served in the SCID-X1 trial, integration site distributions were described for different retro‐ viral vectors being developed for gene therapy. Viral integration sites for HIV-1, MLV and foamy virus (FV) vectors were reported and each exhibits a specific and unique integration profile. HIV-1 based vectors showed preferences for integration within actively transcribed genes [47] whereas MLV vectors tends to integrate within transcription start sites near CpG islands [48-50]. FV vectors also preferentially integrate near transcription start sites and CpG islands but less frequently than MLV vectors and integrate less frequently in genes than HIV vectors. The propensity of MLV-based vectors to integrate preferentially very close to pro‐ moter regions was of significant concern since this may increase the risk of dysregulating

genome, thus affecting their safety for use in HSC gene therapy approaches.

ing to clonal expansion.

regions of preferred or recurrent integration.

**tracking**

**Figure 3.** Clonal Expansion. A) Patient derivedCD34 enriched hematopoietic stem cells (HSC) population prior to ex vivo vector exposure. Untransduced cells are blue. B) Polyclonal proviral integration distribution in vector treated HSCs. This is an ideal proviral distribution, which HSC gene therapy would ideally maintain after infusion into patients. How‐ ever, due to genotoxicity and selection pressures in vivo C) Oligoclonal expansion can be observed, where some clones expand. In some cases, D) individual clones may harbor a proviral integration near genes promoting a proliferative or survival advantage, which may eventually contribute to malignancy.

In summary, our current understanding of genotoxicity is that vector proviruses dysregulate the expression of key regulators of cell cycle, cell survival, genomic stability, proliferative gene networking and cellular differentiation. This leads to an over-representation of genemodified clones with these mutations in the peripheral blood (PB) and bone marrow (BM) which is referred to as clonal expansion (Figure 3). Clonal expansion can lead to additional mutations that can eventually cause frank leukemia. The leukemias observed in the French SCID-X1 trial refocused the gene therapy field to better understand the mechanisms of vector dysregulation of host genes, with the ultimate goal of reducing the risk of genotoxicity.

#### **7. Risk factors for clonal expansion**

It is important to remember that the SCID-X1 trials are one example of clonal expansion in a specific disease setting using a specific vector type with a specific transgene. Several factors can influence clonal expansion including the type of vector, vector design, the therapeutic transgene and the disease setting. For example the transgene in and of itself may provide a selective advantage to cells through its expression. This was true in the SCID-X1 trials, where expression of the corrective γC transgene gave cells a proliferative advantage allow‐ ing reconstitution of the lymphoid cell population from a modest number of gene modified HSCs [5]. To determine the mechanisms behind these expansion events investigators must first characterize the integration sites of the vectors being used to deliver the therapeutic transgene. This allows identification of nearby genes that may have been dysregulated lead‐ ing to clonal expansion.

### **8. Vector integration sites in expanded repopulating clones allow clonal tracking**

To understand the risk of genotoxicity we need to identify where different vectors tend to integrate. Conveniently, integrated vector proviruses serve as molecular tags to identify in‐ tegration sites and to track specific clones in order to study clonal expansion. Sequencing the unique vector-chromosome junctions can identify where in the genome the virus has inte‐ grated. Analysis of vector insertion sites has allowed researchers to compile comprehensive integration profiles for specific virus types and assess the safety of viral vectors based on the regions of preferred or recurrent integration.

Long term tracking of gene-modified cells is necessary to monitor potential adverse events that may occur over time resulting in clonal expansion. By identifying the spectrum of vec‐ tor integration sites in repopulating cells, the clonality of repopulating cells can be estimated and the expansion of specific clones can be monitored. Long-term tracking may also provide insight into specific mechanism of clonal expansion, such as emergent LMO2 expansion in SCID-X1 trials, and will direct novel approaches to reduce genotoxic effects [46]. It has be‐ come an important area of study to understand where retroviruses integrate in the human genome, thus affecting their safety for use in HSC gene therapy approaches.

**Figure 3.** Clonal Expansion. A) Patient derivedCD34-

406 Gene Therapy - Tools and Potential Applications

**7. Risk factors for clonal expansion**

survival advantage, which may eventually contribute to malignancy.

enriched hematopoietic stem cells (HSC) population prior to ex

vivo vector exposure. Untransduced cells are blue. B) Polyclonal proviral integration distribution in vector treated HSCs. This is an ideal proviral distribution, which HSC gene therapy would ideally maintain after infusion into patients. How‐ ever, due to genotoxicity and selection pressures in vivo C) Oligoclonal expansion can be observed, where some clones expand. In some cases, D) individual clones may harbor a proviral integration near genes promoting a proliferative or

In summary, our current understanding of genotoxicity is that vector proviruses dysregulate the expression of key regulators of cell cycle, cell survival, genomic stability, proliferative gene networking and cellular differentiation. This leads to an over-representation of genemodified clones with these mutations in the peripheral blood (PB) and bone marrow (BM) which is referred to as clonal expansion (Figure 3). Clonal expansion can lead to additional mutations that can eventually cause frank leukemia. The leukemias observed in the French SCID-X1 trial refocused the gene therapy field to better understand the mechanisms of vector dysregulation of host genes, with the ultimate goal of reducing the risk of genotoxicity.

It is important to remember that the SCID-X1 trials are one example of clonal expansion in a specific disease setting using a specific vector type with a specific transgene. Several factors can influence clonal expansion including the type of vector, vector design, the therapeutic transgene and the disease setting. For example the transgene in and of itself may provide a

#### **9. The integration profile of retroviruses and its relation to genotoxicity**

Identifying the integration profiles of different vector types has provided important data on the relative genotoxic risk associated with different vectors. Following the leukemias ob‐ served in the SCID-X1 trial, integration site distributions were described for different retro‐ viral vectors being developed for gene therapy. Viral integration sites for HIV-1, MLV and foamy virus (FV) vectors were reported and each exhibits a specific and unique integration profile. HIV-1 based vectors showed preferences for integration within actively transcribed genes [47] whereas MLV vectors tends to integrate within transcription start sites near CpG islands [48-50]. FV vectors also preferentially integrate near transcription start sites and CpG islands but less frequently than MLV vectors and integrate less frequently in genes than HIV vectors. The propensity of MLV-based vectors to integrate preferentially very close to pro‐ moter regions was of significant concern since this may increase the risk of dysregulating proto-oncogenes. The integration profiles were found to be largely independent of the route of entry [47, 51, 52] and target cell type [49, 53, 54] although characteristics such as cell cycle of the target cell can play a minor role in the profile [49,54].

The factors that contribute to the integration profile of viruses and viral vectors are greatly in‐ fluenced by a compliment of host proteins that interact with a poorly defined retroviral pre-in‐ tegration complex (PIC). The PIC is a complex of proteins associated with the viral genome, and during infection, the PIC must migrate to the nucleus to mediate integration of the reversetranscribed viral DNA to generate the vector provirus. This process and the associated proteins that affect it have been studied using various methods [55-57]. Viral Gag and integrase proteins have been shown to interact with chromatin, affectively tethering the PIC to specific chromoso‐ mal regions, thus directing integration [57, 58]. Studies have compared the contributions of the viral integrase and Gag proteins using MLV-HIV chimeras, and shown that both play impor‐ tant roles in integration site specificity [57]. The HIV lens-epithelium-derived growth factor (LEDGF) is a host derived tethering protein that has been demonstrated to associate with the PIC and chromatin affecting HIV-1 integration patterns. This host protein has a strong binding affinity for HIV integrase proteins, which are associated with the lentiviral PIC [59]. The tether‐ ing of the PIC to LEDGF protects the PIC from host enzymatic defenses [56], promotes chroma‐ tin binding [57, 60], and directs integration site distribution [61]. Unique to foamy virus biology, the c-terminal end of the Gag protein contains glycine-arginine motifs known as a GR boxes [62]. These boxes direct viral packaging [63, 64] and nuclear localization [62, 64]. In addi‐ tion to these features, a 13 amino acid motif called the chromatin-binding site (CBS) has been characterized [58]. This CBS contains a functional binding domain for core histones H2A/H2B that is thought to tether the PIC to the chromatin after translocation into the nucleus [58]. Host chromatin tethering proteins often associate with the PIC complex and affect integration site distributions. Better characterization of cell-virus interaction should enhance our understand‐ ing of viral integration patterns. This has potentially led to novel approaches to direct vector in‐ tegrations to "safe harbor" chromosomal regions, that do not have genes that can lead to clonal expansion when dysregulated.

#### **10. Methods for integration site analysis**

Many methods exist for generating retroviral insertion site data. PCR based techniques include ligation mediated PCR (LM-PCR Figure 4A), Linear amplification-mediated PCR (LAM-PCR Figure 4B), and non-restrictive LAM-PCR (nrLAM-PCR Figure 4C). LM-PCR relies on fre‐ quently cutting restriction enzymes to generate fragments that contain the provirus: chromo‐ some junction. These fragments are then ligated to linkers, and after several rounds of PCR, the resulting products are sequenced. LAM-PCR uses an LTR-specific primer in several rounds of 'linear' amplification where the LTR: chromosome junction is amplified. Nested PCR is then used to produce products that can be directly sequenced or transformed into bacteria and se‐ quenced. nrLAM-PCR is similar to LAM-PCR but uses random shearing rather than digestion of DNA with restriction enzymes prior to linker ligation and sequencing, thus avoiding restric‐ tion site bias and is currently the gold standard in the field.

**Figure 4.** PCR Based LTR: Chromosome Junction Sequencing. A) Demonstration of ligation-mediated PCR, where ge‐ nomic DNA is cut by restriction enzyme digestion, ligated to a linker, and amplified before sequencing of oligos with an LTR specific primer. B) Left Panel: Linear-amplification-mediated PCR (LAM-PCR) amplifies regions of genomic DNA containing integrated vector proviruses using an LTR specific primer. The resulting oligos are captured on magnetic beads and double strand synthesis is performed, followed by restriction enzyme digestion and ligation of a double stranded linker. Nested PCR is then used and the resulting products sequenced. Right Panel: Non-restrictive linear-am‐ plification-mediated PCR (nrLAM-PCR) amplifies genomic DNA with integrated vector proviruses with an LTR specific primer. The resulting products are enriched on magnetic beads, followed by single strand linker ligation. Nested PCR is

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then employed and the products are sequenced.

proto-oncogenes. The integration profiles were found to be largely independent of the route of entry [47, 51, 52] and target cell type [49, 53, 54] although characteristics such as cell cycle

The factors that contribute to the integration profile of viruses and viral vectors are greatly in‐ fluenced by a compliment of host proteins that interact with a poorly defined retroviral pre-in‐ tegration complex (PIC). The PIC is a complex of proteins associated with the viral genome, and during infection, the PIC must migrate to the nucleus to mediate integration of the reversetranscribed viral DNA to generate the vector provirus. This process and the associated proteins that affect it have been studied using various methods [55-57]. Viral Gag and integrase proteins have been shown to interact with chromatin, affectively tethering the PIC to specific chromoso‐ mal regions, thus directing integration [57, 58]. Studies have compared the contributions of the viral integrase and Gag proteins using MLV-HIV chimeras, and shown that both play impor‐ tant roles in integration site specificity [57]. The HIV lens-epithelium-derived growth factor (LEDGF) is a host derived tethering protein that has been demonstrated to associate with the PIC and chromatin affecting HIV-1 integration patterns. This host protein has a strong binding affinity for HIV integrase proteins, which are associated with the lentiviral PIC [59]. The tether‐ ing of the PIC to LEDGF protects the PIC from host enzymatic defenses [56], promotes chroma‐ tin binding [57, 60], and directs integration site distribution [61]. Unique to foamy virus biology, the c-terminal end of the Gag protein contains glycine-arginine motifs known as a GR boxes [62]. These boxes direct viral packaging [63, 64] and nuclear localization [62, 64]. In addi‐ tion to these features, a 13 amino acid motif called the chromatin-binding site (CBS) has been characterized [58]. This CBS contains a functional binding domain for core histones H2A/H2B that is thought to tether the PIC to the chromatin after translocation into the nucleus [58]. Host chromatin tethering proteins often associate with the PIC complex and affect integration site distributions. Better characterization of cell-virus interaction should enhance our understand‐ ing of viral integration patterns. This has potentially led to novel approaches to direct vector in‐ tegrations to "safe harbor" chromosomal regions, that do not have genes that can lead to clonal

Many methods exist for generating retroviral insertion site data. PCR based techniques include ligation mediated PCR (LM-PCR Figure 4A), Linear amplification-mediated PCR (LAM-PCR Figure 4B), and non-restrictive LAM-PCR (nrLAM-PCR Figure 4C). LM-PCR relies on fre‐ quently cutting restriction enzymes to generate fragments that contain the provirus: chromo‐ some junction. These fragments are then ligated to linkers, and after several rounds of PCR, the resulting products are sequenced. LAM-PCR uses an LTR-specific primer in several rounds of 'linear' amplification where the LTR: chromosome junction is amplified. Nested PCR is then used to produce products that can be directly sequenced or transformed into bacteria and se‐ quenced. nrLAM-PCR is similar to LAM-PCR but uses random shearing rather than digestion of DNA with restriction enzymes prior to linker ligation and sequencing, thus avoiding restric‐

of the target cell can play a minor role in the profile [49,54].

408 Gene Therapy - Tools and Potential Applications

expansion when dysregulated.

**10. Methods for integration site analysis**

tion site bias and is currently the gold standard in the field.

**Figure 4.** PCR Based LTR: Chromosome Junction Sequencing. A) Demonstration of ligation-mediated PCR, where ge‐ nomic DNA is cut by restriction enzyme digestion, ligated to a linker, and amplified before sequencing of oligos with an LTR specific primer. B) Left Panel: Linear-amplification-mediated PCR (LAM-PCR) amplifies regions of genomic DNA containing integrated vector proviruses using an LTR specific primer. The resulting oligos are captured on magnetic beads and double strand synthesis is performed, followed by restriction enzyme digestion and ligation of a double stranded linker. Nested PCR is then used and the resulting products sequenced. Right Panel: Non-restrictive linear-am‐ plification-mediated PCR (nrLAM-PCR) amplifies genomic DNA with integrated vector proviruses with an LTR specific primer. The resulting products are enriched on magnetic beads, followed by single strand linker ligation. Nested PCR is then employed and the products are sequenced.

One limitation of the above methods is that PCR bias can affect the frequency of detected integration sites [65, 66]. Another method that has been used is shuttle vector rescue tech‐ nology, which eliminates PCR-based bias [67, 68, and 54]. In shuttle vector rescue, vector plasmids encode a bacterial origin of replication and selection gene. DNA fragments that contain the shuttle vector LTR: chromosome junction are ligated and then transformed into bacteria. These bacteria can then be grown as colonies to amplify plasmid clones of each po‐ tential insertion site in the absence of PCR based skewing (Figure 5). Plasmid DNA is then extracted from bacterial colonies and sequenced with an LTR specific primer. In all of the above methods, aligning the genomic sequence immediately next to the proviral LTR to a published genome databases allows for identification of the proviral integration site. It will be interesting to compare the shuttle vector approach to nrLAM-PCR in animal models to provide information on any potential bias from either technique.

models have provided important data on the relative genotoxicity of different vectors systems and have identified genes and gene networks involved in vector-mediated malignant transfor‐ mation [69]. The advantage of tumor prone mice is that the frequency of clonal expansion and tumorigenesis resulting from vector-mediated genotoxic events is increased, thereby allowing

Retroviral Genotoxicity

411

http://dx.doi.org/10.5772/52530

Several studies have focused on gammaretroviral and lentiviral vectors; testing vectors with hybrid LTRs from both viral systems to identify the elements responsible for different geno‐ toxicities. These studies, in conjunction with tumor-prone mouse models, have informed vector design modifications. Enhancer deletion, use of internal housekeeping promoters, and deletions of vector cryptic splice sites can be used to reduce genotoxic events and im‐ prove safety [69]. The tumor prone cdkn2a-/- mouse model has been used to compare retro‐ viral insertional oncogenic potential using MLV and HIV-based vectors in in vivo genotoxicity assays [70]. These assays demonstrated HIV- based lentiviral vectors exhibited an improved safety profile compared to MLV based vectors. The Cdkn2a locus controls cell senescence and has been shown to prevent cell transformation. Inactivation of this gene pro‐ motes malignancy and has been implicated in almost all types of human cancer [71, 72]. These studies have compared the genotoxic contribution of vector components such as strong LTR promoters. They have also shown the ability of self-inactivating LTR designs to

Although these studies primarily identify activated proto-oncogenes, it is also possible to identify dysregulated tumor suppressors using retroviral mutagenesis screens [73-75]. Pro‐ viruses can downregulate nearby host gene transcription via host cell methylation of the proviral LTR that also leads to methylation of the nearby host genes. Identification of the vector provirus location and nearby host genes can be used to identify haplo-insufficien‐ cies related to malignancy. Recent observations of viral LTR methylation causing proviral transgene silencing can now be used to identify down regulation of host genes near the methylated proviral integration sites [75]. The vector LTR methylation events and subse‐ quent silencing of host genes can identify potential tumor suppressors related to vectormediated genotoxicity. A recent study used methylation specific PCR and methylated DNA immunoprecipitation assays to analyze methylated proviral integrations in mutagen‐ ized mouse tumors [75]. In this study the identification of the methylated and downregu‐ lated gene PTP4A3 in MLV vector-mutagenized murine leukemia samples, suggests that haplo-insufficiency may be involved in retroviral genotoxicity [75]. This study also sug‐ gests that future studies may identify vector-mediated haploinsufficiency genes that con‐

Large animal models allow long term monitoring of HSC genotoxicity due to the longer life span of large animals such as dogs and nonhuman primates relative to mice. These models are important to assess the long-term risks associated with malignant transformation follow‐

readout of vector-mediated malignancy within the life span of a mouse.

reduce genotoxicity.

tribute to genotoxicity.

**12. Large animal models of genotoxicity**

**Figure 5.** Plasmid Shuttle Vector Rescue. Genomic DNA is presented with vector integrations showing the proviral 5' LTR, the genetic element encoding a bacterial origin of replications (Ori), and the vector provirus 3'LTR.Vector exposed cells are lysed and genomic DNA harboring integrated vector proviruses collected. The genomic DNA harboring provi‐ ral integrants is fragmented by restriction enzyme digest, and then self-ligated to form plasmids which may contain portions of the provirus encoding a bacterial origin of replication and an antibiotic selection gene. These plasmids are then transformed into E. coli. E. coli transformed with plasmids containing the bacterial origin and antibiotic resistance gene will form colonies. Sequencing the colony plasmids identifies proviral LTR: chromosome junctions.

#### **11. Animal models to study genotoxicity: Tumor prone mouse models**

Animal models allow the in vivo study of the genotoxicity of HSC gene therapy approaches, within specific disease contexts. These studies are critical because while in vitro genotoxicity assays can provide important information on the relative genotoxicity of different vectors [50], only animal models can assess genotoxic effects on in vivo hematopoiesis. Tumor prone mouse models have provided important data on the relative genotoxicity of different vectors systems and have identified genes and gene networks involved in vector-mediated malignant transfor‐ mation [69]. The advantage of tumor prone mice is that the frequency of clonal expansion and tumorigenesis resulting from vector-mediated genotoxic events is increased, thereby allowing readout of vector-mediated malignancy within the life span of a mouse.

One limitation of the above methods is that PCR bias can affect the frequency of detected integration sites [65, 66]. Another method that has been used is shuttle vector rescue tech‐ nology, which eliminates PCR-based bias [67, 68, and 54]. In shuttle vector rescue, vector plasmids encode a bacterial origin of replication and selection gene. DNA fragments that contain the shuttle vector LTR: chromosome junction are ligated and then transformed into bacteria. These bacteria can then be grown as colonies to amplify plasmid clones of each po‐ tential insertion site in the absence of PCR based skewing (Figure 5). Plasmid DNA is then extracted from bacterial colonies and sequenced with an LTR specific primer. In all of the above methods, aligning the genomic sequence immediately next to the proviral LTR to a published genome databases allows for identification of the proviral integration site. It will be interesting to compare the shuttle vector approach to nrLAM-PCR in animal models to

**Figure 5.** Plasmid Shuttle Vector Rescue. Genomic DNA is presented with vector integrations showing the proviral 5' LTR, the genetic element encoding a bacterial origin of replications (Ori), and the vector provirus 3'LTR.Vector exposed cells are lysed and genomic DNA harboring integrated vector proviruses collected. The genomic DNA harboring provi‐ ral integrants is fragmented by restriction enzyme digest, and then self-ligated to form plasmids which may contain portions of the provirus encoding a bacterial origin of replication and an antibiotic selection gene. These plasmids are then transformed into E. coli. E. coli transformed with plasmids containing the bacterial origin and antibiotic resistance

gene will form colonies. Sequencing the colony plasmids identifies proviral LTR: chromosome junctions.

**11. Animal models to study genotoxicity: Tumor prone mouse models**

Animal models allow the in vivo study of the genotoxicity of HSC gene therapy approaches, within specific disease contexts. These studies are critical because while in vitro genotoxicity assays can provide important information on the relative genotoxicity of different vectors [50], only animal models can assess genotoxic effects on in vivo hematopoiesis. Tumor prone mouse

provide information on any potential bias from either technique.

410 Gene Therapy - Tools and Potential Applications

Several studies have focused on gammaretroviral and lentiviral vectors; testing vectors with hybrid LTRs from both viral systems to identify the elements responsible for different geno‐ toxicities. These studies, in conjunction with tumor-prone mouse models, have informed vector design modifications. Enhancer deletion, use of internal housekeeping promoters, and deletions of vector cryptic splice sites can be used to reduce genotoxic events and im‐ prove safety [69]. The tumor prone cdkn2a-/- mouse model has been used to compare retro‐ viral insertional oncogenic potential using MLV and HIV-based vectors in in vivo genotoxicity assays [70]. These assays demonstrated HIV- based lentiviral vectors exhibited an improved safety profile compared to MLV based vectors. The Cdkn2a locus controls cell senescence and has been shown to prevent cell transformation. Inactivation of this gene pro‐ motes malignancy and has been implicated in almost all types of human cancer [71, 72]. These studies have compared the genotoxic contribution of vector components such as strong LTR promoters. They have also shown the ability of self-inactivating LTR designs to reduce genotoxicity.

Although these studies primarily identify activated proto-oncogenes, it is also possible to identify dysregulated tumor suppressors using retroviral mutagenesis screens [73-75]. Pro‐ viruses can downregulate nearby host gene transcription via host cell methylation of the proviral LTR that also leads to methylation of the nearby host genes. Identification of the vector provirus location and nearby host genes can be used to identify haplo-insufficien‐ cies related to malignancy. Recent observations of viral LTR methylation causing proviral transgene silencing can now be used to identify down regulation of host genes near the methylated proviral integration sites [75]. The vector LTR methylation events and subse‐ quent silencing of host genes can identify potential tumor suppressors related to vectormediated genotoxicity. A recent study used methylation specific PCR and methylated DNA immunoprecipitation assays to analyze methylated proviral integrations in mutagen‐ ized mouse tumors [75]. In this study the identification of the methylated and downregu‐ lated gene PTP4A3 in MLV vector-mutagenized murine leukemia samples, suggests that haplo-insufficiency may be involved in retroviral genotoxicity [75]. This study also sug‐ gests that future studies may identify vector-mediated haploinsufficiency genes that con‐ tribute to genotoxicity.

#### **12. Large animal models of genotoxicity**

Large animal models allow long term monitoring of HSC genotoxicity due to the longer life span of large animals such as dogs and nonhuman primates relative to mice. These models are important to assess the long-term risks associated with malignant transformation follow‐ ing insertional mutagenesis from clonal expansion and long-term selection pressures. In two non-human primate studies, the distribution of MLV-based gammaretroviral and SIV and HIV-1 based lentiviral integration sites were evaluated over long periods [76, 77]. Clonal ex‐ pansion or malignant transformation was not observed. However, integrants were observed at higher than expected frequencies near growth promoting genes and proto-oncogenes. This suggests that repopulating cells with integrations near these genes can influence sur‐ vival of those clones. These studies can shed light on potential mechanisms of clonal expan‐ sion and can allow comparison of different vector types. However, these studies in normal animals using vectors with a reporter gene that is not expected to provide a selective growth advantage did not predict the clonal expansion observed in the SCID-X1 trial. This suggests that large animal models of specific disease settings such as the SCID-X1 dog model [34] will be important to test improved vectors designed to reduce genotoxicity in a specific disease setting.

strong need for continued gene-marking studies and clonal tracking of these gene-modified

Retroviral Genotoxicity

413

http://dx.doi.org/10.5772/52530

Chronic Granulomatous disease (CGD) is an x-linked inherited immunodeficiency result‐ ing from a mutation in one of the NADPH oxidase genes [87]. The gp91phox protein ac‐ counts for 70% of cases [81]. The gp91phox transgene has been used in corrective HSC gene therapy clinical trials [81]. Unlike SCID-X1 gene-modified cells, CGD gene-modified cells do not exhibit a proliferative advantage from transgene expression. The lack of condition‐ ing or selection of gene-modified cells contributed to a loss of therapeutic benefit and de‐ tection of gene-modified cells. Patients in the initial trials had low marking with gene expression of the corrected transgene for short periods of therapeutic benefit. Adverse genotoxic events developed 2 ½ years after the initial therapy, with clonal expansion and leukemic transformation [7, 82]. Clonal analysis found activation of MDS1/EVI1, PRDM16, and SETBP1 proto-oncogenes [81]. One of the patients died in treatment from complica‐ tions arising from the leukemia, the other patient survived after receiving an additional al‐ logeneic transplant. In this study there was inefficient engraftment and short-term transgene expression, with vector silencing via vector LTR methylation (Figure 2D) [83]. Further improvements to enhance engraftment and selection of gene-modified cells after

HSC gene therapy has also been used in the treatment of Wiskott-Aldrich syndrome (WAS), an X-linked recessive immune disorder. In this study, patients underwent conditioning with busulfan to enhance the engraftment of gene-modified cells [84] Patients exhibited therapeu‐ tic benefit, with resolution of disease symptoms, although clonal skewing was detected for clones that harbored vector integration sites near CCND2 and MDS1/EVI1 [84]. Despite the high success of WAS HSC gene therapy in nine of the ten patients treated, patient 2 was re‐ ported to have experienced vector-derived genotoxic events after more than 3 years of thera‐ peutic benefit [84], ultimately resulting in T-cell leukemia. The leukemia was a result of proviral integration near the gene LMO2, and this patient has since been treated with che‐

Clonal tracking in vivo has recently been employed in a study where patients with glioblas‐ toma were given gene-modified hematopoietic repopulating repopulating stem cells carry‐ ing a methylguanine methyltransferase mutant (MGMT-P140K) [17]. In this approach genemodified hematopoietic repopulating cells expressing this mutant enzyme are resistant to O6-benzylguanine (O6BG). This allows treatment of the glioblastoma solid tumor with O6BG and an alkylating agent. By protecting the hematopoietic system from chemotherapy-

**14. MDS1/EVI1, PRDM16, SETBP1 in trial for CGD**

infusion are needed for HSC gene therapy to treat CGD.

motherapy resulting in remission [85-87].

**15. CCND2 and MDS1/EVI1 trial for Wiskott-Aldrich**

cells in vivo [80].

Another important contribution of large animal models has been to improve our under‐ standing of the effects of ex vivo culture on clonal expansion. It has been shown in a nonhu‐ man primate model that genotoxicity can be significantly influenced by the culturing conditions of gene-modified cells ex vivo. In this study, only six days of culture increased the incidence of specific clones with gamma retroviral vector integrations near MDS/ EVI1 locus associated with a leukemic phenotype [78]. To monitor the potential effects of ex vivo culturing condition and in vivo selection on gene-modified repopulating cells, clonal track‐ ing methods must be employed.

#### **13. Tracking of genetically modified clones**

The above studies have identified mechanisms of genotoxicity and clonal expansion. During clinical trials, it is important to monitor potential clonal expansion in order to understand genotoxicity and to anticipate potential adverse events [79]. As an example, dysregulation of HMGA2 in a clinical trial for β-thalassemia resulted in clonal expansion of gene-modified cells that has provided a therapeutic benefit without malignancy to date [80]. β-thalassemia is a genetic deficiency that hinders β-globin production and patients with this mutation are reliant upon continued blood transfusions to restore normal blood globin levels. Before HSC gene therapy the only therapeutic available was allogeneic transplant, however the proce‐ dure is high risk and patient limited due to a lack of matched donors. Thus, patients risk transplant rejection or development of graft versus host disease. To achieve therapeutic ben‐ efit using HSC gene therapy, lineage specific transgene expression in erythrocytes is re‐ quired, promoting appropriate β-globin expression. Therapeutic benefit was achieved in two gene therapy patients resulting from a partially dominant clone harboring proviral in‐ sertions near HMGA2 [80]. The authors of this study conclude the clone with HMGA2 may remain homeostatic or eventually progress through multistep leukemogenesis, indicating a strong need for continued gene-marking studies and clonal tracking of these gene-modified cells in vivo [80].

#### **14. MDS1/EVI1, PRDM16, SETBP1 in trial for CGD**

ing insertional mutagenesis from clonal expansion and long-term selection pressures. In two non-human primate studies, the distribution of MLV-based gammaretroviral and SIV and HIV-1 based lentiviral integration sites were evaluated over long periods [76, 77]. Clonal ex‐ pansion or malignant transformation was not observed. However, integrants were observed at higher than expected frequencies near growth promoting genes and proto-oncogenes. This suggests that repopulating cells with integrations near these genes can influence sur‐ vival of those clones. These studies can shed light on potential mechanisms of clonal expan‐ sion and can allow comparison of different vector types. However, these studies in normal animals using vectors with a reporter gene that is not expected to provide a selective growth advantage did not predict the clonal expansion observed in the SCID-X1 trial. This suggests that large animal models of specific disease settings such as the SCID-X1 dog model [34] will be important to test improved vectors designed to reduce genotoxicity in a specific disease

Another important contribution of large animal models has been to improve our under‐ standing of the effects of ex vivo culture on clonal expansion. It has been shown in a nonhu‐ man primate model that genotoxicity can be significantly influenced by the culturing conditions of gene-modified cells ex vivo. In this study, only six days of culture increased the incidence of specific clones with gamma retroviral vector integrations near MDS/ EVI1 locus associated with a leukemic phenotype [78]. To monitor the potential effects of ex vivo culturing condition and in vivo selection on gene-modified repopulating cells, clonal track‐

The above studies have identified mechanisms of genotoxicity and clonal expansion. During clinical trials, it is important to monitor potential clonal expansion in order to understand genotoxicity and to anticipate potential adverse events [79]. As an example, dysregulation of HMGA2 in a clinical trial for β-thalassemia resulted in clonal expansion of gene-modified cells that has provided a therapeutic benefit without malignancy to date [80]. β-thalassemia is a genetic deficiency that hinders β-globin production and patients with this mutation are reliant upon continued blood transfusions to restore normal blood globin levels. Before HSC gene therapy the only therapeutic available was allogeneic transplant, however the proce‐ dure is high risk and patient limited due to a lack of matched donors. Thus, patients risk transplant rejection or development of graft versus host disease. To achieve therapeutic ben‐ efit using HSC gene therapy, lineage specific transgene expression in erythrocytes is re‐ quired, promoting appropriate β-globin expression. Therapeutic benefit was achieved in two gene therapy patients resulting from a partially dominant clone harboring proviral in‐ sertions near HMGA2 [80]. The authors of this study conclude the clone with HMGA2 may remain homeostatic or eventually progress through multistep leukemogenesis, indicating a

setting.

ing methods must be employed.

412 Gene Therapy - Tools and Potential Applications

**13. Tracking of genetically modified clones**

Chronic Granulomatous disease (CGD) is an x-linked inherited immunodeficiency result‐ ing from a mutation in one of the NADPH oxidase genes [87]. The gp91phox protein ac‐ counts for 70% of cases [81]. The gp91phox transgene has been used in corrective HSC gene therapy clinical trials [81]. Unlike SCID-X1 gene-modified cells, CGD gene-modified cells do not exhibit a proliferative advantage from transgene expression. The lack of condition‐ ing or selection of gene-modified cells contributed to a loss of therapeutic benefit and de‐ tection of gene-modified cells. Patients in the initial trials had low marking with gene expression of the corrected transgene for short periods of therapeutic benefit. Adverse genotoxic events developed 2 ½ years after the initial therapy, with clonal expansion and leukemic transformation [7, 82]. Clonal analysis found activation of MDS1/EVI1, PRDM16, and SETBP1 proto-oncogenes [81]. One of the patients died in treatment from complica‐ tions arising from the leukemia, the other patient survived after receiving an additional al‐ logeneic transplant. In this study there was inefficient engraftment and short-term transgene expression, with vector silencing via vector LTR methylation (Figure 2D) [83]. Further improvements to enhance engraftment and selection of gene-modified cells after infusion are needed for HSC gene therapy to treat CGD.

#### **15. CCND2 and MDS1/EVI1 trial for Wiskott-Aldrich**

HSC gene therapy has also been used in the treatment of Wiskott-Aldrich syndrome (WAS), an X-linked recessive immune disorder. In this study, patients underwent conditioning with busulfan to enhance the engraftment of gene-modified cells [84] Patients exhibited therapeu‐ tic benefit, with resolution of disease symptoms, although clonal skewing was detected for clones that harbored vector integration sites near CCND2 and MDS1/EVI1 [84]. Despite the high success of WAS HSC gene therapy in nine of the ten patients treated, patient 2 was re‐ ported to have experienced vector-derived genotoxic events after more than 3 years of thera‐ peutic benefit [84], ultimately resulting in T-cell leukemia. The leukemia was a result of proviral integration near the gene LMO2, and this patient has since been treated with che‐ motherapy resulting in remission [85-87].

Clonal tracking in vivo has recently been employed in a study where patients with glioblas‐ toma were given gene-modified hematopoietic repopulating repopulating stem cells carry‐ ing a methylguanine methyltransferase mutant (MGMT-P140K) [17]. In this approach genemodified hematopoietic repopulating cells expressing this mutant enzyme are resistant to O6-benzylguanine (O6BG). This allows treatment of the glioblastoma solid tumor with O6BG and an alkylating agent. By protecting the hematopoietic system from chemotherapymediated hematopoietic toxicity, higher doses of chemotherapy can be used to treat the glio‐ blastoma. In patients undergoing chemotherapy, gene-modified cells were monitored to track potential clonal expansion and to assess patient safety. Repopulating cells were tracked and their retroviral integration sites monitored at several different time points, preand post-chemotherapeutic treatment. Throughout the course of chemotherapy treatment, over 12,000 unique retroviral insertion sites (RISs) were present in the three treated patients. The heterogeneity of RISs suggests a highly polyclonal engraftment of gene-modified repo‐ pulating cells. During tracking two patients exhibited clonal expansion, with prominent clones appearing with vector proviruses in PRDM16 (PR domain-containing 16), Set binding protein 1(SETBP1), and high-mobility group A2 (HMGA2) genes.

expression [95]. Incorporation of miRNA technologies can improve vector efficacy and safe‐

Retroviral Genotoxicity

415

http://dx.doi.org/10.5772/52530

Chromatin insulators are being developed to reduce the propensity of integrated vector pro‐ viruses to dysregulate host gene expression. Insulators are DNA elements that repress the activity of enhancers on promoters. The chicken hypersensitive site-4 (cHS4] insulator con‐ tains five DNA binding elements within a 250 bp fragment known as the dominant DNase hypersensitive site [96, 97]. A 650 bp cHS4 element has been characterized in conjunction with a 400 bp element from cHS4 that can sufficiently block enhancer activation [98]. Addi‐ tional insulators have been described for sea urchin sns5 insulator and an adeno-associated

The cHS4 insulator has been used in several retroviral vector systems [80,99, 101-106]. Ini‐ tial studies with cHS4 lentiviral vectors were shown to be effective in reducing genotoxici‐ ty [107]. Their use in erythrocytes gave encouraging results, albeit with low titers. In addition, this study also demonstrated the effects of insulator failure after a reduction of cHS4 element repeats, which was reported to have contributed to insertional mutagenesis and expansion of clones harboring HMGA2 mutations [80]. Sea urchin sns5 is a 462 bp in‐ sulator region that was demonstrated to function in gamma retroviral vectors by maintain‐ ing chromatin position affects [100]. This element also contains a previously identified insulator region of 265 bp found to block enhancer-activated directional transcription in human cells [108, 109]. The DHS-S1 viral insulator has been demonstrated to increase transgene expression 1000-fold from an elongation factor 1-alpha (EF1α) promoter in mus‐ cle cells, but was not studied for its ability to block transactivation of host genes [99]. Insu‐ lators can potentially serve several major functions, by protecting against vector silencing, moderating vector variegation or uniformity of expression, and protecting nearby host genes from enhancer activation. Additional studies should help better characterize the effi‐

Incorporation of cell-type specific control elements such as erythrocyte specific enhancerpromoter has been used to control transgene expression [110]. The use of a lineage specific promoter ensures that transgene expression only occurs within the lineage from which the promoter is active. Moreover, avoiding expression of the transgene in other cell types with which the promoter is not active. The premise of lineage-restricted promoters for HSC gene therapy is that they may eliminate or reduce genotoxicity resulting from dysregulation of genes in stem/progenitor cells. This is accomplished by activating transgene expression only in a cell lineage with which transgene expression is required for therapeutic benefit. This ap‐

ty, ultimately reducing or limiting vector-born genotoxic events.

**18. Incorporation of cell-type specific control elements**

**17. Chromatin insulators**

(AAVS1] viral insulator DHS-S1 [99, 100].

cacy of insulated vectors

In summary, it is clear that HSC gene therapy is an efficacious therapeutic approach, able to treat debilitating and often fatal genetic deficiencies. However, the observed clonal expan‐ sion in these early clinical trials presents a major concern in the field. There is a need for vec‐ tors with an improved safety profile that are less likely to dysregulate genes and lead to clonal expansion.

#### **16. Next-generation vectors: Reducing genotoxicity**

Extensive efforts are underway to develop vector systems with safer integration profiles and reduced genotoxic effect. One approach is to retarget vector integration using tethering pro‐ teins that redirect the PIC. Other efforts focus on reducing genotoxicity by producing vec‐ tors less likely to dysregulate nearby genes. Such vectors include self-inactivating LTRs, which have deleted enhancer elements or U3 regions, preventing enhancer mediated expres‐ sion of nearby genes. Newer vectors are also able to regulate context dependent transgene expression using insulators and repressor elements to prevent viral promoters from activat‐ ing genes near the site of insertion [88]. Recently investigators have also identified insertion‐ al effects mediating alternative splicing, producing aberrant splice variants and protein fusion products causing oncogenesis [89, 90]. Modifying the vector-borne cryptic splice sites in vector backbones can create safer vectors reducing aberrant splice variant, reducing post‐ translational dysregulation of gene expression (Figure 2 C), [89, 91-93]. In addition, vector and host miRNAs have recently been explored. An example of miRNA control was demon‐ strated using miRNA let7 control elements, regulating expression of transgenes in stem cells versus somatic cells. Silencing of the transgenes occurs in somatic mature cells by miRNA cleavage sites. When let7 target sequence is matured and expressed, cleavage of the trans‐ gene containing the target sequence occurs [94]. In pluripotent cells, let7 is not expressed, thus the target sequences are not cleaved and full-length transgene is expressed [94]. This technology could potentially direct HSC gene therapy over a major hurdle, by reducing vec‐ tor-born genotoxicity through transgene expression in a highly controlled, cell specific con‐ text. These miRNA technologies have the ability to restrict transgene expression to a specific cell type and are even able to restrict transgene expression within a specific differentiation stage of that cell type, allowing a more specific control of transgene delivery, dosage, and expression [95]. Incorporation of miRNA technologies can improve vector efficacy and safe‐ ty, ultimately reducing or limiting vector-born genotoxic events.

#### **17. Chromatin insulators**

mediated hematopoietic toxicity, higher doses of chemotherapy can be used to treat the glio‐ blastoma. In patients undergoing chemotherapy, gene-modified cells were monitored to track potential clonal expansion and to assess patient safety. Repopulating cells were tracked and their retroviral integration sites monitored at several different time points, preand post-chemotherapeutic treatment. Throughout the course of chemotherapy treatment, over 12,000 unique retroviral insertion sites (RISs) were present in the three treated patients. The heterogeneity of RISs suggests a highly polyclonal engraftment of gene-modified repo‐ pulating cells. During tracking two patients exhibited clonal expansion, with prominent clones appearing with vector proviruses in PRDM16 (PR domain-containing 16), Set binding

In summary, it is clear that HSC gene therapy is an efficacious therapeutic approach, able to treat debilitating and often fatal genetic deficiencies. However, the observed clonal expan‐ sion in these early clinical trials presents a major concern in the field. There is a need for vec‐ tors with an improved safety profile that are less likely to dysregulate genes and lead to

Extensive efforts are underway to develop vector systems with safer integration profiles and reduced genotoxic effect. One approach is to retarget vector integration using tethering pro‐ teins that redirect the PIC. Other efforts focus on reducing genotoxicity by producing vec‐ tors less likely to dysregulate nearby genes. Such vectors include self-inactivating LTRs, which have deleted enhancer elements or U3 regions, preventing enhancer mediated expres‐ sion of nearby genes. Newer vectors are also able to regulate context dependent transgene expression using insulators and repressor elements to prevent viral promoters from activat‐ ing genes near the site of insertion [88]. Recently investigators have also identified insertion‐ al effects mediating alternative splicing, producing aberrant splice variants and protein fusion products causing oncogenesis [89, 90]. Modifying the vector-borne cryptic splice sites in vector backbones can create safer vectors reducing aberrant splice variant, reducing post‐ translational dysregulation of gene expression (Figure 2 C), [89, 91-93]. In addition, vector and host miRNAs have recently been explored. An example of miRNA control was demon‐ strated using miRNA let7 control elements, regulating expression of transgenes in stem cells versus somatic cells. Silencing of the transgenes occurs in somatic mature cells by miRNA cleavage sites. When let7 target sequence is matured and expressed, cleavage of the trans‐ gene containing the target sequence occurs [94]. In pluripotent cells, let7 is not expressed, thus the target sequences are not cleaved and full-length transgene is expressed [94]. This technology could potentially direct HSC gene therapy over a major hurdle, by reducing vec‐ tor-born genotoxicity through transgene expression in a highly controlled, cell specific con‐ text. These miRNA technologies have the ability to restrict transgene expression to a specific cell type and are even able to restrict transgene expression within a specific differentiation stage of that cell type, allowing a more specific control of transgene delivery, dosage, and

protein 1(SETBP1), and high-mobility group A2 (HMGA2) genes.

**16. Next-generation vectors: Reducing genotoxicity**

clonal expansion.

414 Gene Therapy - Tools and Potential Applications

Chromatin insulators are being developed to reduce the propensity of integrated vector pro‐ viruses to dysregulate host gene expression. Insulators are DNA elements that repress the activity of enhancers on promoters. The chicken hypersensitive site-4 (cHS4] insulator con‐ tains five DNA binding elements within a 250 bp fragment known as the dominant DNase hypersensitive site [96, 97]. A 650 bp cHS4 element has been characterized in conjunction with a 400 bp element from cHS4 that can sufficiently block enhancer activation [98]. Addi‐ tional insulators have been described for sea urchin sns5 insulator and an adeno-associated (AAVS1] viral insulator DHS-S1 [99, 100].

The cHS4 insulator has been used in several retroviral vector systems [80,99, 101-106]. Ini‐ tial studies with cHS4 lentiviral vectors were shown to be effective in reducing genotoxici‐ ty [107]. Their use in erythrocytes gave encouraging results, albeit with low titers. In addition, this study also demonstrated the effects of insulator failure after a reduction of cHS4 element repeats, which was reported to have contributed to insertional mutagenesis and expansion of clones harboring HMGA2 mutations [80]. Sea urchin sns5 is a 462 bp in‐ sulator region that was demonstrated to function in gamma retroviral vectors by maintain‐ ing chromatin position affects [100]. This element also contains a previously identified insulator region of 265 bp found to block enhancer-activated directional transcription in human cells [108, 109]. The DHS-S1 viral insulator has been demonstrated to increase transgene expression 1000-fold from an elongation factor 1-alpha (EF1α) promoter in mus‐ cle cells, but was not studied for its ability to block transactivation of host genes [99]. Insu‐ lators can potentially serve several major functions, by protecting against vector silencing, moderating vector variegation or uniformity of expression, and protecting nearby host genes from enhancer activation. Additional studies should help better characterize the effi‐ cacy of insulated vectors

#### **18. Incorporation of cell-type specific control elements**

Incorporation of cell-type specific control elements such as erythrocyte specific enhancerpromoter has been used to control transgene expression [110]. The use of a lineage specific promoter ensures that transgene expression only occurs within the lineage from which the promoter is active. Moreover, avoiding expression of the transgene in other cell types with which the promoter is not active. The premise of lineage-restricted promoters for HSC gene therapy is that they may eliminate or reduce genotoxicity resulting from dysregulation of genes in stem/progenitor cells. This is accomplished by activating transgene expression only in a cell lineage with which transgene expression is required for therapeutic benefit. This ap‐ proach might protect primitive cells from dysregulation, as the promoter is not expressed until differentiation into the target cell type. This is an attractive area of research for diseases that characteristically are exhibited in one lineage of the blood system such as hemaglobino‐ pathies. This approach was used in a thalassemia trial, where a β-locus-control-region-de‐ rived promoter was used [80]. The transgene is delivered to long term repopulating HSCs, but the promoter is not active. Only after erythroid differentiation would the enhancer be‐ come active, resulting in transgene expression in erythrocytes. This may reduce the occur‐ rence of proto-oncogene activation in stem/progenitor cells. Other lineage specific promoters are being studied, including B cell lineage specific promoters [111]. When a lineage specific promoter is not a viable option, vectors may need to be targeted to specific regions of the chromatin, where vector insertion is at a much lower risk of causing malignancy.

longevity and survival of HSC gene-modified clones after infusion. Several conditional pro‐ moter systems such as TET on/off and pro-drug inducible expression cassettes have been used to target cancer cells harboring dangerous integrations through vector silencing and

Retroviral Genotoxicity

417

http://dx.doi.org/10.5772/52530

The tetracycline (Tet) on/off gene expression system utilizes a pro-drug to regulate trans‐ gene expression by modifying a Tet repressor protein (TetR). TetR is constitutively ex‐ pressed and depending on its conformation will either be bound to the tetracycline operator (TetO), or unbound. In a Tet on system, TetR does not bind TetO until administration of the pro-drug, typically doxycycline. Once the pro-drug is administered, TetR actively binds Te‐ tO and silences transcription of nearby transgenes. In the absence of the pro-drug TetR can‐ not bind TetO, and this region of the genome is no longer blocked from transcription, and gene expression resumes. Alternatively, modifications have been made to TetR, allowing it to bind to repressor sequences until it is deactivated by a pro-drug; this system is called Tet off. The Tet on/off system may be used in conjunction with suicide genes to ablate undesired

In gene suicide approaches, HSC gene therapy delivers an active transgene in conjunction with a pro-drug induced suicide gene such as Thymidine Kinase [115]. In the event that transformations result from insertional mutagenesis and clonal expansion, clones harboring integrations can be eliminated or reduced by activating expression of the suicide gene, in‐ ducing apoptosis and eliminating clones harboring proviral integrations [26]. Recent clinical trials using an inducible caspase 9 (iCasp9), which remains in an inactivated state until di‐ merization following treatment with AP1903 small molecule, was reported in four patients with graft versus host disease after gene-modified hematopoietic transfusion [116]. In four patients, a single infusion of AP1903 was reported to have eliminated 90% of gene modified T cells within 30 minutes of administration of the inducing drug AP1903. GVHD and other associated illnesses typically observed after allogeneic bone marrow transplants were not detected up to a year after AP1903 treatment [116]. Thymidine kinase and iCasp9 present effective safety switches to control an array of genotoxic effects arising from HSC gene ther‐ apy [26, 117]. Utilizing these safety mechanisms in new vector designs will aid in furthering safety and reducing genotoxic events, and allow for selective ablation of expanded gene-

The use of HSC gene therapy in clinical trials is expanding, and the therapeutic potential is enormous. Following the initial successes with ADA SCID and SCID-X1 additional effica‐ cious therapies were reported for WAS, β-thalassemia, and CGD. Seymour et al. reported that the majority of over 90 patients receiving HSC gene therapy exhibit prolonged clinical benefit, with greater than 90% survival rate despite the occurrence of genotoxic events [46]. Current studies that are underway aim to characterize and reduce genotoxicity. Several ap‐ proaches have reduced genotoxic events in preclinical studies. With ongoing technological

suicide gene activation [114].

clones.

modified clones in vivo.

**21. Concluding summary**

#### **19. Re-targeting of retroviral vectors**

Efforts have been made to target retroviral proviruses to specific chromosomal locations. LEDGF, a host cell protein that interacts with HIV Gag has been used to effect tethering and targeting of viral integration Gijsbers et al [112]. In this study, cells were modified to express LEDGF protein containing a chromatin-interacting domain of chromobox homolog 1 (CBX1), which binds di- and tri- methylated regions of histone 3 (H3) in heterochromatic re‐ gions of the genome [112]. H3s are located pericentric to regions of heterochromatin, which is safer in terms of insertional mutagenesis as genes in these regions are normally silent. However, the reporting of significant retargeting of integration sites to heterochromatin is encouraging.

In addition, authors reported that transgene expression was not affected by targeting to these transcriptionally unfavorable heterochromatic sites [112]. These exciting experiments have demonstrated that vectors containing Gag and Pol C termini with adapted or unique binding domains could direct insertional distribution [113]. However, LEDGF cannot be modified in HSC gene therapy and alternative tethering approaches must be devised.

Additional tethering proteins have been studied and need to be fully characterized to ex‐ pand targeted integration locations in in vivo approaches [113]. In future studies use of ap‐ propriate tethers for modified integration site preference may reduce genotoxicity and may provide a better understanding of virus and host interactions affecting viral integration. In addition, even with vector systems that have incorporated these safety mechanisms, geno‐ toxic events may arise and methods to ablate the gene-modified cells will be useful to avoid malignancy.

#### **20. Approaches to ablate expanded cell clones**

Several approaches exist to ablate or control expanded clones after insertionally activated oncogenesis has occurred. Conditional selection systems have been employed to control the longevity and survival of HSC gene-modified clones after infusion. Several conditional pro‐ moter systems such as TET on/off and pro-drug inducible expression cassettes have been used to target cancer cells harboring dangerous integrations through vector silencing and suicide gene activation [114].

The tetracycline (Tet) on/off gene expression system utilizes a pro-drug to regulate trans‐ gene expression by modifying a Tet repressor protein (TetR). TetR is constitutively ex‐ pressed and depending on its conformation will either be bound to the tetracycline operator (TetO), or unbound. In a Tet on system, TetR does not bind TetO until administration of the pro-drug, typically doxycycline. Once the pro-drug is administered, TetR actively binds Te‐ tO and silences transcription of nearby transgenes. In the absence of the pro-drug TetR can‐ not bind TetO, and this region of the genome is no longer blocked from transcription, and gene expression resumes. Alternatively, modifications have been made to TetR, allowing it to bind to repressor sequences until it is deactivated by a pro-drug; this system is called Tet off. The Tet on/off system may be used in conjunction with suicide genes to ablate undesired clones.

In gene suicide approaches, HSC gene therapy delivers an active transgene in conjunction with a pro-drug induced suicide gene such as Thymidine Kinase [115]. In the event that transformations result from insertional mutagenesis and clonal expansion, clones harboring integrations can be eliminated or reduced by activating expression of the suicide gene, in‐ ducing apoptosis and eliminating clones harboring proviral integrations [26]. Recent clinical trials using an inducible caspase 9 (iCasp9), which remains in an inactivated state until di‐ merization following treatment with AP1903 small molecule, was reported in four patients with graft versus host disease after gene-modified hematopoietic transfusion [116]. In four patients, a single infusion of AP1903 was reported to have eliminated 90% of gene modified T cells within 30 minutes of administration of the inducing drug AP1903. GVHD and other associated illnesses typically observed after allogeneic bone marrow transplants were not detected up to a year after AP1903 treatment [116]. Thymidine kinase and iCasp9 present effective safety switches to control an array of genotoxic effects arising from HSC gene ther‐ apy [26, 117]. Utilizing these safety mechanisms in new vector designs will aid in furthering safety and reducing genotoxic events, and allow for selective ablation of expanded genemodified clones in vivo.

#### **21. Concluding summary**

proach might protect primitive cells from dysregulation, as the promoter is not expressed until differentiation into the target cell type. This is an attractive area of research for diseases that characteristically are exhibited in one lineage of the blood system such as hemaglobino‐ pathies. This approach was used in a thalassemia trial, where a β-locus-control-region-de‐ rived promoter was used [80]. The transgene is delivered to long term repopulating HSCs, but the promoter is not active. Only after erythroid differentiation would the enhancer be‐ come active, resulting in transgene expression in erythrocytes. This may reduce the occur‐ rence of proto-oncogene activation in stem/progenitor cells. Other lineage specific promoters are being studied, including B cell lineage specific promoters [111]. When a lineage specific promoter is not a viable option, vectors may need to be targeted to specific regions of the

chromatin, where vector insertion is at a much lower risk of causing malignancy.

Efforts have been made to target retroviral proviruses to specific chromosomal locations. LEDGF, a host cell protein that interacts with HIV Gag has been used to effect tethering and targeting of viral integration Gijsbers et al [112]. In this study, cells were modified to express LEDGF protein containing a chromatin-interacting domain of chromobox homolog 1 (CBX1), which binds di- and tri- methylated regions of histone 3 (H3) in heterochromatic re‐ gions of the genome [112]. H3s are located pericentric to regions of heterochromatin, which is safer in terms of insertional mutagenesis as genes in these regions are normally silent. However, the reporting of significant retargeting of integration sites to heterochromatin is

In addition, authors reported that transgene expression was not affected by targeting to these transcriptionally unfavorable heterochromatic sites [112]. These exciting experiments have demonstrated that vectors containing Gag and Pol C termini with adapted or unique binding domains could direct insertional distribution [113]. However, LEDGF cannot be modified in HSC gene therapy and alternative tethering approaches must be devised.

Additional tethering proteins have been studied and need to be fully characterized to ex‐ pand targeted integration locations in in vivo approaches [113]. In future studies use of ap‐ propriate tethers for modified integration site preference may reduce genotoxicity and may provide a better understanding of virus and host interactions affecting viral integration. In addition, even with vector systems that have incorporated these safety mechanisms, geno‐ toxic events may arise and methods to ablate the gene-modified cells will be useful to avoid

Several approaches exist to ablate or control expanded clones after insertionally activated oncogenesis has occurred. Conditional selection systems have been employed to control the

**19. Re-targeting of retroviral vectors**

416 Gene Therapy - Tools and Potential Applications

**20. Approaches to ablate expanded cell clones**

encouraging.

malignancy.

The use of HSC gene therapy in clinical trials is expanding, and the therapeutic potential is enormous. Following the initial successes with ADA SCID and SCID-X1 additional effica‐ cious therapies were reported for WAS, β-thalassemia, and CGD. Seymour et al. reported that the majority of over 90 patients receiving HSC gene therapy exhibit prolonged clinical benefit, with greater than 90% survival rate despite the occurrence of genotoxic events [46]. Current studies that are underway aim to characterize and reduce genotoxicity. Several ap‐ proaches have reduced genotoxic events in preclinical studies. With ongoing technological refinement, newer and safer HSC gene therapy vectors are entering, or will soon enter, the clinical arena. These advances are crucial for HSC gene therapy to enter mainstream medi‐ cine as an effective and safe therapeutic approach.

[6] Segal BH, Veys P, Malech H, Cowan MJ. Chronic Granulomatous Disease: Lessons from a Rare Disorder. Biol Blood Marrow Transplant. 2011; 17(1): p. s123-s131.

Retroviral Genotoxicity

419

http://dx.doi.org/10.5772/52530

[7] Stein S, Ott M, Schultze-Strasser S, Jauch A, Burwinkel B, Kinner A, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med. 2010; 16(2): p. 198-204. [8] Kang EM, Malech HL. Gene Therapy for Chronic Granulomatous Disease. In Fried‐ mann T, editor. Methods in Enzymology. San Diego, CA: Academic Press; ELSEVI‐

[9] Aiuti A, Cattaneo F, Galimberti S, Benninghoff U, Cassani B, Callegaro L, et al. Gene therapy for severe combined immunodeficiency due to adenosine deaminase defi‐

[10] Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Sci‐

[11] Aiuti A, Cassani B, Andolfi G, Mirolo M, Biasco L, Recchia A, et al. Multilineage hematopoietic reconstituion without clonal selection in ADA-SCID patients treated

[12] Blaese RM, Culver KW, Miller AD, Carter CS, Fleisher T, Clerici M, et al. T lympho‐ cyte-directed gene therapy for ADA- SCID: initial trial results after 4 years. Science.

[13] Charrier S, Dupré L, Scaramuzza S, Jeanson-Leh L, Blundell M, Danos O, et al. Lenti‐ viral vectors targeting WASp expression to hematopoietic cells, efficiently transduce and correct cells from WAS patients. Nature: Gene Therapy. 2007; 14(5): p. 415-428.

[14] Zanta-Boussif M, Charrier S, Brice-Ouzet A, Martin S, Opolon P, Thrasher A, et al. Validation of a mutated PRE sequence allowing high and sustained transgene ex‐ pression while abrogating WHV-X protein synthesis: application to the gene therapy

[15] Cartier N, Hacein-Bey-Abina S, Bartholomae C, Veres G, Schmidt M, Kutschera I, et al. Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adreno‐

[16] Biffi A, De Palma M, Quattrini A, Del Carro U, Amadio S, Visigalli I, et al. Correction of metachromatic leukodystrophy in the mouse model by transplantation of geneti‐ cally modified hematopoietic stem cells. J. Clin. Invest. 2004; 113(8): p. 1118-1129. [17] Adair JE, Beard BC, Trobridge Gd, Neff T, Rockhill JK, Daniel L. Silbergeld MMM, et al. Extended Survival of Glioblastom Patients After Chemoprotective HSC Gene

[18] Booth C, Gaspar BH, Thrasher AJ. Gene therapy for primary immunodeficiency.

with stem cell gene therapy. J Clin Invest. 2007; 117(8): p. 2233-2240.

of WAS. Nature; Gene Therapy. 2009; 16(5): p. 605-619.

leukodystrophy. Science. 2009; 326(5954): p. 818-823.

Therapy. Sci. Transl. Med. 2012; 4(133): p. 133ra57.

Curr. Opin. Pediatr. 2011; 23(6): p. 659-666.

ER; 2012. p. 125-154.

ciency. N. Engl.J. med. 2009; 360(5): p. 447-458.

ence. 2002; 296(5577): p. 2410-2413.

1995; 270(5235): p. 475–480.

#### **Acknowledgements**

G.D.T. was supported in part from the National Institutes of Health (AI097100), by funds provided for medical and biological research by the State of Washington Initiative Measure No. 171, and by the Department of Defense Peer Reviewed Cancer Research Program under award number W81XWH-11-1-0576. Views and opinions of, and endorsements by the au‐ thor(s) do not reflect those of the US Army or the Department of Defense.

#### **Author details**

Dustin T. Rae1 and Grant D. Trobridge1,2

\*Address all correspondence to: grant.trobridge@wsu.edu

1 Department of Pharmaceutical Sciences, Washington State University, Pullman, Washing‐ ton, USA

2 School of Molecular Biosciences, Washington State University, Pullman, Washington, USA

#### **References**


[6] Segal BH, Veys P, Malech H, Cowan MJ. Chronic Granulomatous Disease: Lessons from a Rare Disorder. Biol Blood Marrow Transplant. 2011; 17(1): p. s123-s131.

refinement, newer and safer HSC gene therapy vectors are entering, or will soon enter, the clinical arena. These advances are crucial for HSC gene therapy to enter mainstream medi‐

G.D.T. was supported in part from the National Institutes of Health (AI097100), by funds provided for medical and biological research by the State of Washington Initiative Measure No. 171, and by the Department of Defense Peer Reviewed Cancer Research Program under award number W81XWH-11-1-0576. Views and opinions of, and endorsements by the au‐

1 Department of Pharmaceutical Sciences, Washington State University, Pullman, Washing‐

2 School of Molecular Biosciences, Washington State University, Pullman, Washington, USA

[1] Malech H, Maples P, Whiting-Theobald N, Linton G, Sekhsaria S, Vowells S, et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene therapy of chronic granulomatous disease. Proc. Natl. Acad. Sci. 1997; 94(22): p. 12133-12138.

[2] Malech H, Horwitz M, Linton G. Extended production of oxidase normal neutrophils in X-linked chronic granulomatous disease (CGD) following gene therapy with

[3] Barese CN, Goebel WS, Dinauer MC. Gene therapy for chronic granulomatous dis‐

[4] Bleijs DA. Gene Therapy Net. [Online].; 2012 [cited 2012 June 18. Available from:

[5] Hacein-Bey-Abina S, Garrigue A, Wang G, Soulier J, Lim A, Morillon E, et al. Inser‐ tional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J. Clin. Invest. 118:3132–3142 (2008). doi:10.1172/JCI35700. 2008; 118(9): p. 3132-3142.

gp91phos transduced CD34+ cells. Blood. 1998; 92((suppl 10)): p. 690a.

ease. Expert Opinion on Biological Therapy. 2004; 4(9): p. 1423-1434.

thor(s) do not reflect those of the US Army or the Department of Defense.

cine as an effective and safe therapeutic approach.

and Grant D. Trobridge1,2

http://www.genetherapynet.com/.

\*Address all correspondence to: grant.trobridge@wsu.edu

**Acknowledgements**

418 Gene Therapy - Tools and Potential Applications

**Author details**

Dustin T. Rae1

ton, USA

**References**


[19] Hacein-Bey-Abina S, Von KC, Schmidt M, McCormack M, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science. 2003; 302(5644): p. 415-419.

[33] Ting-De Ravin S, Kennedy D, Naumann N, Kennedy J, Choi U, Hartnett B, et al. Cor‐ rection of canine X-linked severe combined immunodeficiency by in vivo retroviral

Retroviral Genotoxicity

421

http://dx.doi.org/10.5772/52530

[34] Kennedy D, Hartnett B, Kennedy J, Vernau W, Moore P, O'Malley T, et al. Ex vivo γretroviral gene therapy of dogs with X-linked severe combined immunodeficiency and the development of a thymic T cell lymphoma. Veterinary Immunology and Im‐

[35] Oram S, Thoms J, Pridans C, Janes M, Kinston S, Anand S, et al. A previously unrec‐ ognised promoter of LMO2 forms part of a transcriptional regulatory circuit media‐ ting LMO2 expression in a subset of T-acute lymphoblastic leukemia patients.

[36] Kool J, Berns A. High-throughput insertional mutagenesis screens in mice to identify

[37] Suzuki T, Minehata K, Akagi K, Jenkins N, Copeland N. Tumor supresor gene identi‐ fication using retroviral insertional mutagenesis in Blm-deficient mice. Embo. J. 2006;

[38] Kustikova O, Fehse B, Modlich U, Yang M, Düllmann J, Kamino K, et al. Clonal Dominance of Hematopoietic Stem Cells Triggered by Retroviral Gene Marking. Sci‐

[39] Warren A, Colledge W, Carlton M, Evans M, Smith A, Rabbitts T. The oncogenic cys‐ teine-rich LIM domain protein rbtn2 is essential for erythroid development. Cell.

[40] Lessard J, Sauvageau G. Bmi-1 determines the proliferative capacity of normal and

[41] Park I, Qian D, Kiel M, Becker M, Pihalja M, Weissman I, et al. Bmi-1 is required for maintenance of adult self-renewing hematopoietic stem cells. Nature. 2003;

[42] Kazuhiko I, Philip J, Bessler M, Bessler M. 3¢UTR-truncated Hmga2 cDNA causes MPN-like hematopoiesis by conferring a clonal growth advantage at the level of HSC

[43] Peterson EA, Stanbery L, Li C, Kocak H, Makarova O, Petty EM. SEPT9\_i1 and Ge‐ nomic Instability: Mechanistic Insights and Relevance to Tumorigenesis. GENES,

[44] Cerveira N, Santos J, Teixeira M. Structural and Expression Changes of Septins in Myeloid Neoplasia. Critical Re3views in Oncogenesis. 2009; 15(1-2): p. 91-115. [45] Kuo YH,. Zaidi SK, Gornostaeva S, Komori T, Stein GS, and Lucio H. Runx2 induces acute myeloid leukemia in cooperation with Cbfb -SMMHC in mice. Blood. 2009;

oncogenic networks. Nat. Rev. Cancer. 2009; 9(6): p. 389-399.

leukemic stem cells. Nature. 2003; 423(6937): p. 255-260.

CHROMOSOMES & CANCER. 2011; 50(11): p. 940–949.

in mice. Blood. 2011; 117(22): p. 5860-5869.

gene therapy. Blood. 2006; 107(8): p. 3091-3097.

munopathology. 2011; 142(1-2): p. 36-48.

Oncogene. 2010; 29(43): p. 5796-5808.

25(14): p. 3422-3431.

1994; 78(1): p. 45-57.

423(6937): p. 302-305.

113(14): p. 3323-3332.

ence. 2005; 308(5725): p. 1171.


[33] Ting-De Ravin S, Kennedy D, Naumann N, Kennedy J, Choi U, Hartnett B, et al. Cor‐ rection of canine X-linked severe combined immunodeficiency by in vivo retroviral gene therapy. Blood. 2006; 107(8): p. 3091-3097.

[19] Hacein-Bey-Abina S, Von KC, Schmidt M, McCormack M, Wulffraat N, Leboulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene therapy

[20] Scobie L, Hector R, Grant L, Bell M, Nielsen A, Meikle S, et al. A novel model of SCID-X1 reconstitution reveals predisposition to retrovirus-induced lymphoma but

[21] Nienhuis AW. Development of gene therapy for blood disorders. American Society

[22] Olga K, Martijn B, Baum C. The Genomic risk of somatic gene therapy. Seminars in

[23] Rous R. Transmission of a malignant new growth by means of a cell-free filtrate. JA‐

[24] Varmus H, Weiss R, Friis R, Levinson W, Bishop J. Detection of avian tumor virusspecific nucleotide sequences in avian cell DNAs (reassociation kinetics-RNA tumor viruses-gas antigen-Rous sarcoma virus, chick cells). Proc. Natl. Acad. Sci. USA.

[25] Sheridan C. Gene therapy finds its niche. Nature biotechnology. 2011; 29(2): p.

[26] Rivie're I, Dunbar CE, Sadelain M. Hematopoietic stem cell engineering at a cross‐

[27] Nielsen J, McNagny K. Novel functions of the CD34 family. J of Cell Science. 2008;

[28] Brenner M, Rill D, Holladay M, Heslop H, Moen R, Buschle M, et al. Gene marking to determine whether autologus marrow infusion restores long-term haemopoiesis in

[29] Brenner M, Rill D, Moen R, Krance R, Mirro JJ, Anderson W, et al. Gene-marking to trace origin of relapse after autologous bone-marrow transplant. Lancet. 1993;

[30] Horn PA, Morris JC, Neff T, Hiem HP. Stem Cell Gene Transfer- Efficacy and Safety

[31] Di Santo JP, Kuhn R, Muller W. Common Cytokine Receptor Y chain (Yc)- Depend‐ ent Cytokines: Understanding in vivo Functions by Gene Targetting. immunological

[32] Kiem H, Andrews R, Morris J, Peterson L, Heyward S, Allen J, et al. Improved gene transfer in baboon marrow repopulating cells using recombinant human fibronectin fragment in combination with interleukin-6, stem cell factor, FLT-3 ligand, and meg‐

akaryocyte growth and evelopment factor. Blood. 1998; 92(6): p. 1878-1886.

no evidence of gamma C gene oncogenicity. Mol Ther. 2009; 17(8): p. 1483.

for SCID-X1. Science. 2003; 302(5644): p. 415-419.

of Hematology; BLOOD. 2008; 111(9): p. 4431-4444.

Cancer Biology. 2010; 20(4): p. 269-278.

roads. BLOOD. 2012; 119(520): p. 1107-1116.

cancer patients. Lancet. 1993; 342(8880): p. 1134-1137.

in Large Animal Studies. Mol Ther. 2004; 10(3): p. 417-431.

MA. 1983; 250(11): p. 1445-1449.

1972; 69(1): p. 20-24.

420 Gene Therapy - Tools and Potential Applications

121(Pt 22): p. 3682-3692.

341(8837): p. 85-86.

Reviews. 1995; 148.

121-128.


[46] Seymour LW, Thrasher AJ. Gene therapy matures in the clinic. Nature Biotechnolo‐ gy. 2012; 30(7): p. 588-593.

[59] Cherepanov P, Maertens G, Proost P, Devreese B, Van Beeumen J, Engelborghs Y, et al. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in

Retroviral Genotoxicity

423

http://dx.doi.org/10.5772/52530

[60] Emiliani S, Mousnier A, Busschots K, Maroun M, Van Maele B, Tempé D, et al. Inte‐ grase mutants defective for interaction with LEGF/p75 are impaired in chromosome

[61] Ciuffi A, Llano M, Poeschla E, Hoffmann C, Leipzig J, Shinn P, et al. A role of LEGF/p75 in targetting HIV DNA integration. Nat Med. 2005; 11(12): p. 1287-1289. [62] Schliephake A, Rethwilm A. Nuclear localization of foamy virus Gag precursor pro‐

[63] Stenbak C, Linial M. Role of the C terminus of foamy virus Gag in RNA packaging

[64] Yu S, Edelmann K, Strong R, Moebes A, Rethwilm A, Linial M. The carboxyl termi‐ nus of the human foamy virus Gag protein contains separable nucleic acid binding

[65] Gabriel R, Eckenberg R, Paruzynski A, Bartholomae C, Nowrouzi A, Arens A, et al. Comprehensive genomic access to vector integration in clinical gene therapy. Nat

[66] Biasco L, Baricordi C, Aiuti A. Retroviral Integrations in Gene Therapy Trials. Molec‐

[67] Berger SA, Bernstein A. Characterization of a Retrovirus Shuttle Vector Capable of Either Proviral Integration or Extrachromosomal Replication in Mouse Cells. MO‐

[68] Cepko CL, Roberts BE, Mulligan RC. Construction and Applications of a Highly Transmissible Murine Retrovirus Shuttle Vector. Cell. 1984; 37(3): p. 1053-1062. [69] Montini E, Cesana D, Schmidt M, Sanvito F, Bartholomae C, Ranzani M, et al. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J. Clin. Invest. 2009;

[70] Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C, et al. Hema‐ topoietic stem cell gene transfer in a tumor-prone mouse model uncovers low geno‐

toxicity of lentiviral vector integration. Nat. Biotechnol. 2006; 24(6): p. 687-696.

[72] Sherr CJ. The INK4a/ARF network in tumour supression. Nat. Rev. Mol. Cell Biol.

[73] Greenman CD. Haploinsufficient Gene Selection in Cancer. Science. 2012; 337(6090):

[71] Sherr CJ. Principles of tumour supression. Cell. 2004; 116(2): p. 235-246.

tethering and HIV-1 replication. J Biol Chem. 2005; 280(27): p. 25517-25523.

human cells. J Biol Chem. 2003; 278(1): p. 373-381.

and Pol expression. J Virol. 2004; 78(17): p. 9423–9430.

and nuclear transport domains. J Virol. 1996; 70(12): p. 8255–8262.

LECULAR AND CELLULAR BIOLOGY. 1975; 5(2): p. 305-312.

tein. J Virol. 1994; 68(8): p. 4946–4954.

Med. 2009; 15(12): p. 1431-1436.

119(4): p. 964-975.

2001; 2(10): p. 731-737.

p. 47-48.

ular Therapy. 2012; 20(4): p. 709-716.


[59] Cherepanov P, Maertens G, Proost P, Devreese B, Van Beeumen J, Engelborghs Y, et al. HIV-1 integrase forms stable tetramers and associates with LEDGF/p75 protein in human cells. J Biol Chem. 2003; 278(1): p. 373-381.

[46] Seymour LW, Thrasher AJ. Gene therapy matures in the clinic. Nature Biotechnolo‐

[47] Schröder A, Shinn P, Chen H, Berry C, Ecker J, Bushman F. HIV-1 integration in the human genome favors active genes and local hotspots. Cell. 2002; 110(4): p. 521-529.

[48] Tsukahara T, Agawa H, Matsumoto S, Matsuda M, Ueno S, Yamashita Y, et al. Mur‐ ine leukemia virus vector integration favors promoter regions and regional hot spots in human T-cell line. Biochemical and Biophysical Research Communications. 2006;

[49] Mitchell R, Beitzel B, Schroder A, Shinn P, Chen H, Berry C, et al. Retroviral DNA integration: ASLV, HIV, and MLV show distinct target site preferences. PLoS Biol.

[50] Wu X, Li Y, Crise B, Burgess S. Trasncription start regions in the human genome are favored targets for MLV integration. Science. 2003; 300(5626): p. 1749-1751.

[51] Barr S, Ciuffi A, Leipzig J, Shinn P, Ecker J, Bushman F. HIV integration site selec‐ tion: targeting in macrophages and the effects of different routes of viral entry. Mol

[52] Aiken C. Pseudotyping human immunodeficiency virus type 1 (HIV-1) by the glyco‐ protein of vestcular stomattits virus targets HIV-1 entry to an endocytic pathway and suppresses both the requirment for Nef and the sensitivity to cyclosporin. A. J Virol.

[53] Narezkina A, Taganov K, Litwin S, Stoyanova R, Hayashi J, Seeger C, et al. Genomewide analyses of avian sarcoma virus integration sites. J Virol. 2004; 78(21): p.

[54] Trobridge G, Miller D, Jacobs M, Allen J, Kiem H, Kaul R, et al. Foamy virus vector integration sites in normal human cells. Proc Natl Acad SCI USA. 2006; 103(5): p.

[55] Brass A, Dykxhoorn D, Benita Y, Yan N, Engelman A, Xavier R, et al. Identification of host proteins required for HIV infection through a functional genomic screen. Sci‐

[56] Llano M, Delgado S, Vanegas M, Poeschla E. Lens epithelium-derived growth factor/P75 prevents proteasomal degredation of HIV-1 integrase. J Biol Chem. 2004;

[57] Lewinski M, Yamashita M, Emerman M, Ciuffi A, Marshall H, Crawford G, et al. Ret‐ roviral DNA integration: viral and cellular determinants of target-site selection. PLoS

[58] Tobaly-Tapiero J, Bittoun P, Lehmann-Che J, Delelis O, Giron M, de Thé H, et al. Chromatin Tethering of Incoming Foamy Virus by the Structural Gag Protein. Traffic

gy. 2012; 30(7): p. 588-593.

422 Gene Therapy - Tools and Potential Applications

345(3): p. 1099-1107.

2004; 2(8): p. E234.

Ther. 2006; 14(2): p. 218-225.

1997; 71(8): p. 5871-5877.

ence. 2008; 319(5865): p. 921-926.

279(53): p. 55570-55577.

pathog. 2006; 2(6): p. e60.

2008. 2008; 9(10): p. 1717-1727.

11656-11663.

1498-1503.


[74] Solimini N, Xu Q, Mermel C, Liang A, Schlabach M, Luo J, et al. Recurrent Hemizy‐ gous Deletions in Cancers May Optimize Proliferative Potential. Science. 2012; 337(6090): p. 104-108.

[86] Galya A, Thrasher AJ. Gene therapy for the Wiskott–Aldrich syndrome. Current

Retroviral Genotoxicity

425

http://dx.doi.org/10.5772/52530

[87] Stefan Zorn. Effective Gene Therapy for Children with Wiskott-Aldrich-Syndrome, a

[88] Bushey A, Dorman E, Corces V. Chromatin insulators; regulatory mechanisms and

[89] Moiani A, Paleari Y, Sartori D, Mezzadra R, Miccio A, Cattoglio C, et al. Lentiviral vector integration in the human genome induces alternative splicing and generates

[90] Nilsen T, Maroney P, Goodwin R, Rottman F, Crittenden L, Raines M, et al. c-ervB activation in ALV-induced erythroblastosis: novel RNA processing and promoter in‐ sertion result in expression of aminotruncated EGF receptor. Cell. 1985; 41(3): p.

[91] Cattoglio C, Pellin D, Rizzi E, Maruggi G, Corti G, Miselli F, et al. High-definition mapping of retroviral integration sites identifies avtice regulatory elements in human

[92] Maruggi G, Porcellini S, Facchini G, Perna S, Cattoglio C, Sartori D, et al. Transcrip‐ tional enhancers induce insertional gene deregulation independantly from the vector

[93] Almarza D, Bussadori G, Navarro M, Mavilio F, Larcher F, Murillas R. Risk assess‐ ment in skin gene therapy: viral-celluar fusion transcripts generated by proviral tran‐ scriptional read-through in Keratinocytes transduced with self-inactivating lentiviral

[94] Di Stefano B, Maffioletti S, Gentner B, Ungaro F, Schira G, Naldini L, et al. A miRNAbased System for Selecting and Maintaining the Pluripotent State in Human Induced

[95] Brown B, Naldini L. Exploiting and antagonizing microRNA regulation for therapeu‐ tic and experimental applications. Nat. Rev. Genet. 2009; 10(8): p. 578-585.

[96] Wallace J, Felsenfeld G. We gather together: insulators and genome organization.

[97] Gaszner M, Felsenfeld G. Insulators: exploiting transcriptional and epigenetic mecha‐

[98] Arumugam P, Urbinati F, Velu C, higashimoto T, Grimes H, Malik P. The 3' region of the chicken hypersensitive site-4 insulator has properties similar to its core and is re‐

[99] Ogata T, Kozuka T, Kanda T. Identification of an insulator in AAVS1, a preferred re‐ gion for integration of adeno-associated virus DNA.. Journal of virology. 2003;

Current opinion in genetics & development. 2007; 17(5): p. 400-407.

quired for fill insulator activity. PloS one. 2009; 4(9): p. e6995.

multipotent hematopoietic progenitors. Blood. 2010; 116(25): p. 5507-5517.

Opinion in Allergy and Clinical Immunology. 2011; 11(6): p. 545-50.

Severe Inborn Immunodeficiency Disease. 2010..

epigentic inheritance. MOl Cell. 2008; 32(1): p. 1-9.

type and design. Mol. Ther. 2009; 17(5): p. 851-856.

Pluripotent Stem Cells. Stem Cells. 2011; 29(11): p. 1-18.

vectors. Gene Ther. 2011; 18(7): p. 674-681.

nisms. Nat Rev Genet. 2006; 7(9): p. 703-713.

77(16): p. 9000-9007.

719-726.

aberrant transcripts. J. Clin. Invest. 2012; 122(5): p. 1653-1666.


[86] Galya A, Thrasher AJ. Gene therapy for the Wiskott–Aldrich syndrome. Current Opinion in Allergy and Clinical Immunology. 2011; 11(6): p. 545-50.

[74] Solimini N, Xu Q, Mermel C, Liang A, Schlabach M, Luo J, et al. Recurrent Hemizy‐ gous Deletions in Cancers May Optimize Proliferative Potential. Science. 2012;

[75] Beekman R, Valkhof M, Erkeland S, Taskesen E, Rockova V, Peeters J, et al. Retrovi‐ ral Integration Mutagenesis in Mice and Comparative analysis in Human AML Iden‐ tify Reduced PTP4A3 Expression as a Prognostic Indicator. PLoS one. 2011; 6(10): p.

[76] Beard B, Dickerson D, Beebe K, Gooch C, Fletcher J, Okbinoglu T, et al. Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in

[77] Hematti P, Hong B, Ferguson C, Adler R, Hanawa H, Sellers S, et al. Distinct genom‐ ic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor

[78] Sellers S, Gomes T, Larochelle A, Lopez R, Adler R, Krouse A, et al. Ex vivo expan‐ sion of retrovirally transcuded primate CD34+ cells results in over representation of clones with MDS1/EVI1 insertion sties in the myeloid lineage after tranplantation.

[79] Tey SK, Brenner Mk. The Continuing Contribution of Gene Marking to Cell and

[80] Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F, et al. Transfusion independence and HMGA2 activation after gene therapy of human beta-thalassae‐

[81] Kang H, Bartholomae C, Paruzynski A, Arens A, Kim S, Yu S, et al. Retroviral gene therapy for X-linked Chronic Granulomatus Disease: Results from Phase I/II Trial.

[82] Ott M, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med. 2006; 12(4): p. 401-409.

[83] Segal BH, Veys P, Malech H, Cowan aMJ. Chronic Granulomatous Disease: Lessons from a Rare Disorder. Biol Blood Marrow Transplant. 2011; 17(1 suppl): p. S123-S131.

[84] Boztug K, Schmidt M, Schwarzer A, P B, Avedillo Díez I, Dewey RA, et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med. 2010; 363: p.

[85] Braun CJ, Boztug K, Schmidt M, Albert MH, Schwarzer A, Paruzynski A, et al. 165 Efficacy of Gene Therapy for Wiskott-Aldrich-Syndrome. In 53 rd ASH Anual Meet‐ ing and Exposition; 2011; Elizabeth Ballroom DE (Manchester Grand Hyatt San Die‐

primate repopulating cells. J mole Ther. 2007; 15(7): p. 1356-1365.

Gene Therapy. Molecular Therapy. 2007; 15(4): p. 666-677.

337(6090): p. 104-108.

424 Gene Therapy - Tools and Potential Applications

cells. PLoS Biol. 2004; 2(12): p. e423.

Mol Ther. 2010; 18(9): p. 1633-1639.

mia. Nature. 2010; 467(7313): p. 318-322.

Mol.Ther. 2011; 19(11): p. 2092-2101.

1918-1927.

go).

e26537-e26537.


[100] D'Apolito D, Baiamonte E, Bagliesi M, Di Marzo R, Calzolari R, Ferro L, et al. The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure. Mol Ther. 2009; 17(8): p. 1434-1441.

[112] Gijsbers R, Ronen K, Vets S, Malani N, De Rijck J, McNeely M, et al. LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin. Mol ther. 2010; 18(3):

Retroviral Genotoxicity

427

http://dx.doi.org/10.5772/52530

[113] Yi Y, Hahm S, Lee K. Retroviral Gene Therapy: Safety Issues and Possible Solutions.

[114] Curtin J, Candolfi M, Xiong W, Lowenstein P, Castro M. Turning the gene tap off; implications of regulating gene expression for cancer therapeutics. Mol Cancer Ther.

[115] Lupo-Stanghellini M, Provasi E, Bondanza A, Ciceri F, Bordignon C, Bonini C. Clini‐ cal impact of suicide gene therapy in allogeneic hematopoietic stem cell transplanta‐

[116] Sadelain M. Eliminating Cells Gone Astray. n engl j med. 2011; 365(18): p. 1735-1736.

[117] Ellis J, Baum C, Benvenisty N, Mostoslavsky G, Okano H, Stanford W, et al. Benefits of utilizing genemodified iPSCs for clinical applications. Cell Stem Cell. 2010; 7(4): p.

p. 552-560.

429-430.

2008; 7(3): p. 439-449.

Current Gene Therapy. 2005; 5(1): p. 25-35.

tion. Hum Gene Ther. 2010; 21(3): p. 241-250.


[112] Gijsbers R, Ronen K, Vets S, Malani N, De Rijck J, McNeely M, et al. LEDGF hybrids efficiently retarget lentiviral integration into heterochromatin. Mol ther. 2010; 18(3): p. 552-560.

[100] D'Apolito D, Baiamonte E, Bagliesi M, Di Marzo R, Calzolari R, Ferro L, et al. The sea urchin sns5 insulator protects retroviral vectors from chromosomal position effects by maintaining active chromatin structure. Mol Ther. 2009; 17(8): p. 1434-1441. [101] Li C, Emery D. The cSH4 chromatin insulator reduced gammaretroviral vector si‐ lencing by epigenetic modification of integrated provirus. Gene Therapy. 2008; 15(1):

[102] Evans-Galea M, Wielgosz M, Hanawa H, Srivastava D, Nienhuis A. Supression of clonal dominance in cultured human lymphoid cells by addition of the cHS4 insula‐

[103] Li C, Xiong D, Stamatoyannopoulos G, Emery D. Genomic and functional assays demonstrate reduced gammaretroviral vector genotoxicity associated with use of the

[104] Aker M, Tubb J, Groth A, Bukovsky A, Bell A, Felsenfeld G, et al. Extended corse se‐ quences from the cSH4 insulator are necessary for protecting retroviral vectors for si‐

[105] Arumugam P, Scholes J, Perelman N, Xia P, Yee J, Malik P. Improved human betaglobin expression from self-inactivating lentiviral vectors carrying the chicken hyper‐

sensitive site-4 (cSH4) insulator element. Mol Ther. 2007; 15(10): p. 1863-1871. [106] Malik P, Arumugam P, Yee J, Puthenveetil G. Successfull correction of the human Cooley's anemia beta-thalassemia major phenotype using a lentiviral vector flanked by the chicken hypersensitive site 4 chromatin insulator. Ann N Y Acad Sci. 2005;

[107] Hanawa H, Yamamoto M, Zhao H, Shimada T, Persons DA. Optimized lentiviral vector improves titers and transgene expression of vectors containing the chicken B-

[108] Palla F, Melfi R, Anello L, Di Bernardo M, Spinelli G. Enhancer blocking activity lo‐ cated near the 3' end of the sea urchin early H2A histone gene. Proceedings of the National Academy of Sciences of the United States of America. 1997; 94(6): p.

[109] Melfi R, Palla F, Di Simone P, Alessandro C, Cali L, Anello L, et al. Functional charac‐ terization of the enhancer blocking element of the sea urchin early histone gene clus‐ ter reveals insulator properties and three essential cis-acting sequence. J Mol Biol.

[110] Cassani B, Montini E, Maruggi G, Ambrosi A, Mirolo M, Selleri S, et al. Integration of retroviral vectors induces minor changes in the transcriptional activity of T cells from ADA-SCID patients treated with gene therapy. Blood. 2009; 114(17): p. 3546-3456. [111] Sather B, Ryu B, Stirling B, Garibov M, Kerns H, Humblet-Baron S, et al. Develop‐ ment of B-lineage predominant lentiviral vectors for use in genetic therapies for B

globin locus HS4 insulator element. Mol. Ther. 2009; 17(4): p. 667-674.

lencing position effects. Human Gene Therapy. 2008; 18(4): p. 333-343.

tor to a lentiviral vector. Mol Ther. 2007; 15(4): p. 801-809.

cHS4 chromatin insulator. Mol Ther. 2009; 17(4): p. 716-724.

p. 49-53.

426 Gene Therapy - Tools and Potential Applications

1054: p. 238-249.

2272-2277.

2000; 304(5): p. 753-763.

cell disorders. Mol Ther. 2011; 19(3).


**Chapter 17**

**Efficient AAV Vector Production System: Towards Gene**

Successful gene therapy requires an adequate level of long-term transgene expression in the target tissues. While various viral vectors have been considered for the delivery of genes *in vivo*, an adeno-associated virus (AAV)-based vector is emerging as the gene transfer vehicle with the most potential for use in the neuromuscular gene therapies. The advantages of the AAV vector include the lack of disease associated with a wild-type vi‐ rus, the ability to transduce non-dividing cells, and the long-term expression of the deliv‐ ered transgenes.[1] Some serotypes of recombinant AAV (rAAV) exhibit a potent tropism for striated muscles.[2] Therefore, a supplementation of secretory protein can be achieved with this vector to use intramuscular injection.[3] Since a 5-kb genome is considered to be the upper limit for a single AAV virion, various truncated genes could be provided to

Due to ingenious cloning and preparation techniques, adenovirus vectors are efficient deliv‐ ery systems of episomal DNA into eukaryotic cell nuclei.[5] The utility of adenovirus vectors has been increased by capsid modifications that alter tropism, and by the generation of hy‐ brid vectors that promote chromosomal insertion.[6] Also, gutted adenovirus vectors devoid of all adenoviral genes allow for the insertion of large transgenes, and trigger fewer cytotox‐ ic and immunogenic effects than do those only deleted in the E1 regions of the adenovirus early genes.[7] Human artificial chromosomes (HACs) have the capacity to deliver genes in any size into host cells without integrating the gene into the host genome, thereby prevent‐

> © 2013 Okada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

ing the possibility of insertional mutagenesis and genomic instability.[8]

**Therapy For Duchenne Muscular Dystrophy**

Additional information is available at the end of the chapter

Takashi Okada

**1. Introduction**

**1.1. Choice of vector**

http://dx.doi.org/10.5772/53023

meet size capacity, if nessessarry.[4]

### **Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy**

Takashi Okada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53023

#### **1. Introduction**

#### **1.1. Choice of vector**

Successful gene therapy requires an adequate level of long-term transgene expression in the target tissues. While various viral vectors have been considered for the delivery of genes *in vivo*, an adeno-associated virus (AAV)-based vector is emerging as the gene transfer vehicle with the most potential for use in the neuromuscular gene therapies. The advantages of the AAV vector include the lack of disease associated with a wild-type vi‐ rus, the ability to transduce non-dividing cells, and the long-term expression of the deliv‐ ered transgenes.[1] Some serotypes of recombinant AAV (rAAV) exhibit a potent tropism for striated muscles.[2] Therefore, a supplementation of secretory protein can be achieved with this vector to use intramuscular injection.[3] Since a 5-kb genome is considered to be the upper limit for a single AAV virion, various truncated genes could be provided to meet size capacity, if nessessarry.[4]

Due to ingenious cloning and preparation techniques, adenovirus vectors are efficient deliv‐ ery systems of episomal DNA into eukaryotic cell nuclei.[5] The utility of adenovirus vectors has been increased by capsid modifications that alter tropism, and by the generation of hy‐ brid vectors that promote chromosomal insertion.[6] Also, gutted adenovirus vectors devoid of all adenoviral genes allow for the insertion of large transgenes, and trigger fewer cytotox‐ ic and immunogenic effects than do those only deleted in the E1 regions of the adenovirus early genes.[7] Human artificial chromosomes (HACs) have the capacity to deliver genes in any size into host cells without integrating the gene into the host genome, thereby prevent‐ ing the possibility of insertional mutagenesis and genomic instability.[8]

© 2013 Okada; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Long-term correction of genetic diseases requires permanent integration of therapeutic genes into chromosomes of the affected cells. However, retrovirus vector integration can trigger deregulated premalignant cell proliferation with unexpected frequency, most like‐ ly driven by retrovirus enhancer activity on the LMO2 gene promoter. [9] A goal in clin‐ ical gene therapy is to develop gene transfer vehicles that can integrate exogenous therapeutic genes at specific chromosomal loci as a safe harbor, so that insertional onco‐ genesis is prevented. AAV can insert its genome into a specific locus, designated AAVS1, on chromosome 19 of the human genome.[10] The AAV Rep78/68 proteins and the Rep78/68-binding sequences are the trans- and cis-acting elements needed for this reac‐ tion. A dual high-capacity adenovirus-AAV hybrid vector with full-length human dystro‐ phin-coding sequences flanked by AAV integration-enhancing elements was tested for targeted integration.[11]

#### **1.2. AAV biology**

AAV is a small (20-26nm) non-enveloped dependent parvovirus with a single-stranded line‐ ar genome that contains two open reading frames (*rep* and *cap*).[12] The viral genome is characterized by the inverted terminal repeats (ITRs) to flank these open reading frames (Figure 1A). The genome encodes four replication proteins (Rep78, Rep68, Rep52, and Rep40) and three capsid proteins (Cap: VP1, VP2, and VP3). The large Rep (Rep78 and Rep68) proteins regulate AAV gene expression and hold nicking activity at the terminal res‐ olution site as well as binding activity at Rep binding elements to process AAV replication (Figure 1B). The small Rep proteins (Rep52 and Rep40) are used for the accumulation of sin‐ gle-stranded viral genome followed by packaging within AAV capsids.

The minimum sets of regions in helper adenovirus that mediate AAV vector replication are the E1, E2A, E4, and VA.[13] A human embryonic kidney cell line 293 encodes the E1 region of the Ad5 genome.[14] The helper plasmid assembling E2A, E4, and VA regions (Ad-helper plasmid) is cotransfected into the 293 cells, along with plasmids encoding the AAV vector genome (vector plasmid) as well as *rep* and *cap* genes (AAV-helper plasmid). AAV vector is produced as efficiently as when adenovirus infection is employed as a helper virus. Further‐ more, contamination of most adenovirus proteins can be avoided in AAV vector stock made by this helper virus-free method.

**Figure 1.** A) The *rep* and *cap* genes flanked by ITRs. The large Rep proteins (Rep78 and Rep68) are produced from transcripts using p5 promoter, while small Rep (Rep52 and Rep40) are produced from p19 promoter. (B) Recombinant AAV production. AAV has productive infection in the presence of adenovirus helper regions (E1, E2A, E4, and VA). This process is characterized by genome replication, assembly of the capsid proteins (VP1, VP2, and VP3), and packaging

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

431

We found that choice of AAV serotypes and promoters could be quite useful for targeted transgene expression. For instance, the transgene expression of rAAV5 with the Rous sarco‐ ma virus (RSV) promoter was preferentially found in the granular cells of the gerbil hippo‐ campus, whereas transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells.[16] Since AAV3 vector can specifically transduce cochlear in‐ ner hair cells with high efficiency *in vivo*, rAAV-mediated transduction might be promising for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutation.[17] Also, there is a significant difference in transgene expression by various AAV serotypes transduced into muscle. We observed that intramuscular injection of AAV5-IL-10 promoted a much higher serum level of secreted transgene product, as compared to AAV2-

leading to virion production along with exosome releasing.

#### **1.3. Vector application using various serotypes**

The preparation of AAV vector for gene therapy study of neuromuscular diseases is greatly facilitated. Although AAV2 has been the serotype most extensively studied in preclinical and clinical trials, recently we have focused on the use of AAV vectors pseudotyped with capsid protein of alternative serotypes. A number of primate AAV serotypes have been characterized in the literature and are designated. There is divergence in homology and tropism for various AAV serotypes. For instance, the homology with capsid protein is only about 60% between AAV2 and AAV5[15], therefore the capsid structure could be responsi‐ ble for the improved transduction efficiency.

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy http://dx.doi.org/10.5772/53023 431

Long-term correction of genetic diseases requires permanent integration of therapeutic genes into chromosomes of the affected cells. However, retrovirus vector integration can trigger deregulated premalignant cell proliferation with unexpected frequency, most like‐ ly driven by retrovirus enhancer activity on the LMO2 gene promoter. [9] A goal in clin‐ ical gene therapy is to develop gene transfer vehicles that can integrate exogenous therapeutic genes at specific chromosomal loci as a safe harbor, so that insertional onco‐ genesis is prevented. AAV can insert its genome into a specific locus, designated AAVS1, on chromosome 19 of the human genome.[10] The AAV Rep78/68 proteins and the Rep78/68-binding sequences are the trans- and cis-acting elements needed for this reac‐ tion. A dual high-capacity adenovirus-AAV hybrid vector with full-length human dystro‐ phin-coding sequences flanked by AAV integration-enhancing elements was tested for

AAV is a small (20-26nm) non-enveloped dependent parvovirus with a single-stranded line‐ ar genome that contains two open reading frames (*rep* and *cap*).[12] The viral genome is characterized by the inverted terminal repeats (ITRs) to flank these open reading frames (Figure 1A). The genome encodes four replication proteins (Rep78, Rep68, Rep52, and Rep40) and three capsid proteins (Cap: VP1, VP2, and VP3). The large Rep (Rep78 and Rep68) proteins regulate AAV gene expression and hold nicking activity at the terminal res‐ olution site as well as binding activity at Rep binding elements to process AAV replication (Figure 1B). The small Rep proteins (Rep52 and Rep40) are used for the accumulation of sin‐

The minimum sets of regions in helper adenovirus that mediate AAV vector replication are the E1, E2A, E4, and VA.[13] A human embryonic kidney cell line 293 encodes the E1 region of the Ad5 genome.[14] The helper plasmid assembling E2A, E4, and VA regions (Ad-helper plasmid) is cotransfected into the 293 cells, along with plasmids encoding the AAV vector genome (vector plasmid) as well as *rep* and *cap* genes (AAV-helper plasmid). AAV vector is produced as efficiently as when adenovirus infection is employed as a helper virus. Further‐ more, contamination of most adenovirus proteins can be avoided in AAV vector stock made

The preparation of AAV vector for gene therapy study of neuromuscular diseases is greatly facilitated. Although AAV2 has been the serotype most extensively studied in preclinical and clinical trials, recently we have focused on the use of AAV vectors pseudotyped with capsid protein of alternative serotypes. A number of primate AAV serotypes have been characterized in the literature and are designated. There is divergence in homology and tropism for various AAV serotypes. For instance, the homology with capsid protein is only about 60% between AAV2 and AAV5[15], therefore the capsid structure could be responsi‐

gle-stranded viral genome followed by packaging within AAV capsids.

targeted integration.[11]

430 Gene Therapy - Tools and Potential Applications

by this helper virus-free method.

**1.3. Vector application using various serotypes**

ble for the improved transduction efficiency.

**1.2. AAV biology**

**Figure 1.** A) The *rep* and *cap* genes flanked by ITRs. The large Rep proteins (Rep78 and Rep68) are produced from transcripts using p5 promoter, while small Rep (Rep52 and Rep40) are produced from p19 promoter. (B) Recombinant AAV production. AAV has productive infection in the presence of adenovirus helper regions (E1, E2A, E4, and VA). This process is characterized by genome replication, assembly of the capsid proteins (VP1, VP2, and VP3), and packaging leading to virion production along with exosome releasing.

We found that choice of AAV serotypes and promoters could be quite useful for targeted transgene expression. For instance, the transgene expression of rAAV5 with the Rous sarco‐ ma virus (RSV) promoter was preferentially found in the granular cells of the gerbil hippo‐ campus, whereas transgene expression of rAAV2 with the RSV promoter was found in the pyramidal and granular cells.[16] Since AAV3 vector can specifically transduce cochlear in‐ ner hair cells with high efficiency *in vivo*, rAAV-mediated transduction might be promising for gene replacement strategies to correct recessive genetic hearing loss due to monogenic mutation.[17] Also, there is a significant difference in transgene expression by various AAV serotypes transduced into muscle. We observed that intramuscular injection of AAV5-IL-10 promoted a much higher serum level of secreted transgene product, as compared to AAV2mediated transfer.[18] We further demonstrated that AAV1 could more efficiently transduce the muscle than AAV5. Intramuscular single injection of modest doses of rAAV1 expressing IL-10 (6x1010 g.c. per rat) introduced therapeutic levels of the transgene expression over the long-term to treat pulmonary arterial hypertension.[3] rAAV1-mediated sustained IL-10 ex‐ pression also significantly ameliorated hypertensive organ damage to improve survival rate of Dahl salt-sensitive rats.[19] Furthermore, this protein supplementation therapy by rAAV1-mediated muscle transduction was quite effective to prevent vascular remodeling and end-organ damage in the stroke-prone spontaneously hypertensive rat.[20] Interesting‐ ly, alpha-sarcoglycan expression with single intramuscular injection of rAAV8 was widely distributed in the hind limb muscle as well as cardiac muscle, and persisted for 7 months with a reversal of the muscle pathology and improvement in the contractile force in the al‐ pha-sarcoglycan-deficient mice.[21] Intravenous administration of rAAV8 into the hind limb in dogs resulted in improved transgene expression in the skeletal muscles lasting over a pe‐ riod of 8 weeks.[22] Moreover, rAAV9 would be administered systemically with excellent cardiac tropism.[23] Further strategies have been attempted to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.

cells along with plasmids encoding the AAV vector genome and *rep*-*cap* genes, the AAV vec‐ tor is produced as efficiently as when using adenovirus infection. Importantly, contamina‐ tion of most adenovirus proteins can be avoided in AAV vector stock made by this helper

> **delayed/inefficient expression**

**Figure 2.** DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector genome of both polarities are rescued from the vector plasmid and individu‐ ally packaged into the AAV capsids. As a genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes through the inter-molecular annealing or second strand synthesis. In contrast, a scAAV vector with half the size of the ssAAV genome has a mutation in the terminal resolution site (TRS) to form a vector genome with wild-type ITRs at the both ends and mutated ITR at the center of symmetry. After uncoating in the target cell nucleus, this DNA structure can readily fold into transcriptionally active double-stranded form through

Although various subtypes of the 293 cells harbor the E1 region of the adenovirus type 5 ge‐ nome, to utilize a 293 cell stably expressing Bcl-xL (293B) has great advantage to support E1B19K function and protect cells from apoptosis.[26] Despite improvements in vector pro‐ duction, including the development of packaging cell lines expressing Rep/Cap or methods to regulate Rep/Cap,[27] maintaining such cell lines remains difficult, as the early expression

We developed a large-scale transfection method of producing AAV vectors with an active gassing system that uses large culture vessels to process labor-effective transfection in a closed system.[28] This vector production system achieved reasonable production efficiency by improving gas exchange to prevent pH drop in the culture medium. Also, vector purifi‐ cation with the dual ion-exchange membrane adsorbers was effective and allowed higher levels of gene transfer *in vivo*.[29] Furthermore, the membrane adsorbers enabled the effec‐ tive recovery of the AAV vector in the supernatant exosomes of the transduced cells culture.

**inter-molecular annealing**

**second strand synthesis**

**ssAAV scAAV**

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

**DNA rescue/packaging**

**genome conversion**

**immediate/efficient expression**

**intra-molecular annealing**

**mutated TRS**

http://dx.doi.org/10.5772/53023

433

virus-free method.

intra-molecular annealing.

of Rep proteins is toxic to cells.

**Vector plasmid**

**Vector genome**

**Transgene (target cell nucleus)**

#### **1.4. scAAV**

Clinical gene therapy often requires rapid transduction with reasonable efficiency. In the case of AAV, second strand synthesis of the vector genome in the nucleus is the rate-limiting step for efficient transduction. Therefore, self-complementary AAV (scAAV) vector would be quite promising to promote efficient transduction regardless of DNA synthesis or annealing. [24] The scAAV vectors can bypass the inter-molecular annealing or second-strand synthesis by using intra-molecular annealing to immediately form transcriptionally active doublestranded DNA (Figure 2). Although immediate and efficient transduction could be observed with scAAV, the maximal insert size of the transgene cassette is reduced to 3.3 kb.[25]

#### **2. Effective production strategies of rAAV**

#### **2.1. Principle of production**

To gain acceptance as a medical treatment with a dose of over 1x1013 genome copies (g.c.)/kg body weight, therapeutic strategies with AAV vectors require a scalable and provident pro‐ duction method. However, the production and purification of recombinant virus stocks with conventional techniques entails cumbersome procedures not suited to the clinical setting. Therefore, development of effective large-scale culture and purification steps are required to meet end-product specifications.

A production protocol of AAV vectors in the absence of a helper virus[13] is widely em‐ ployed for triple plasmid transduction of human embryonic kidney 293 cells.[1] The adeno‐ virus regions that mediate AAV vector replication (namely, the VA, E2A and E4 regions) were assembled into a helper plasmid. When this helper plasmid is co-transfected into 293 cells along with plasmids encoding the AAV vector genome and *rep*-*cap* genes, the AAV vec‐ tor is produced as efficiently as when using adenovirus infection. Importantly, contamina‐ tion of most adenovirus proteins can be avoided in AAV vector stock made by this helper virus-free method.

mediated transfer.[18] We further demonstrated that AAV1 could more efficiently transduce the muscle than AAV5. Intramuscular single injection of modest doses of rAAV1 expressing IL-10 (6x1010 g.c. per rat) introduced therapeutic levels of the transgene expression over the long-term to treat pulmonary arterial hypertension.[3] rAAV1-mediated sustained IL-10 ex‐ pression also significantly ameliorated hypertensive organ damage to improve survival rate of Dahl salt-sensitive rats.[19] Furthermore, this protein supplementation therapy by rAAV1-mediated muscle transduction was quite effective to prevent vascular remodeling and end-organ damage in the stroke-prone spontaneously hypertensive rat.[20] Interesting‐ ly, alpha-sarcoglycan expression with single intramuscular injection of rAAV8 was widely distributed in the hind limb muscle as well as cardiac muscle, and persisted for 7 months with a reversal of the muscle pathology and improvement in the contractile force in the al‐ pha-sarcoglycan-deficient mice.[21] Intravenous administration of rAAV8 into the hind limb in dogs resulted in improved transgene expression in the skeletal muscles lasting over a pe‐ riod of 8 weeks.[22] Moreover, rAAV9 would be administered systemically with excellent cardiac tropism.[23] Further strategies have been attempted to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with

Clinical gene therapy often requires rapid transduction with reasonable efficiency. In the case of AAV, second strand synthesis of the vector genome in the nucleus is the rate-limiting step for efficient transduction. Therefore, self-complementary AAV (scAAV) vector would be quite promising to promote efficient transduction regardless of DNA synthesis or annealing. [24] The scAAV vectors can bypass the inter-molecular annealing or second-strand synthesis by using intra-molecular annealing to immediately form transcriptionally active doublestranded DNA (Figure 2). Although immediate and efficient transduction could be observed

with scAAV, the maximal insert size of the transgene cassette is reduced to 3.3 kb.[25]

To gain acceptance as a medical treatment with a dose of over 1x1013 genome copies (g.c.)/kg body weight, therapeutic strategies with AAV vectors require a scalable and provident pro‐ duction method. However, the production and purification of recombinant virus stocks with conventional techniques entails cumbersome procedures not suited to the clinical setting. Therefore, development of effective large-scale culture and purification steps are required to

A production protocol of AAV vectors in the absence of a helper virus[13] is widely em‐ ployed for triple plasmid transduction of human embryonic kidney 293 cells.[1] The adeno‐ virus regions that mediate AAV vector replication (namely, the VA, E2A and E4 regions) were assembled into a helper plasmid. When this helper plasmid is co-transfected into 293

a greater diversity of tissue tropism for clinical gene therapy.

**2. Effective production strategies of rAAV**

**2.1. Principle of production**

meet end-product specifications.

**1.4. scAAV**

432 Gene Therapy - Tools and Potential Applications

**Figure 2.** DNA rescue and transduction of a conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector genome of both polarities are rescued from the vector plasmid and individu‐ ally packaged into the AAV capsids. As a genome conversion in the transduced cell nucleus, the single-to-double stranded conversion of the DNA goes through the inter-molecular annealing or second strand synthesis. In contrast, a scAAV vector with half the size of the ssAAV genome has a mutation in the terminal resolution site (TRS) to form a vector genome with wild-type ITRs at the both ends and mutated ITR at the center of symmetry. After uncoating in the target cell nucleus, this DNA structure can readily fold into transcriptionally active double-stranded form through intra-molecular annealing.

Although various subtypes of the 293 cells harbor the E1 region of the adenovirus type 5 ge‐ nome, to utilize a 293 cell stably expressing Bcl-xL (293B) has great advantage to support E1B19K function and protect cells from apoptosis.[26] Despite improvements in vector pro‐ duction, including the development of packaging cell lines expressing Rep/Cap or methods to regulate Rep/Cap,[27] maintaining such cell lines remains difficult, as the early expression of Rep proteins is toxic to cells.

We developed a large-scale transfection method of producing AAV vectors with an active gassing system that uses large culture vessels to process labor-effective transfection in a closed system.[28] This vector production system achieved reasonable production efficiency by improving gas exchange to prevent pH drop in the culture medium. Also, vector purifi‐ cation with the dual ion-exchange membrane adsorbers was effective and allowed higher levels of gene transfer *in vivo*.[29] Furthermore, the membrane adsorbers enabled the effec‐ tive recovery of the AAV vector in the supernatant exosomes of the transduced cells culture. This rapid and scalable viral purification protocol is particularly promising for considerable *in vivo* experimentation and clinical investigations (Figure 3).

utilized per flask is 1120 ml. Subsequently, cells are grown for 48-72 h until reaching 70-90% confluence and are consequently transfected with appropriate triple plasmids. An aquarium pump (Nisso, Tokyo, Japan) should be used to circulate the gas through the CF10 with 5%

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

Half of the medium in the CF10 tissue culture flask are exchanged with fresh D-MEM/F-12 containing 10% FBS, 1 h before transfection of the 293 cells. Subsequently, the cells are cotransfected with 650 *µ*g of each plasmid: a proviral vector plasmid, an AAV helper plasmid, as well as an adenoviral helper plasmid, using calcium phosphate co-precipitation. Each plasmid was added to 112 ml of 300 mM CaCl2. This solution was gently added to the same volume of 2 x HBS (290 mM NaCl, 50 mM HEPES buffer, 1.5 mM Na2HPO4, pH 7.0) and gently inverted 3 times to form a uniform solution. This solution was immediately mixed with fresh D-MEM/F-12 containing 10% FBS to produce a homogeneous plasmid solution mixture. Subsequently, the medium in the culture flask was replaced with this plasmid solu‐ tion mixture. At the end of a 6-12 h incubation, the plasmid solution mixture in the culture

The culture supernatant sample for the ion-exchange procedure is processed by centrifuga‐ tion and filtration. The culture supernatant fluid 72-96 h after the transduction is sampled and then clarified with an appropriate amount of the activated charcoal (Wako Pure Chemi‐ cal Industries, Osaka, Japan). Insoluble debris is removed by a centrifugation at 3,000 *g* for 15 min and filtration. The elucidated culture supernatant is enriched with a hollow fiber cross flow membrane (100,000 NMWC, GE Healthcare, Pittsburgh, PA). For the material ob‐ tained from a CF10, 5 mM MgCl2 (final concentration) with 2,500-5,000 units of Benzonase

tion) is added to terminate the reaction. Place 38 ml of the sample solution in a semi-sterile ultracentrifuge tube (Ultrabottle #3430-3870; Nalge Nunc, Rochester, NY) and remove the

centrifugation for 3 h and then the vector fraction is dialyzed in the MHA buffer (3.3 mM

Chromatography can be performed using an appropriate FPLC system, such as AKTA ex‐ plorer 10S (Amersham Biosciences, Piscataway, NJ, USA) equipped with a 50 ml Superloop. The sample which passed through the MustangTM S membrane (optional treatment, PALL corporation, NY) is dialyzed against MHA buffer and further loaded onto an anion-ex‐ change membrane (acrodisc unit with MustangTM Q membrane, PALL corporation, equili‐ brated with MHA buffer) at a rate of 3 ml/min. The membrane is then washed with 10 column volumes of MHA buffer. Bound virus on the MustangTM Q membrane is eluted over a 50 column volume span with a 0-2 M linear NaCl gradient in MHA buffer and 0.5-1 ml fractions are collected. Recombinant rAAV particle number is determined by quantitative PCR of DNase I-treated stocks with plasmid standards. The final titer of the purified vectors

sample is quickly concentrated by the brief two-tier CsCl (1.25 and 1.60 g/cm3

C. Sequentially, 5 mM EDTA (final concentra‐

C to achieve cleared lysates. The

http://dx.doi.org/10.5772/53023

435

) step gradient

flask was replaced with pre-warmed fresh D-MEM/F-12 containing 2% FBS.

CO2 and humidity in an incubator.

**2.3. Purification phase**

nuclease is added to incubate for 30 min at 37 o

MES 3.3 mM HEPES [pH 8.0], 3.3 mM NaOAc).

cell debris by centrifugation at 10,000*g* for 15 minutes at 4 o

Recent developments also suggest that AAV vector production in insect cells would be com‐ patible with current good manufacturing practice production on an industrial scale.[30]

**Figure 3.** A scalable triple plasmid transfection using active gassing. When (1) a vector plasmid encoding the trans‐ gene cassette flanked by ITRs is co-transfected into human embryonic kidney 293 cells with (2) an AAV packaging plasmid harboring *rep*-*cap* genes and (3) an adenovirus helper plasmid, the AAV vector is produced as efficiently as when using adenovirus infection. A large-scale transduction method to produce AAV vectors with an active gassing system makes use of large culture vessels for labor- and cost-effective vector production in a closed system. Samples containing vector particles are further purified with a quick two-tier CsCl gradient centrifugation and an ion-exchange chromatography to obtain highly purified vector stocks.

#### **2.2. Large-scale production with active gassing**

Our protocol utilizes the transfection of 293B cells in one 10-Tray flask (CF10; Nalge Nunc International, Rochester, NY) with a surface area of 6320 cm2 by using an active gassing at 500 ml/min. Typical transduction procedure is conducted with one or two CF10 to meet downstream purification protocol. Although previous protocols for recombinant virus pro‐ duction in a large culture vessel had the problem of insufficient transduction efficiency be‐ cause of inadequate gas exchange, this method to use active gassing significantly improves productivity of the vectors and is linearly scalable from the small 225-cm2 flask.[3]

The 293B cells are cultured in Dulbecco's modified Eagle's medium and Nutrient Mixture F-12 (D-MEM/F-12, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (SIGMA-ALDRICH, St Louis, MO), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 o C in a 5% CO2 incubator. Cells are initially plated at 8 x 107 cells per CF10 to achieve a monolayer of 20 to 40% confluency when cells attached to surface of the flask. The volume of medium utilized per flask is 1120 ml. Subsequently, cells are grown for 48-72 h until reaching 70-90% confluence and are consequently transfected with appropriate triple plasmids. An aquarium pump (Nisso, Tokyo, Japan) should be used to circulate the gas through the CF10 with 5% CO2 and humidity in an incubator.

Half of the medium in the CF10 tissue culture flask are exchanged with fresh D-MEM/F-12 containing 10% FBS, 1 h before transfection of the 293 cells. Subsequently, the cells are cotransfected with 650 *µ*g of each plasmid: a proviral vector plasmid, an AAV helper plasmid, as well as an adenoviral helper plasmid, using calcium phosphate co-precipitation. Each plasmid was added to 112 ml of 300 mM CaCl2. This solution was gently added to the same volume of 2 x HBS (290 mM NaCl, 50 mM HEPES buffer, 1.5 mM Na2HPO4, pH 7.0) and gently inverted 3 times to form a uniform solution. This solution was immediately mixed with fresh D-MEM/F-12 containing 10% FBS to produce a homogeneous plasmid solution mixture. Subsequently, the medium in the culture flask was replaced with this plasmid solu‐ tion mixture. At the end of a 6-12 h incubation, the plasmid solution mixture in the culture flask was replaced with pre-warmed fresh D-MEM/F-12 containing 2% FBS.

#### **2.3. Purification phase**

This rapid and scalable viral purification protocol is particularly promising for considerable

Recent developments also suggest that AAV vector production in insect cells would be com‐ patible with current good manufacturing practice production on an industrial scale.[30]

> **Exosome releasing**

**Large-scale transfection with active gassing**

**Figure 3.** A scalable triple plasmid transfection using active gassing. When (1) a vector plasmid encoding the trans‐ gene cassette flanked by ITRs is co-transfected into human embryonic kidney 293 cells with (2) an AAV packaging plasmid harboring *rep*-*cap* genes and (3) an adenovirus helper plasmid, the AAV vector is produced as efficiently as when using adenovirus infection. A large-scale transduction method to produce AAV vectors with an active gassing system makes use of large culture vessels for labor- and cost-effective vector production in a closed system. Samples containing vector particles are further purified with a quick two-tier CsCl gradient centrifugation and an ion-exchange

Our protocol utilizes the transfection of 293B cells in one 10-Tray flask (CF10; Nalge Nunc

500 ml/min. Typical transduction procedure is conducted with one or two CF10 to meet downstream purification protocol. Although previous protocols for recombinant virus pro‐ duction in a large culture vessel had the problem of insufficient transduction efficiency be‐ cause of inadequate gas exchange, this method to use active gassing significantly improves

The 293B cells are cultured in Dulbecco's modified Eagle's medium and Nutrient Mixture F-12 (D-MEM/F-12, Invitrogen, Grand Island, NY) with 10% fetal bovine serum (SIGMA-ALDRICH, St Louis, MO), 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 o

of 20 to 40% confluency when cells attached to surface of the flask. The volume of medium

**AAV vector**

by using an active gassing at

flask.[3]

cells per CF10 to achieve a monolayer

C in a

**Purification with ion-exchange**

*in vivo* experimentation and clinical investigations (Figure 3).

**3. Adenovirus helper plasmid**

chromatography to obtain highly purified vector stocks.

**2.2. Large-scale production with active gassing**

International, Rochester, NY) with a surface area of 6320 cm2

5% CO2 incubator. Cells are initially plated at 8 x 107

productivity of the vectors and is linearly scalable from the small 225-cm2

**E4**

**VA RNA E2A**

**2. AAV**

**Rep Cap**

**1. Vector plasmid**

**Transgene ITR ITR**

434 Gene Therapy - Tools and Potential Applications

**packaging plasmid**

The culture supernatant sample for the ion-exchange procedure is processed by centrifuga‐ tion and filtration. The culture supernatant fluid 72-96 h after the transduction is sampled and then clarified with an appropriate amount of the activated charcoal (Wako Pure Chemi‐ cal Industries, Osaka, Japan). Insoluble debris is removed by a centrifugation at 3,000 *g* for 15 min and filtration. The elucidated culture supernatant is enriched with a hollow fiber cross flow membrane (100,000 NMWC, GE Healthcare, Pittsburgh, PA). For the material ob‐ tained from a CF10, 5 mM MgCl2 (final concentration) with 2,500-5,000 units of Benzonase nuclease is added to incubate for 30 min at 37 o C. Sequentially, 5 mM EDTA (final concentra‐ tion) is added to terminate the reaction. Place 38 ml of the sample solution in a semi-sterile ultracentrifuge tube (Ultrabottle #3430-3870; Nalge Nunc, Rochester, NY) and remove the cell debris by centrifugation at 10,000*g* for 15 minutes at 4 o C to achieve cleared lysates. The sample is quickly concentrated by the brief two-tier CsCl (1.25 and 1.60 g/cm3 ) step gradient centrifugation for 3 h and then the vector fraction is dialyzed in the MHA buffer (3.3 mM MES 3.3 mM HEPES [pH 8.0], 3.3 mM NaOAc).

Chromatography can be performed using an appropriate FPLC system, such as AKTA ex‐ plorer 10S (Amersham Biosciences, Piscataway, NJ, USA) equipped with a 50 ml Superloop. The sample which passed through the MustangTM S membrane (optional treatment, PALL corporation, NY) is dialyzed against MHA buffer and further loaded onto an anion-ex‐ change membrane (acrodisc unit with MustangTM Q membrane, PALL corporation, equili‐ brated with MHA buffer) at a rate of 3 ml/min. The membrane is then washed with 10 column volumes of MHA buffer. Bound virus on the MustangTM Q membrane is eluted over a 50 column volume span with a 0-2 M linear NaCl gradient in MHA buffer and 0.5-1 ml fractions are collected. Recombinant rAAV particle number is determined by quantitative PCR of DNase I-treated stocks with plasmid standards. The final titer of the purified vectors from a CF10 usually ranges around 5 x 1013 genome copies (g.c.), although it depends on the vector constructs and transgene.

*micro-dystrophin* transduction of *mdx* mice accomplished prevention of cardiac fibrosis as well as heart failure.[23] The transduction efficiency achieved with rAAV9 was nearly com‐ plete, with persistent expression for 74 weeks after transduction (Figure 4BC). Both the strong affinity of the rAAV9 for cardiac tissue and the therapeutic effect of the expressed mi‐ cro-dystrophin might be involved in the prevention of the degeneration of the cardiomyo‐

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

437

**Figure 4.** AAV9 vector-mediated *cardiac* transduction. (A) Structures of full-length and truncated dystrophin. Helperdependent adenovirus vector can package 14-kb of full-length dystrophin cDNA because of the large-sized deletion in its genome. A mini-dystrophin is cloned from a patient with Becker muscular dystrophy, which is caused by in-frame deletions resulting in the synthesis of partially functional protein. A truncated micro-dystrophin cDNAs harboring only four rod repeats with hinge 1, 2, and 4 and a deleted C-terminal domain (delta CS1) is constructed to be packaged in the AAV vector. (B) Transverse section of *mdx* mouse heart at mid-ventricular level 24 weeks after transduction of *micro-dystrophin*, stained with anti-dystrophin antibody NCL-DysB. Scale bar, 500 µm. (C) Expression of dystrophin in C57BL10 hearts at the sarcolemma (a), while it is absent in *mdx* hearts (b). Magnified views of sections from the center of the left ventricle at 28 weeks (c-e) show micro-dystrophin expression in the areas indicated in B (scale bar, 100 µm).

The impact of codon usage optimization on micro-dystrophin expression and function in the *mdx* mouse was demonstrated to compare the function of two different configurations of co‐ don-optimized *micro-dystrophin* genes under the control of a muscle-restrictive promoter

At 74 weeks after transduction, *mdx* mice still retain extensive expression of micro-dystrophin (f).

cytes and cardiac fibrosis.

#### **3. AAV-mediated therapeutic approach to neuromuscular disease**

#### **3.1. DMD gene replacement therapy**

Duchenne muscular dystrophy (DMD) is the most common form of childhood muscular dystrophy and is an X-linked recessive disorder with an incidence of one in 3500 live male births.[31] DMD causes progressive degeneration and regeneration of skeletal and cardiac muscles due to mutations in the *dystrophin* gene, which encodes a 427-kDa subsarcolemmal cytoskeletal protein.[32] DMD is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. Due to recent advances in res‐ piratory care, much attention is now focused on treating the cardiac conditions suffered by DMD patients. The approximately 2.5-megabase *dystrophin* gene is the largest gene identi‐ fied to date, and because of its size, it is susceptible to a high sporadic mutation rate. Ab‐ sence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting. Whereas DMD is characterized by the absence of functional protein, Becker muscular dystrophy, which is commonly caused by in-frame deletions of the *dystrophin* gene, results in the synthesis of an incompletely functional protein.

Successful therapy for DMD requires the restoration of dystrophin protein in skeletal and cardiac muscles. While various viral vectors have been considered for the delivery of genes to muscle fibers, the AAV-based vector is emerging as an appropriate gene transfer vehicle with the most potential for use in DMD gene therapies. As for another candidate vehicle, the gutted adenovirus vector can package 14-kb of full-length *dystrophin* cDNA due to the large deletion in virus genome. Multiple proximal muscles of seven-day-old utrophin/dystrophin double knockout mice (*dko* mice), which typically show symptoms similar to human DMD, were effectively transduced with the gutted adenovirus bearing full-length murine *dystro‐ phin* cDNA.[33] However, further improvements are needed to regulate the virus-associated host immune response before clinical trials can be performed.

A series of truncated *dystrophin* cDNAs containing rod repeats with hinge 1, 2, and 4 were constructed (Figure 4A).[4] Although AAV vectors are too small to package the full-length *dystrophin* cDNA, AAV vector-mediated gene therapy using a rod-truncated *dystrophin* gene provides a promising appraoch.[34] The structure and, particularly, the length of the rod are crucial for the function of micro-dystrophin.[35] An AAV type 2 vector expressing microdystrophin (DeltaCS1) under the control of a muscle-specific MCK promoter was injected into the tibialis anterior (TA) muscles of dystrophin-deficient *mdx* mice,[36] and resulted in extensive and long-term expression of micro-dystrophin that exhibited improved force gen‐ eration. Likewise, AAV6 vector-mediated systemic *micro-dystrophin* gene transfer was effec‐ tive in treating *dko* mice.[37] The potential for ameliorating the pathology of advanced-stage muscular dystrophy by systemic administration of AAV6 vectors encoding a micro-dystro‐ phin expression construct was also demonstrated.[38] Furthermore, AAV9 vector-mediated *micro-dystrophin* transduction of *mdx* mice accomplished prevention of cardiac fibrosis as well as heart failure.[23] The transduction efficiency achieved with rAAV9 was nearly com‐ plete, with persistent expression for 74 weeks after transduction (Figure 4BC). Both the strong affinity of the rAAV9 for cardiac tissue and the therapeutic effect of the expressed mi‐ cro-dystrophin might be involved in the prevention of the degeneration of the cardiomyo‐ cytes and cardiac fibrosis.

from a CF10 usually ranges around 5 x 1013 genome copies (g.c.), although it depends on the

Duchenne muscular dystrophy (DMD) is the most common form of childhood muscular dystrophy and is an X-linked recessive disorder with an incidence of one in 3500 live male births.[31] DMD causes progressive degeneration and regeneration of skeletal and cardiac muscles due to mutations in the *dystrophin* gene, which encodes a 427-kDa subsarcolemmal cytoskeletal protein.[32] DMD is associated with severe, progressive muscle weakness and typically leads to death between the ages of 20 and 35 years. Due to recent advances in res‐ piratory care, much attention is now focused on treating the cardiac conditions suffered by DMD patients. The approximately 2.5-megabase *dystrophin* gene is the largest gene identi‐ fied to date, and because of its size, it is susceptible to a high sporadic mutation rate. Ab‐ sence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting. Whereas DMD is characterized by the absence of functional protein, Becker muscular dystrophy, which is commonly caused by in-frame deletions of the

Successful therapy for DMD requires the restoration of dystrophin protein in skeletal and cardiac muscles. While various viral vectors have been considered for the delivery of genes to muscle fibers, the AAV-based vector is emerging as an appropriate gene transfer vehicle with the most potential for use in DMD gene therapies. As for another candidate vehicle, the gutted adenovirus vector can package 14-kb of full-length *dystrophin* cDNA due to the large deletion in virus genome. Multiple proximal muscles of seven-day-old utrophin/dystrophin double knockout mice (*dko* mice), which typically show symptoms similar to human DMD, were effectively transduced with the gutted adenovirus bearing full-length murine *dystro‐ phin* cDNA.[33] However, further improvements are needed to regulate the virus-associated

A series of truncated *dystrophin* cDNAs containing rod repeats with hinge 1, 2, and 4 were constructed (Figure 4A).[4] Although AAV vectors are too small to package the full-length *dystrophin* cDNA, AAV vector-mediated gene therapy using a rod-truncated *dystrophin* gene provides a promising appraoch.[34] The structure and, particularly, the length of the rod are crucial for the function of micro-dystrophin.[35] An AAV type 2 vector expressing microdystrophin (DeltaCS1) under the control of a muscle-specific MCK promoter was injected into the tibialis anterior (TA) muscles of dystrophin-deficient *mdx* mice,[36] and resulted in extensive and long-term expression of micro-dystrophin that exhibited improved force gen‐ eration. Likewise, AAV6 vector-mediated systemic *micro-dystrophin* gene transfer was effec‐ tive in treating *dko* mice.[37] The potential for ameliorating the pathology of advanced-stage muscular dystrophy by systemic administration of AAV6 vectors encoding a micro-dystro‐ phin expression construct was also demonstrated.[38] Furthermore, AAV9 vector-mediated

**3. AAV-mediated therapeutic approach to neuromuscular disease**

*dystrophin* gene, results in the synthesis of an incompletely functional protein.

host immune response before clinical trials can be performed.

vector constructs and transgene.

436 Gene Therapy - Tools and Potential Applications

**3.1. DMD gene replacement therapy**

**Figure 4.** AAV9 vector-mediated *cardiac* transduction. (A) Structures of full-length and truncated dystrophin. Helperdependent adenovirus vector can package 14-kb of full-length dystrophin cDNA because of the large-sized deletion in its genome. A mini-dystrophin is cloned from a patient with Becker muscular dystrophy, which is caused by in-frame deletions resulting in the synthesis of partially functional protein. A truncated micro-dystrophin cDNAs harboring only four rod repeats with hinge 1, 2, and 4 and a deleted C-terminal domain (delta CS1) is constructed to be packaged in the AAV vector. (B) Transverse section of *mdx* mouse heart at mid-ventricular level 24 weeks after transduction of *micro-dystrophin*, stained with anti-dystrophin antibody NCL-DysB. Scale bar, 500 µm. (C) Expression of dystrophin in C57BL10 hearts at the sarcolemma (a), while it is absent in *mdx* hearts (b). Magnified views of sections from the center of the left ventricle at 28 weeks (c-e) show micro-dystrophin expression in the areas indicated in B (scale bar, 100 µm). At 74 weeks after transduction, *mdx* mice still retain extensive expression of micro-dystrophin (f).

The impact of codon usage optimization on micro-dystrophin expression and function in the *mdx* mouse was demonstrated to compare the function of two different configurations of co‐ don-optimized *micro-dystrophin* genes under the control of a muscle-restrictive promoter (Spc5-12).[39] Codon optimization of micro-dystrophin significantly increased micro-dystro‐ phin mRNA and protein levels after intramuscular and systemic administration of plasmid DNA or rAAV8. By randomly assembling myogenic regulatory elements into synthetic pro‐ moter recombinant libraries, several artificial promoters were isolated whose transcriptional potencies greatly exceed those of natural myogenic and viral gene promoters.[40]

phenolate mofetil (MMF) was attempted to improve rAAV2-mediated transduction. The AAV2 capsids can induce a cellular immune response via MHC class I antigen presentation with a cross-presentation pathway,[49] and rAAV2 could also stimulate human dendritic cells (DCs).[50] Whereas the non-immunogenic nature of AAV6 in murine studies, rAAV6 also elicited robust cellular immune responses in dogs.[51] In contrast, other serotypes, such as rAAV8, induce T-cell activation to a lesser degree.[42] The rAAV8-injected muscles

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

and CD8+

endosome

**Figure 5.** rAAV-mediated transduction of dog. (A) Intravascular vector administration by limb perfusion. A blood pres‐ sure cuff is applied just above the knee of an anesthetized CXMDJ dog. A 24-gauge intravenous catheter is inserted into the lateral saphenous vein, connected to a three-way stopcock, and flushed with saline. With a blood pressure cuff inflated to over 300 mmHg, saline (2.6 ml/kg) containing papaverine (0.44 mg/kg, Sigma-Aldrich, St. Louis, MO) and heparin (16 U/kg) is injected by hand over a 10 second period. The three-way stopcock is connected to a syringe containing rAAV8 (1 x 1014 vg/kg, 3.8 ml/kg). The syringe is placed in a PHD 2000 syringe pump (Harvard Apparatus, Edenbridge, UK). Five minutes after the papaverine/heparin injection, rAAV8-LacZ is injected at a rate of 0.6 ml/sec. Two minutes after the rAAV injection, the blood pressure cuff is released and the catheter is removed. Four weeks after the transduction, the expression slightly fell off. (B) AAV-mediated stimulation of innate immune response via TLR9/MyD88 pathway. Bone marrow (BM)-derived dendritic cells (DCs) were obtained from humerus bones and cul‐ tured in RPMI (10% FCS, p/s) for 7 days with canine GM-CSF and IL-4. DCs were transduced with rAAV2- or rAAV8-*lacZ* (1x106 vg/cell for 4 hours, and mRNA levels of MyD88 and IFN-ß were analyzed. Untransduced cells were used as a normalization standard to demonstrate relative value of expression. Results are representative of two independent

Normalized expression

BM

0 2 4

Retrovirus (ssRNA)

Adenovirus

MyD88

TLR7/ 8 TLR9

Innate immune response

(dsDNA) AAV

rAAV2 rAAV8 rAAV2 rAAV8

0

40

DC differentiation (GM-CSF, IL-4)

20

MyD88

(ssDNA)

IFN-ß

2 days

rAAV2 rAAV8

qRT-PCR

IFNs Cytokines

Resident antigen-presenting cells, such as DCs, myoblasts, myotubes and regenerating imma‐ ture myofibers, should play a substantial role in the immune response against rAAV. Our study also showed that MyD88 and co-stimulating factors, such as CD80, CD86 and type I in‐ terferon, are up-regulated in both rAAV2- and rAAV8-transduced dog DCs (Figure 5B).[42]

T lymphocytes in the endomysium

http://dx.doi.org/10.5772/53023

439

showed lowed rates of infiltration of CD4+

rAAV8-Lac Z

**A B**

4 weeks

Beagle dog, ECR, Limb-perfusion

experiments. Error bars represent s.e.m., n = 3.

**200 µm**

2 weeks

than the rAAV2-injected muscles.[42]

#### **3.2. Intravascular vector administration by limb perfusion**

Although recent studies suggest that vectors based on AAV are capable of body-wide trans‐ duction in rodents,[21] translating the characteristics into large animals with advanced im‐ mune system remains a lot of challenges. Intravascular delivery can be performed as a form of limb perfusion, which might bypass the immune activation of DCs in the injected muscle. [41] We performed limb perfusion-assisted intravenous administration of rAAV8-lacZ into the hind limb of Beagle dogs (Figure 5A).[42] Administration of rAAV8 by limb perfusion demonstrated extensive transgene expression in the distal limb muscles of canine X-linked muscular dystrophy in Japan (CXMDJ ) dogs without obvious immune responses for the du‐ ration of the experiment over four weeks after injection.

#### **3.3. Systemic transduction and immunological issues**

In comparison with fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, paradoxically elicits a five-fold increase in cardiac injury and dilated cardiomyopathy.[43] Because the dystrophin-deficient heart is highly sensitive to increased stress, increased activity by the repaired skeletal muscle provides the stimulus for heightened cardiac injury and heart remodeling. In contrast, a single intravenous injec‐ tion of AAV9 vector expressing micro-dystrophin efficiently transduces the entire heart in neonatal *mdx* mice, thereby ameliorating cardiomyopathy.[44]

Since a number of muscular dystrophy patients can be identified through newborn screening in future, neonatal transduction may lead to an effective early intervention in DMD patients. After a single intravenous injection, robust skeletal muscle transduction with AAV9 vector throughout the body was observed in neonatal dogs.[45] Systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and lasted for at least six months, whereas rAAV9 was barely transduced into the cardiac muscle of dogs. Likewise, *in utero* gene delivery of full-length murine *dystrophin* to *mdx* mice using a high-capacity adeno‐ viral vector resulted in effective protection from cycles of degeneration and regeneration.[46]

Neo-antigens introduced by AAV vectors evoke significant immune reactions in DMD mus‐ cle, since increased permeability of the DMD muscle allows leakage of the transgene prod‐ ucts from the dystrophin-deficient sarcolemma of muscle fibers.[47] rAAV2 transfer into skeletal muscles of normal dogs resulted in low levels of transient expression, together with intense cellular infiltration, and the marked activation of cellular and humoral immune re‐ sponses.[48] Furthermore, an *in vitro* interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more strongly than do murine spleno‐ cytes. Therefore, co-administration of immunosuppressants, cyclosporine (CSP) and myco‐ phenolate mofetil (MMF) was attempted to improve rAAV2-mediated transduction. The AAV2 capsids can induce a cellular immune response via MHC class I antigen presentation with a cross-presentation pathway,[49] and rAAV2 could also stimulate human dendritic cells (DCs).[50] Whereas the non-immunogenic nature of AAV6 in murine studies, rAAV6 also elicited robust cellular immune responses in dogs.[51] In contrast, other serotypes, such as rAAV8, induce T-cell activation to a lesser degree.[42] The rAAV8-injected muscles showed lowed rates of infiltration of CD4+ and CD8+ T lymphocytes in the endomysium than the rAAV2-injected muscles.[42]

(Spc5-12).[39] Codon optimization of micro-dystrophin significantly increased micro-dystro‐ phin mRNA and protein levels after intramuscular and systemic administration of plasmid DNA or rAAV8. By randomly assembling myogenic regulatory elements into synthetic pro‐ moter recombinant libraries, several artificial promoters were isolated whose transcriptional

Although recent studies suggest that vectors based on AAV are capable of body-wide trans‐ duction in rodents,[21] translating the characteristics into large animals with advanced im‐ mune system remains a lot of challenges. Intravascular delivery can be performed as a form of limb perfusion, which might bypass the immune activation of DCs in the injected muscle. [41] We performed limb perfusion-assisted intravenous administration of rAAV8-lacZ into the hind limb of Beagle dogs (Figure 5A).[42] Administration of rAAV8 by limb perfusion demonstrated extensive transgene expression in the distal limb muscles of canine X-linked

In comparison with fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, paradoxically elicits a five-fold increase in cardiac injury and dilated cardiomyopathy.[43] Because the dystrophin-deficient heart is highly sensitive to increased stress, increased activity by the repaired skeletal muscle provides the stimulus for heightened cardiac injury and heart remodeling. In contrast, a single intravenous injec‐ tion of AAV9 vector expressing micro-dystrophin efficiently transduces the entire heart in

Since a number of muscular dystrophy patients can be identified through newborn screening in future, neonatal transduction may lead to an effective early intervention in DMD patients. After a single intravenous injection, robust skeletal muscle transduction with AAV9 vector throughout the body was observed in neonatal dogs.[45] Systemic transduction was achieved in the absence of pharmacological intervention or immune suppression and lasted for at least six months, whereas rAAV9 was barely transduced into the cardiac muscle of dogs. Likewise, *in utero* gene delivery of full-length murine *dystrophin* to *mdx* mice using a high-capacity adeno‐ viral vector resulted in effective protection from cycles of degeneration and regeneration.[46]

Neo-antigens introduced by AAV vectors evoke significant immune reactions in DMD mus‐ cle, since increased permeability of the DMD muscle allows leakage of the transgene prod‐ ucts from the dystrophin-deficient sarcolemma of muscle fibers.[47] rAAV2 transfer into skeletal muscles of normal dogs resulted in low levels of transient expression, together with intense cellular infiltration, and the marked activation of cellular and humoral immune re‐ sponses.[48] Furthermore, an *in vitro* interferon-gamma release assay showed that canine splenocytes respond to immunogens or mitogens more strongly than do murine spleno‐ cytes. Therefore, co-administration of immunosuppressants, cyclosporine (CSP) and myco‐

) dogs without obvious immune responses for the du‐

potencies greatly exceed those of natural myogenic and viral gene promoters.[40]

**3.2. Intravascular vector administration by limb perfusion**

ration of the experiment over four weeks after injection.

**3.3. Systemic transduction and immunological issues**

neonatal *mdx* mice, thereby ameliorating cardiomyopathy.[44]

muscular dystrophy in Japan (CXMDJ

438 Gene Therapy - Tools and Potential Applications

Resident antigen-presenting cells, such as DCs, myoblasts, myotubes and regenerating imma‐ ture myofibers, should play a substantial role in the immune response against rAAV. Our study also showed that MyD88 and co-stimulating factors, such as CD80, CD86 and type I in‐ terferon, are up-regulated in both rAAV2- and rAAV8-transduced dog DCs (Figure 5B).[42]

**Figure 5.** rAAV-mediated transduction of dog. (A) Intravascular vector administration by limb perfusion. A blood pres‐ sure cuff is applied just above the knee of an anesthetized CXMDJ dog. A 24-gauge intravenous catheter is inserted into the lateral saphenous vein, connected to a three-way stopcock, and flushed with saline. With a blood pressure cuff inflated to over 300 mmHg, saline (2.6 ml/kg) containing papaverine (0.44 mg/kg, Sigma-Aldrich, St. Louis, MO) and heparin (16 U/kg) is injected by hand over a 10 second period. The three-way stopcock is connected to a syringe containing rAAV8 (1 x 1014 vg/kg, 3.8 ml/kg). The syringe is placed in a PHD 2000 syringe pump (Harvard Apparatus, Edenbridge, UK). Five minutes after the papaverine/heparin injection, rAAV8-LacZ is injected at a rate of 0.6 ml/sec. Two minutes after the rAAV injection, the blood pressure cuff is released and the catheter is removed. Four weeks after the transduction, the expression slightly fell off. (B) AAV-mediated stimulation of innate immune response via TLR9/MyD88 pathway. Bone marrow (BM)-derived dendritic cells (DCs) were obtained from humerus bones and cul‐ tured in RPMI (10% FCS, p/s) for 7 days with canine GM-CSF and IL-4. DCs were transduced with rAAV2- or rAAV8-*lacZ* (1x106 vg/cell for 4 hours, and mRNA levels of MyD88 and IFN-ß were analyzed. Untransduced cells were used as a normalization standard to demonstrate relative value of expression. Results are representative of two independent experiments. Error bars represent s.e.m., n = 3.

#### **4. Safety and potential impact of clinical trials**

#### **4.1. Clinical trials for muscle transduction**

While low immunogenicity was considered a major strength supporting the use of rAAV in clinical trials, a number of observations have recently provided a more balanced view of this procedure.[52] An obvious barrier to AAV transduction is the presence of circulating neu‐ tralizing antibodies that prevent the virion from binding to its cellular receptor.[53] This po‐ tential threat can be reduced by prescreening patients for AAV serotype-specific neutralizing antibodies or by performing therapeutic procedures such as plasmapheresis be‐ fore gene transfer. Another challenge recently revealed is the development of a cell-mediat‐ ed cytotoxic T-cell (CTL) response to AAV capsid peptides. In the human factor IX gene therapy trial in which rAAV was delivered to the liver, only short-term transgene expres‐ sion was achieved and levels of therapeutic protein declined to baseline levels 10 weeks af‐ ter vector infusion.[52] This was accompanied by elevation of serum transaminase levels and a CTL response toward specific AAV capsid peptides. To overcome this response, tran‐ sient immunosuppression may be required until AAV capsids are completely cleared. Addi‐ tional findings suggest that T-cell activation requires AAV2 capsid binding to the heparan sulfate proteoglycan (HSPG) receptor, which would permit virion shuttling into a DC path‐ way, as cross-presentation.[54] Exposure to vectors from other AAV clades, such as AAV8, did not activate capsid-specific T-cells.

(http://www.ema.europa.eu/ema) The European Medicines Agency's Committee for Medici‐ nal Products for Human Use has recommended the authorization of Glybera (rAAV1-ex‐ pressing LPL S447X variant) for marketing in the European Union. It is intended to treat lipoprotein lipase deficiency in patients with severe or multiple pancreatitis attacks, despite

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

441

To regulate host immune response against vectors and transgene products, treatments in‐ volving immunosuppressants and other strategies have been attempted in the animal models. A brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), CSP and MMF was effective in permitting AAV6-mediated, long-term and robust expression of a canine micro-dystrophin in the skeletal muscle of a dog DMD model.[58] To establish the feasibility of multiple AAV1 injections for extending the treat‐ ment to whole body muscles, the dystrophic *mdx* mouse was repeatedly transduced with AAV1 vector, and the immune response was characterized.[59] By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, a five-day-long immunomo‐ dulation treatment was found to be sufficient for totally abrogating the formation of anti-

There have been numerous reports to develop the therapeutic potential of mesenchymal stem cells (or mesenchymal multipotent stromal cells MSCs).[60] Because of their immuno‐ modulatory properties, increasing experimental and early clinical observations indicate that allogeneic, and even xenogeneic, MSCs may be useful for tissue transplantation.[61] In fact, the immune tolerance with MSCs is well investigated in various animal studies. Infusion of syngeneic MSCs into a sensitized mouse model of kidney transplantation resulted in the ex‐ pansion of donor-specific T- regulatory cells into lymphoid organs, prolonged allograft sur‐

The use of a histone deacetylase (HDAC) inhibitor depsipeptide effectively enhances the utility of rAAV-mediated gene therapy.[63] In contrast to adenovirus-mediated transduc‐ tion, the improved transduction with rAAV induced by the depsipeptide is due to enhanced transgene expression rather than to increased viral entry. The enhanced transduction is re‐ lated to the histone-associated chromatin form of the rAAV concatemer in the transduced cells. Since various HDAC inhibitors are approved in clinical usage for many diseases to achieve therapeutic benefits, the application of such inhibitors to the rAAV-mediated gene

dietary fat restrictions.

AAV1 antibodies.

**5. Challenges and future perspectives**

**5.1. Immunomodulation to augment clinical benefits**

vival and promoted the development of tolerance.[62]

therapy is theoretically and practically reasonable.

**5.2. Pharmacological intervention**

The initial clinical studies lay the foundation for future studies, providing important infor‐ mation about vector dose, viral serotype selection, and immunogenicity in humans. The first virus-mediated gene transfer for muscle disease was carried out for limb-girdle muscular dystrophy type 2D using rAAV1. The study, consisting of intramuscular injection of virus into a single muscle, was limited in scope and the main conclusion was to establish the safe‐ ty of this procedure in phase I clinical trials. The first clinical gene therapy trial for DMD began in March 2006.[55] This was a Phase I/IIa study in which an AAV vector was used to deliver micro-dystrophin to the biceps of boys with DMD. The study was conducted on six boys with DMD, each of whom received an injection of mini-dystrophin-expressing rAAV2.5 in a muscle of one arm and a placebo in the other arm. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle.[56] The potential for T-cell immuni‐ ty to self and non-self dystrophin epitopes should be considered in designing and monitor‐ ing experimental therapies for this disease. Basically, this issue is in common with the treatment of genetic diseases. Although concerns regarding risk of an immune response to the transgene product limited the ability to achieve therapeutic efficacy, rAAV2-mediated gene transfer to human skeletal muscle can persist for up to a decade.[57]

#### **4.2. Gene therapy medicine**

After more than two decades of expectations, the field of gene therapy appears close to reaching a regulatory approval by proposing rAAV-mediated muscle transduction. Europe‐ an medicine agency eventually recommends first gene therapy medicine for approval. (http://www.ema.europa.eu/ema) The European Medicines Agency's Committee for Medici‐ nal Products for Human Use has recommended the authorization of Glybera (rAAV1-ex‐ pressing LPL S447X variant) for marketing in the European Union. It is intended to treat lipoprotein lipase deficiency in patients with severe or multiple pancreatitis attacks, despite dietary fat restrictions.

#### **5. Challenges and future perspectives**

**4. Safety and potential impact of clinical trials**

While low immunogenicity was considered a major strength supporting the use of rAAV in clinical trials, a number of observations have recently provided a more balanced view of this procedure.[52] An obvious barrier to AAV transduction is the presence of circulating neu‐ tralizing antibodies that prevent the virion from binding to its cellular receptor.[53] This po‐ tential threat can be reduced by prescreening patients for AAV serotype-specific neutralizing antibodies or by performing therapeutic procedures such as plasmapheresis be‐ fore gene transfer. Another challenge recently revealed is the development of a cell-mediat‐ ed cytotoxic T-cell (CTL) response to AAV capsid peptides. In the human factor IX gene therapy trial in which rAAV was delivered to the liver, only short-term transgene expres‐ sion was achieved and levels of therapeutic protein declined to baseline levels 10 weeks af‐ ter vector infusion.[52] This was accompanied by elevation of serum transaminase levels and a CTL response toward specific AAV capsid peptides. To overcome this response, tran‐ sient immunosuppression may be required until AAV capsids are completely cleared. Addi‐ tional findings suggest that T-cell activation requires AAV2 capsid binding to the heparan sulfate proteoglycan (HSPG) receptor, which would permit virion shuttling into a DC path‐ way, as cross-presentation.[54] Exposure to vectors from other AAV clades, such as AAV8,

The initial clinical studies lay the foundation for future studies, providing important infor‐ mation about vector dose, viral serotype selection, and immunogenicity in humans. The first virus-mediated gene transfer for muscle disease was carried out for limb-girdle muscular dystrophy type 2D using rAAV1. The study, consisting of intramuscular injection of virus into a single muscle, was limited in scope and the main conclusion was to establish the safe‐ ty of this procedure in phase I clinical trials. The first clinical gene therapy trial for DMD began in March 2006.[55] This was a Phase I/IIa study in which an AAV vector was used to deliver micro-dystrophin to the biceps of boys with DMD. The study was conducted on six boys with DMD, each of whom received an injection of mini-dystrophin-expressing rAAV2.5 in a muscle of one arm and a placebo in the other arm. Dystrophin-specific T cells were detected after treatment, providing evidence of transgene expression even when the functional protein was not visualized in skeletal muscle.[56] The potential for T-cell immuni‐ ty to self and non-self dystrophin epitopes should be considered in designing and monitor‐ ing experimental therapies for this disease. Basically, this issue is in common with the treatment of genetic diseases. Although concerns regarding risk of an immune response to the transgene product limited the ability to achieve therapeutic efficacy, rAAV2-mediated

gene transfer to human skeletal muscle can persist for up to a decade.[57]

After more than two decades of expectations, the field of gene therapy appears close to reaching a regulatory approval by proposing rAAV-mediated muscle transduction. Europe‐ an medicine agency eventually recommends first gene therapy medicine for approval.

**4.1. Clinical trials for muscle transduction**

440 Gene Therapy - Tools and Potential Applications

did not activate capsid-specific T-cells.

**4.2. Gene therapy medicine**

#### **5.1. Immunomodulation to augment clinical benefits**

To regulate host immune response against vectors and transgene products, treatments in‐ volving immunosuppressants and other strategies have been attempted in the animal models. A brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), CSP and MMF was effective in permitting AAV6-mediated, long-term and robust expression of a canine micro-dystrophin in the skeletal muscle of a dog DMD model.[58] To establish the feasibility of multiple AAV1 injections for extending the treat‐ ment to whole body muscles, the dystrophic *mdx* mouse was repeatedly transduced with AAV1 vector, and the immune response was characterized.[59] By blocking the T-B crosstalk with anti-CD40 Abs and CTLA4/Fc fusion protein, a five-day-long immunomo‐ dulation treatment was found to be sufficient for totally abrogating the formation of anti-AAV1 antibodies.

There have been numerous reports to develop the therapeutic potential of mesenchymal stem cells (or mesenchymal multipotent stromal cells MSCs).[60] Because of their immuno‐ modulatory properties, increasing experimental and early clinical observations indicate that allogeneic, and even xenogeneic, MSCs may be useful for tissue transplantation.[61] In fact, the immune tolerance with MSCs is well investigated in various animal studies. Infusion of syngeneic MSCs into a sensitized mouse model of kidney transplantation resulted in the ex‐ pansion of donor-specific T- regulatory cells into lymphoid organs, prolonged allograft sur‐ vival and promoted the development of tolerance.[62]

#### **5.2. Pharmacological intervention**

The use of a histone deacetylase (HDAC) inhibitor depsipeptide effectively enhances the utility of rAAV-mediated gene therapy.[63] In contrast to adenovirus-mediated transduc‐ tion, the improved transduction with rAAV induced by the depsipeptide is due to enhanced transgene expression rather than to increased viral entry. The enhanced transduction is re‐ lated to the histone-associated chromatin form of the rAAV concatemer in the transduced cells. Since various HDAC inhibitors are approved in clinical usage for many diseases to achieve therapeutic benefits, the application of such inhibitors to the rAAV-mediated gene therapy is theoretically and practically reasonable.

#### **5.3. In situ gene therapy**

Transplantation of genetically modified vector-producing cells is a possible future treatment for genetic diseases as an *in situ* gene therapy. MSCs are known to accumulate at the site of inflammation or tumors, and therefore can be utilized as a platform for the targeted delivery of therapeutic agents.[64] The MSCs-based targeted gene therapy should enhance the thera‐ peutic efficacy, since MSCs would deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the reagents act locally without elevating their systemic concentrations. We developed the genetically-modi‐ fied MSCs that produce viral vectors to augment therapeutic efficacy of systemic gene thera‐ py.[65] MSCs isolated from the SD rats bone marrow were transfected with retroviral vector components by nucleofection. As a result, the injection of luciferase-expressing vector-pro‐ ducing MSCs caused significantly stronger signal of bioluminescence at the site of subcuta‐ neous tumors in mice compared with luciferease-expressing non-vector-producing MSCs. [66] Furthermore, tumor-bearing nude mice were treated with the vector-producing MSCs combined with HSV-*tk*/GCV system to demonstrate improved anti-tumor effects. This study suggests the effectiveness of vector-producing MSCs in systemic gene therapy. The thera‐ peutic benefit of this strategy should be further examined by using rAAV-producing MSCs in the various animal models of inflammatory diseases including neuromuscular disorders.

translate gene transduction technologies into clinical practice, development of an effective delivery system with improved vector constructs as well as efficient immunological modu‐ lation must be established. A novel protocol that considers all of these issues would help im‐

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

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443

This work was supported by the Grant for Research on Nervous and Mental Disorders, Health Science Research Grants for Research on the Human Genome and Gene Therapy; and the Grant for Research on Brain Science from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). We would like to

Department of Molecular Therapy, National Institute of Neuroscience, National Center of

[1] Okada, T., K. Shimazaki, T. Nomoto, T. Matsushita, H. Mizukami, M. Urabe, et al. (2002). "Adeno-associated viral vector-mediated gene therapy of ischemia-induced

[2] Inagaki, K., S. Fuess, T. A. Storm, G. A. Gibson, C. F. McTiernan, M. A. Kay, et al. (2006). "Robust systemic transduction with AAV9 vectors in mice: efficient global

[3] Ito, T., T. Okada, H. Miyashita, T. Nomoto, M. Nonaka-Sarukawa, R. Uchibori, et al. (2007). "Interleukin-10 expression mediated by an adeno-associated virus vector pre‐ vents monocrotaline-induced pulmonary arterial hypertension in rats." Circ Res 101:

[4] Yuasa, K., Y. Miyagoe, K. Yamamoto, Y. Nabeshima, G. Dickson and S. Takeda (1998). "Effective restoration of dystrophin-associated proteins in vivo by adenovi‐ rus-mediated transfer of truncated dystrophin cDNAs." FEBS Lett 425: 329-336.

cardiac gene transfer superior to that of AAV8." Mol Ther 14: 45-53.

thank Dr. James M. Wilson for providing p5E18-VD2/8 and pAAV2/9.

Neurology and Psychiatry, Ogawa-Higashi, Kodaira, Tokyo, Japan

neuronal death." Methods Enzymol 346: 378-393.

prove the therapeutic benefits of clinical gene therapy.

Address all correspondence to: t-okada@ncnp.go.jp

**Acknowledgments**

**Author details**

Takashi Okada

**References**

734-741.

#### **5.4. Capsid modification**

A DNA shuffling-based approach for developing cell type-specific vectors is an intriguing possibility to achieve altered tropism. Capsid genomes of AAV serotypes 1-9 were random‐ ly reassembled using PCR to generate a chimeric capsid library.[67] A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the ico‐ sahedral threefold axis of symmetry, while AAV1 and 9 contribute to two-fold and five-fold symmetry interactions, respectively.

A versatile rAAV targeting system to redirect rAAV-mediated transduction to specific cell surface receptors would be useful. Insertion of an IgG binding domain of protein A into the AAV2 capsid at amino acid position 587 could permit antibody-mediated vector retargeting, although producing mosaic particles is required to avoid low particle yields.[68] Alterna‐ tively, a targeting system using the genetic fusion of short biotin acceptor peptide along with the metabolic biotinylation via a biotin ligase was developed for the purification and targeting of multiple AAV serotypes.[69]

#### **6. Conclusions and outlook**

Although an increasing number of scalable methods for purification of rAAV have been de‐ scribed, in order to generate sufficient clinical-grade vector to support clinical trials we need to further improve a large-scale GMP-compatible system for production and purification. To translate gene transduction technologies into clinical practice, development of an effective delivery system with improved vector constructs as well as efficient immunological modu‐ lation must be established. A novel protocol that considers all of these issues would help im‐ prove the therapeutic benefits of clinical gene therapy.

#### **Acknowledgments**

**5.3. In situ gene therapy**

442 Gene Therapy - Tools and Potential Applications

**5.4. Capsid modification**

symmetry interactions, respectively.

targeting of multiple AAV serotypes.[69]

**6. Conclusions and outlook**

Transplantation of genetically modified vector-producing cells is a possible future treatment for genetic diseases as an *in situ* gene therapy. MSCs are known to accumulate at the site of inflammation or tumors, and therefore can be utilized as a platform for the targeted delivery of therapeutic agents.[64] The MSCs-based targeted gene therapy should enhance the thera‐ peutic efficacy, since MSCs would deliver therapeutic molecules in a concentrated fashion. This targeted therapy can also reduce systemic adverse side effects, because the reagents act locally without elevating their systemic concentrations. We developed the genetically-modi‐ fied MSCs that produce viral vectors to augment therapeutic efficacy of systemic gene thera‐ py.[65] MSCs isolated from the SD rats bone marrow were transfected with retroviral vector components by nucleofection. As a result, the injection of luciferase-expressing vector-pro‐ ducing MSCs caused significantly stronger signal of bioluminescence at the site of subcuta‐ neous tumors in mice compared with luciferease-expressing non-vector-producing MSCs. [66] Furthermore, tumor-bearing nude mice were treated with the vector-producing MSCs combined with HSV-*tk*/GCV system to demonstrate improved anti-tumor effects. This study suggests the effectiveness of vector-producing MSCs in systemic gene therapy. The thera‐ peutic benefit of this strategy should be further examined by using rAAV-producing MSCs in the various animal models of inflammatory diseases including neuromuscular disorders.

A DNA shuffling-based approach for developing cell type-specific vectors is an intriguing possibility to achieve altered tropism. Capsid genomes of AAV serotypes 1-9 were random‐ ly reassembled using PCR to generate a chimeric capsid library.[67] A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the ico‐ sahedral threefold axis of symmetry, while AAV1 and 9 contribute to two-fold and five-fold

A versatile rAAV targeting system to redirect rAAV-mediated transduction to specific cell surface receptors would be useful. Insertion of an IgG binding domain of protein A into the AAV2 capsid at amino acid position 587 could permit antibody-mediated vector retargeting, although producing mosaic particles is required to avoid low particle yields.[68] Alterna‐ tively, a targeting system using the genetic fusion of short biotin acceptor peptide along with the metabolic biotinylation via a biotin ligase was developed for the purification and

Although an increasing number of scalable methods for purification of rAAV have been de‐ scribed, in order to generate sufficient clinical-grade vector to support clinical trials we need to further improve a large-scale GMP-compatible system for production and purification. To This work was supported by the Grant for Research on Nervous and Mental Disorders, Health Science Research Grants for Research on the Human Genome and Gene Therapy; and the Grant for Research on Brain Science from the Ministry of Health, Labor and Welfare of Japan. This work was also supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology (MEXT). We would like to thank Dr. James M. Wilson for providing p5E18-VD2/8 and pAAV2/9.

#### **Author details**

Takashi Okada

Address all correspondence to: t-okada@ncnp.go.jp

Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Ogawa-Higashi, Kodaira, Tokyo, Japan

#### **References**


[5] Okada, T., J. Ramsey, J. Munir, O. Wildner and M. Blaese (1998). "Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex." Nucleic Acids Res. 26: 1947-1950.

[18] Yoshioka, T., T. Okada, Y. Maeda, U. Ikeda, M. Shimpo, T. Nomoto, et al. (2004). "Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits athe‐

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

445

[19] Nonaka-Sarukawa, M., T. Okada, T. Ito, K. Yamamoto, T. Yoshioka, T. Nomoto, et al. (2008). "Adeno-associated virus vector-mediated systemic interleukin-10 expression ameliorates hypertensive organ damage in Dahl salt-sensitive rats." J Gene Med 10:

[20] Nomoto, T., T. Okada, K. Shimazaki, T. Yoshioka, M. Nonaka-Sarukawa, T. Ito, et al. (2009). "Systemic delivery of IL-10 by an AAV vector prevents vascular remodeling and end-organ damage in stroke-prone spontaneously hypertensive rat." Gene Ther

[21] Nishiyama, A., B. N. Ampong, S. Ohshima, J. H. Shin, H. Nakai, M. Imamura, et al. (2008). "Recombinant adeno-associated virus type 8-mediated extensive therapeutic gene delivery into skeletal muscle of alpha-sarcoglycan-deficient mice." Hum Gene

[22] Ohshima, S., J. H. Shin, K. Yuasa, A. Nishiyama, J. Kira, T. Okada, et al. (2009). "Transduction Efficiency and Immune Response Associated With the Administration

[23] Shin, J. H., Y. Nitahara-Kasahara, H. Hayashita-Kinoh, S. Ohshima-Hosoyama, K. Ki‐ noshita, T. Chiyo, et al. (2011). "Improvement of cardiac fibrosis in dystrophic mice

[24] McCarty, D. M., P. E. Monahan and R. J. Samulski (2001). "Self-complementary re‐ combinant adeno-associated virus (scAAV) vectors promote efficient transduction in‐

[25] Wu, J., W. Zhao, L. Zhong, Z. Han, B. Li, W. Ma, et al. (2007). "Self-complementary recombinant adeno-associated viral vectors: packaging capacity and the role of rep

[26] Yamaguchi, T., T. Okada, K. Takeuchi, T. Tonda, M. Ohtaki, S. Shinoda, et al. (2003). "Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS, a

[27] Okada, T., H. Mizukami, M. Urabe, T. Nomoto, T. Matsushita, Y. Hanazono, et al. (2001). "Development and characterization of an antisense-mediated prepackaging cell line for adeno-associated virus vector production." Biochem Biophys Res Com‐

[28] Okada, T., T. Nomoto, T. Yoshioka, M. Nonaka-Sarukawa, T. Ito, T. Ogura, et al. (2005). "Large-scale production of recombinant viruses by use of a large culture ves‐

[29] Okada, T., M. Nonaka-Sarukawa, R. Uchibori, K. Kinoshita, H. Hayashita-Kinoh, Y. Nitahara-Kasahara, et al. (2009). "Scalable purification of adeno-associated virus sero‐

by rAAV9-mediated microdystrophin transduction" Gene Ther. 18: 910-919.

of AAV8 Vector Into Dog Skeletal Muscle." Mol Ther 17: 73-91.

dependently of DNA synthesis." Gene Ther 8: 1248-1254.

proteins in vector purity." Hum Gene Ther 18: 171-182.

BH3-only cell death activator." Gene Ther 10: 375-385.

sel with active gassing." Hum Gene Ther 16: 1212-1218.

rosclerosis in apolipoprotein E-deficient mice." Gene Ther 11: 1772-1779.

368-374.

16: 383-391.

Ther 19: 719-730.

mun 288: 62-68.


[18] Yoshioka, T., T. Okada, Y. Maeda, U. Ikeda, M. Shimpo, T. Nomoto, et al. (2004). "Adeno-associated virus vector-mediated interleukin-10 gene transfer inhibits athe‐ rosclerosis in apolipoprotein E-deficient mice." Gene Ther 11: 1772-1779.

[5] Okada, T., J. Ramsey, J. Munir, O. Wildner and M. Blaese (1998). "Efficient directional cloning of recombinant adenovirus vectors using DNA-protein complex." Nucleic

[6] Okada, T., N. J. Caplen, W. J. Ramsey, M. Onodera, K. Shimazaki, T. Nomoto, et al. (2004). "In situ generation of pseudotyped retroviral progeny by adenovirus-mediat‐ ed transduction of tumor cells enhances the killing effect of HSV-tk suicide gene

[7] Hammerschmidt, D. E. (1999). "Development of a gutless vector." J Lab Clin Med 134:

[8] Hoshiya, H., Y. Kazuki, S. Abe, M. Takiguchi, N. Kajitani, Y. Watanabe, et al. (2008). "A highly Stable and Nonintegrated Human Artificial Chromosome (HAC) Contain‐

[9] Hacein-Bey-Abina, S., A. Garrigue, G. P. Wang, J. Soulier, A. Lim, E. Morillon, et al. (2008). "Insertional oncogenesis in 4 patients after retrovirus-mediated gene therapy

[10] Kotin, R. M., R. M. Linden and K. I. Berns (1992). "Characterization of a preferred site on human chromosome 19q for integration of adeno-associated virus DNA by non-

[11] Goncalves, M. A., G. P. van Nierop, M. R. Tijssen, P. Lefesvre, S. Knaan-Shanzer, I. van der Velde, et al. (2005). "Transfer of the full-length dystrophin-coding sequence into muscle cells by a dual high-capacity hybrid viral vector with site-specific inte‐

[12] Srivastava, A., E. W. Lusby and K. I. Berns (1983). "Nucleotide sequence and organi‐

[13] Matsushita, T., S. Elliger, C. Elliger, G. Podsakoff, L. Villarreal, G. J. Kurtzman, et al. (1998). "Adeno-associated virus vectors can be efficiently produced without helper

[14] Graham, F. L., J. Smiley, W. C. Russell and R. Nairn (1977). "Characteristics of a hu‐ man cell line transformed by DNA from human adenovirus type 5." J Gen Virol 36:

[15] Chiorini, J. A., F. Kim, L. Yang and R. M. Kotin (1999). "Cloning and characterization

[16] Nomoto, T., T. Okada, K. Shimazaki, H. Mizukami, T. Matsushita, Y. Hanazono, et al. (2003). "Distinct patterns of gene transfer to gerbil hippocampus with recombinant

[17] Liu, M., Y. Yue, S. Q. Harper, R. W. Grange, J. S. Chamberlain and D. Duan (2005). "Adeno-associated virus-mediated microdystrophin expression protects young mdx

adeno-associated virus type 2 and 5." Neuroscience Letters 340: 153-157.

muscle from contraction-induced injury." Mol Ther 11: 245-256.

zation of the adeno-associated virus 2 genome." J Virol 45: 555-564.

of adeno-associated virus type 5." J Virol 73: 1309-1319.

therapy in vitro and in vivo." J Gene Med 6: 288-299.

ing the 2.4 Mb Entire Human Dystrophin Gene." Mol Ther.

of SCID-X1." J Clin Invest 118: 3132-3142.

gration ability." J Virol 79: 3146-3162.

virus." Gene Ther 5: 938-945.

59-74.

homologous recombination." Embo J 11: 5071-5078.

Acids Res. 26: 1947-1950.

444 Gene Therapy - Tools and Potential Applications

C3.


type 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes." Hum Gene Ther 20: 1013-1021.

[41] Hagstrom, J. E., J. Hegge, G. Zhang, M. Noble, V. Budker, D. L. Lewis, et al. (2004). "A facile nonviral method for delivering genes and siRNAs to skeletal muscle of

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

447

[42] Ohshima, S., J. H. Shin, K. Yuasa, A. Nishiyama, J. Kira, T. Okada, et al. (2008). "Transduction Efficiency and Immune Response Associated With the Administration

[43] Townsend, D., S. Yasuda, S. Li, J. S. Chamberlain and J. M. Metzger (2008). "Emer‐ gent dilated cardiomyopathy caused by targeted repair of dystrophic skeletal mus‐

[44] Bostick, B., Y. Yue, Y. Lai, C. Long, D. Li and D. Duan (2008). "Adeno-associated vi‐ rus serotype-9 microdystrophin gene therapy ameliorates electrocardiographic ab‐

[45] Yue, Y., A. Ghosh, C. Long, B. Bostick, B. F. Smith, J. N. Kornegay, et al. (2008). "A single intravenous injection of adeno-associated virus serotype-9 leads to whole

[46] Reay, D. P., R. Bilbao, B. M. Koppanati, L. Cai, T. L. O'Day, Z. Jiang, et al. (2008). "Full-length dystrophin gene transfer to the mdx mouse in utero." Gene Ther 15:

[47] Yuasa, K., M. Sakamoto, Y. Miyagoe-Suzuki, A. Tanouchi, H. Yamamoto, J. Li, et al. (2002). "Adeno-associated virus vector-mediated gene transfer into dystrophin-defi‐ cient skeletal muscles evokes enhanced immune response against the transgene

[48] Yuasa, K., M. Yoshimura, N. Urasawa, S. Ohshima, J. M. Howell, A. Nakamura, et al. (2007). "Injection of a recombinant AAV serotype 2 into canine skeletal muscles

[49] Li, C., M. Hirsch, A. Asokan, B. Zeithaml, H. Ma, T. Kafri, et al. (2007). "Adeno-asso‐ ciated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo." J Virol 81: 7540-7547.

[50] Zhang, Y., N. Chirmule, G. Gao and J. Wilson (2000). "CD40 ligand-dependent activa‐ tion of cytotoxic T lymphocytes by adeno-associated virus vectors in vivo: role of im‐

[51] Wang, Z., J. M. Allen, S. R. Riddell, P. Gregorevic, R. Storb, S. J. Tapscott, et al. (2007). "Immunity to adeno-associated virus-mediated gene transfer in a random-bred ca‐

[52] Manno, C. S., G. F. Pierce, V. R. Arruda, B. Glader, M. Ragni, J. J. Rasko, et al. (2006). "Successful transduction of liver in hemophilia by AAV-Factor IX and limitations im‐

nine model of Duchenne muscular dystrophy." Hum Gene Ther 18: 18-26.

posed by the host immune response." Nat Med 12: 342-347.

evokes strong immune responses against transgene products." Gene Ther.

body skeletal muscle transduction in dogs." Mol Ther 16: 1944-1952.

mammalian limbs." Mol Ther 10: 386-398.

cle." Mol Ther 16: 832-835.

product." Gene Ther 9: 1576-1588.

mature dendritic cells." J Virol 74: 8003-8010.

531-536.

of AAV8 Vector Into Dog Skeletal Muscle." Mol Ther.

normalities in mdx mice." Hum Gene Ther 19: 851-856.


[41] Hagstrom, J. E., J. Hegge, G. Zhang, M. Noble, V. Budker, D. L. Lewis, et al. (2004). "A facile nonviral method for delivering genes and siRNAs to skeletal muscle of mammalian limbs." Mol Ther 10: 386-398.

type 1 (AAV1) and AAV8 vectors, using dual ion-exchange adsorptive membranes."

[30] Cecchini, S., A. Negrete and R. M. Kotin (2008). "Toward exascale production of re‐ combinant adeno-associated virus for gene transfer applications." Gene Ther 15:

[31] Emery, A. E. (1991). "Population frequencies of inherited neuromuscular diseases--a

[32] Hoffman, E. P., R. H. Brown, Jr. and L. M. Kunkel (1987). "Dystrophin: the protein

[33] Kawano, R., M. Ishizaki, Y. Maeda, Y. Uchida, E. Kimura and M. Uchino (2008). "Transduction of full-length dystrophin to multiple skeletal muscles improves motor performance and life span in utrophin/dystrophin double knockout mice." Mol Ther

[34] Wang, B., J. Li and X. Xiao (2000). "Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse

[35] Sakamoto, M., K. Yuasa, M. Yoshimura, T. Yokota, T. Ikemoto, M. Suzuki, et al. (2002). "Micro-dystrophin cDNA ameliorates dystrophic phenotypes when intro‐ duced into mdx mice as a transgene." Biochem Biophys Res Commun 293: 1265-1272.

[36] Yoshimura, M., M. Sakamoto, M. Ikemoto, Y. Mochizuki, K. Yuasa, Y. Miyagoe-Su‐ zuki, et al. (2004). "AAV vector-mediated microdystrophin expression in a relatively small percentage of mdx myofibers improved the mdx phenotype." Mol Ther 10:

[37] Gregorevic, P., J. M. Allen, E. Minami, M. J. Blankinship, M. Haraguchi, L. Meuse, et al. (2006). "rAAV6-microdystrophin preserves muscle function and extends lifespan

[38] Gregorevic, P., M. J. Blankinship, J. M. Allen and J. S. Chamberlain (2008). "Systemic microdystrophin gene delivery improves skeletal muscle structure and function in

[39] Foster, H., P. S. Sharp, T. Athanasopoulos, C. Trollet, I. R. Graham, K. Foster, et al. (2008). "Codon and mRNA sequence optimization of microdystrophin transgenes im‐ proves expression and physiological outcome in dystrophic mdx mice following

[40] Li, X., E. M. Eastman, R. J. Schwartz and R. Draghia-Akli (1999). "Synthetic muscle promoters: activities exceeding naturally occurring regulatory sequences." Nat Bio‐

product of the Duchenne muscular dystrophy locus." Cell 51: 919-928.

Hum Gene Ther 20: 1013-1021.

446 Gene Therapy - Tools and Potential Applications

world survey." Neuromuscul Disord 1: 19-29.

model." Proc Natl Acad Sci U S A 97: 13714-13719.

in severely dystrophic mice." Nat Med 12: 787-789.

old dystrophic mdx mice." Mol Ther 16: 657-664.

AAV2/8 gene transfer." Mol Ther 16: 1825-1832.

823-830.

16: 825-831.

821-828.

technol 17: 241-245.


[53] Scallan, C. D., H. Jiang, T. Liu, S. Patarroyo-White, J. M. Sommer, S. Zhou, et al. (2006). "Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice." Blood 107: 1810-1817.

[65] Okada, T. and K. Ozawa (2008). "Vector-producing tumor-tracking multipotent mes‐ enchymal stromal cells for suicide cancer gene therapy." Front Biosci 13: 1887-1891.

Efficient AAV Vector Production System: Towards Gene Therapy For Duchenne Muscular Dystrophy

http://dx.doi.org/10.5772/53023

449

[66] Uchibori, R., T. Okada, T. Ito, M. Urabe, H. Mizukami, A. Kume, et al. (2009). "Retro‐ viral vector-producing mesenchymal stem cells for targeted suicide cancer gene ther‐

[67] Li, W., A. Asokan, Z. Wu, T. Van Dyke, N. DiPrimio, J. S. Johnson, et al. (2008). "Engi‐ neering and selection of shuffled AAV genomes: a new strategy for producing target‐

[68] Muzyczka, N. and K. H. Warrington, Jr. (2005). "Custom adeno-associated virus cap‐ sids: the next generation of recombinant vectors with novel tropism." Hum Gene

[69] Arnold, G. S., A. K. Sasser, M. D. Stachler and J. S. Bartlett (2006). "Metabolic biotiny‐ lation provides a unique platform for the purification and targeting of multiple AAV

apy." J Gene Med 11: 373-381.

vector serotypes." Mol Ther 14: 97-106.

Ther 16: 408-416.

ed biological nanoparticles." Mol Ther 16: 1252-1260.


[65] Okada, T. and K. Ozawa (2008). "Vector-producing tumor-tracking multipotent mes‐ enchymal stromal cells for suicide cancer gene therapy." Front Biosci 13: 1887-1891.

[53] Scallan, C. D., H. Jiang, T. Liu, S. Patarroyo-White, J. M. Sommer, S. Zhou, et al. (2006). "Human immunoglobulin inhibits liver transduction by AAV vectors at low

[54] Vandenberghe, L. H., L. Wang, S. Somanathan, Y. Zhi, J. Figueredo, R. Calcedo, et al. (2006). "Heparin binding directs activation of T cells against adeno-associated virus

[55] Rodino-Klapac, L. R., L. G. Chicoine, B. K. Kaspar and J. R. Mendell (2007). "Gene therapy for duchenne muscular dystrophy: expectations and challenges." Arch Neu‐

[56] Mendell, J. R., K. Campbell, L. Rodino-Klapac, Z. Sahenk, C. Shilling, S. Lewis, et al. (2010). "Dystrophin immunity in Duchenne's muscular dystrophy." N Engl J Med

[57] Buchlis, G., G. M. Podsakoff, A. Radu, S. M. Hawk, A. W. Flake, F. Mingozzi, et al. (2012). "Factor IX expression in skeletal muscle of a severe hemophilia B patient 10

[58] Wang, Z., C. S. Kuhr, J. M. Allen, M. Blankinship, P. Gregorevic, J. S. Chamberlain, et al. (2007). "Sustained AAV-mediated Dystrophin Expression in a Canine Model of Duchenne Muscular Dystrophy with a Brief Course of Immunosuppression." Mol

[59] Lorain, S., D. A. Gross, A. Goyenvalle, O. Danos, J. Davoust and L. Garcia (2008). "Transient immunomodulation allows repeated injections of AAV1 and correction of

[60] Nitahara-Kasahara, Y., H. Hayashita-Kinoh, S. Ohshima-Hosoyama, H. Okada, M. Wada-Maeda, A. Nakamura, et al. (2012). "Long-term engraftment of multipotent mesenchymal stromal cells that differentiate to form myogenic cells in dogs with

[61] Chiu, R. C. (2008). "MSC immune tolerance in cellular cardiomyoplasty." Semin

[62] Casiraghi, F., N. Azzollini, M. Todeschini, R. A. Cavinato, P. Cassis, S. Solini, et al. (2012). "Localization of Mesenchymal Stromal Cells Dictates Their Immune or Proin‐

[63] Okada, T., R. Uchibori, M. Iwata-Okada, M. Takahashi, T. Nomoto, M. Nonaka-Saru‐ kawa, et al. (2006). "A histone deacetylase inhibitor enhances recombinant adeno-as‐ sociated virus-mediated gene expression in tumor cells." Mol Ther 13: 738-746.

[64] Studeny, M., F. C. Marini, J. L. Dembinski, C. Zompetta, M. Cabreira-Hansen, B. N. Bekele, et al. (2004). "Mesenchymal stem cells: potential precursors for tumor stroma and targeted-delivery vehicles for anticancer agents." J Natl Cancer Inst 96:

flammatory Effects in Kidney Transplantation." Am J Transplant. (in press)

AAV2 neutralizing titers in SCID mice." Blood 107: 1810-1817.

years after AAV-mediated gene transfer." Blood 119: 3038-3041.

muscular dystrophy in multiple muscles." Mol Ther 16: 541-547.

Duchenne muscular dystrophy." Mol Ther 20: 168-177.

Thorac Cardiovasc Surg 20: 115-118.

serotype 2 capsid." Nat Med 12: 967-971.

rol 64: 1236-1241.

448 Gene Therapy - Tools and Potential Applications

363: 1429-1437.

Ther 15: 1160-1166.

1593-1603.


**Section 4**

**Applications: Inhereted Diseases**

**Applications: Inhereted Diseases**

**Chapter 18**

**Gene Therapy for Primary Immunodeficiencies**

Primary immunodeficiencies (PID) are caused by mutations in genes involved in the normal development or activity of the immune system [1, 2]. PIDs include B- and T-cell defects, phagocytic disorders, and complement deficiencies with the common feature of frequent lifethreatening infections. The phenotypes vary from asymptomatic (IgA deficiency) to severe PIDs (such as Severe combined immunodeficiencies). Treatment of patients with severe PIDs relies in intravenous injection of immunoglobulins, bone marrow transplantation (BMT) and antibiotics. Identical and haploidentical BMT are the only curative treatment, however, the lack of a HLA-matched donor in over 70% of the patients make necessary the development of new therapeutic strategies [3, 4]. Gene therapy (GT) could be the best alternative for the treatment of patients with severe PID that lack a HLA-matched donor [5]. The aim of GT strategies is the stable correction of the mutated gene on the patient's own haematopoietic stem

The first successful gene therapy clinical trial used gamma-retroviral derived vectors express‐ ing common cytokine-receptor gamma chain (γc) cDNA in HSCs from X-linked severe combined immunodeficiency (SCID-X1) patients [6]. So far, using a very similar vector platform, over 50 PID patients treated with GT can been considered "cured" from SCID-X1, adenosine deaminase deficiency (ADA) and Wiskott-Aldrich syndrome (WAS) PID [7-13]. However, in six children, GT treatment resulted in clonal T-cell proliferation (leukaemia-like

The results obtained in the SCID-X1, ADA and WAS clinical trials clearly showed the impor‐ tance to improve vector's safety and efficiency [8,14, 15]. Lentiviral-based vectors have been the vector of choice to enhance efficiency and, at the same time, reduce the side effects of gammaretroviral vectors (see below). Several GT clinical trials for SCID-X1, chronic granu‐

> © 2013 Martin et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Francisco Martin, Alejandra Gutierrez-Guerrero and

Additional information is available at the end of the chapter

Karim Benabdellah

**1. Introduction**

cells (HSCs).

disease) [9].

http://dx.doi.org/10.5772/55594

### **Gene Therapy for Primary Immunodeficiencies**

Francisco Martin, Alejandra Gutierrez-Guerrero and Karim Benabdellah

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55594

#### **1. Introduction**

Primary immunodeficiencies (PID) are caused by mutations in genes involved in the normal development or activity of the immune system [1, 2]. PIDs include B- and T-cell defects, phagocytic disorders, and complement deficiencies with the common feature of frequent lifethreatening infections. The phenotypes vary from asymptomatic (IgA deficiency) to severe PIDs (such as Severe combined immunodeficiencies). Treatment of patients with severe PIDs relies in intravenous injection of immunoglobulins, bone marrow transplantation (BMT) and antibiotics. Identical and haploidentical BMT are the only curative treatment, however, the lack of a HLA-matched donor in over 70% of the patients make necessary the development of new therapeutic strategies [3, 4]. Gene therapy (GT) could be the best alternative for the treatment of patients with severe PID that lack a HLA-matched donor [5]. The aim of GT strategies is the stable correction of the mutated gene on the patient's own haematopoietic stem cells (HSCs).

The first successful gene therapy clinical trial used gamma-retroviral derived vectors express‐ ing common cytokine-receptor gamma chain (γc) cDNA in HSCs from X-linked severe combined immunodeficiency (SCID-X1) patients [6]. So far, using a very similar vector platform, over 50 PID patients treated with GT can been considered "cured" from SCID-X1, adenosine deaminase deficiency (ADA) and Wiskott-Aldrich syndrome (WAS) PID [7-13]. However, in six children, GT treatment resulted in clonal T-cell proliferation (leukaemia-like disease) [9].

The results obtained in the SCID-X1, ADA and WAS clinical trials clearly showed the impor‐ tance to improve vector's safety and efficiency [8,14, 15]. Lentiviral-based vectors have been the vector of choice to enhance efficiency and, at the same time, reduce the side effects of gammaretroviral vectors (see below). Several GT clinical trials for SCID-X1, chronic granu‐

© 2013 Martin et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

lomatous disease (CGD) and WAS PID using lentiviral vectors (LVs) have started in the last few years.

recover from ongoing infections with poor prognosis (disseminated infections) and live in a

Gene Therapy for Primary Immunodeficiencies

http://dx.doi.org/10.5772/55594

455

However, 5 of the 20 patients with SCID-X1 on GT trials developed leukaemia 3-6 years after treatment. Four patients were successfully treated with chemotherapy and they are alive and doing well. However the other patient died from chemotherapy-refractory leukemia [26]. This leukaemia-like disease was a result of vector-mediated up-regulation of host cellular onco‐ genes (i.e. LMO2) [8, 27]. Several studies have demonstrated that MLV-derived vectors integration favour transcriptionally active genes near transcription start sites (TSSs) [28-30]. Leukemogenesis could also be the result of insertional mutagenesis (activation of the LMO2 oncogene) combined with the acquisition of genetic abnormalities unrelated to vector inser‐

However, in spite of the secondary effects observed, the results obtained with GT using first generation MLV-based vectors are comparable to those obtained with HLA-identical HSC transplant (HSCT). It is expected that next generation vectors will certainly improve these

**2.3. Adenosine Deaminase (ADA) Severe Combined Immunodeficiency (ADA-SCID)**

ADA-deficiency has been also considered an important target for GT. The *ADA* gene codify for an enzyme that is expressed in all tissues and catalyses the deamination of 2'-deoxyade‐ nosine and adenosine to 2'deoxyinosine and inosine. Its absence or malfunction cause the accumulation of purine metabolites that are toxic to the cells. Although the *ADA* gene is expressed in all tissues, the accumulation of purine metabolites in the immune cells is the main problem. As consequence, ADA patients suffer from lymphopenia, reduced (or absent) cellular and humoral immunity, failure to thrive and recurrent infections. Additionally, the accumu‐ lation of purine metabolites in other tissues also produces skeletal, hepatic, renal, lung, and neurologic abnormalities [31, 32]. Like for SCID-X1, bone marrow transplantation (BMT) is the best therapeutic alternative. However, contrary to SCID-X1, there are other treatment options that allow ADA patients to have near-normal lives: Enzyme replacement therapy (ERT) with polyethylene-glycol-conjugated bovine ADA (PEG-ADA). However, although ERT treatment is well tolerated and can partially restore immune function, its effect decline over time and, in

ADA deficiency has been successfully treated by GT using a similar approach to that for SCID-X1, but requiring mild bone-marrow chemoablation [34]. The authors showed immunological

expressing-MLV based vectors. The selective growth advantage of ADA-expressing lympho‐ cytes played an important role in the success of this trial. Similar findings have been reported by Gaspar *et. al.* [23] and again by Aiuti *et al*[10]. In total, over 40 patients with ADA have been treated in Italy, UK and USA. At present all patients are alive and 29 of them do not require

It is important to remark that no leukaemia-like disease have been observed in the ADA-SCID GT trial. The author propose that the differences between SCID-X1 and ADA might be related

using ADA-

and metabolic reconstitution after transplantation of gene-modified CD34+

normal environment without evidence of increased susceptibility to infection.

tion, such as the increase activity of NOTCH1 or the deletion of CDKN2A gene [8].

results as it will discussed later.

addition, lifelong treatment is very expensive[33].

ERT [9, 10, 23, 25, 34-36].

This chapter intend to illustrate the past, present and near future of GT for the treatment of severe PIDs

#### **2. Gamma-retroviral vector based gene therapy clinical trials for primary immunodeficiencies**

#### **2.1. Gammaretrovirus-based vectors**

Gammaretrovirus, also named oncoretrovirus, are efficient, integrative, easy to manipulate and poorly immunogenic. Vector derived for these retroviruses are often named "retroviral vectors" and "oncoretroviral vectors". All the clinical data that will be presented in this section was obtained using a similar gammaretroviral backbone: **LTR---ψ-----transgene------LTR**. As consequence the therapeutic gene is expressed through the promoter and enhancer sequences present at the viral LTR. Another common aspect of all the GT strategies presented in this section is the modification of the patient´s hematopoietic stem cells (HSCs). However HSCs are quiescent or very slowly dividing cells and gammaretroviral-based vectors require active cell division for transduction [16]. Therefore HSCs transduction protocols require cytokine "pre-stimulation" to induce cell proliferation [17], a process that can modify the characteristics of the haematopoietic precursors [18]. However, since LTR-driven gammaretroviral vectors were the only integrative vectors available at the time, several clinical trials started on SCID-X1, ADA CGD and WAS. An overall conclusion of these clinical trials was that GT is as efficient and safe as haploidentical BMT. However it was also evident the necessity of improving the vector system before GT of PID could be of general use in clinic.

#### **2.2. X-linked Severe Combined Immunodeficiency (SCID-X1)**

SCID-X1 is a monogenic disease caused by mutations in the interleukin-2 receptor gamma chain gene (γc). Patients with SCID-X1 deficiency do not have T nor NK cells, consequently B-lymphocyte function is also intrinsically compromised [19]. SCID-X1 has been an attractive GT target because patient's cells expressing the transgene have a growth advantage over nonexpressing cells [20, 21]. Therefore, GT could, in theory, achieved complete immune reconsti‐ tution with a relatively low number of gene-corrected cells. The Fischer group at the "Unité d'Immunologie et d'Hématologie Pédiatriques, Hôpital Necker" in France achieved the first unequivocal success of gene therapy in the two patients treated [6]. The authors transduced patients HSCs (CD34+ ) with a Murine Leukaemia Virus (MLV) based vector expressing the γc cDNA following pre-activation with stem cell factor (SCF), polyethylene glycol-megakaryo‐ cyte differentiation factor (PG-MDF), IL-3 and Flt3-L. The continuation of this work and other clinical trials in other countries enrolled a total of 20 SCID-X1 patients [7, 8, 22, 23]. Between 5 and 12 years after GT, 17 of the 20 treated patients are alive and display full or nearly full correction of the T cell deficiency [24, 25]. The GT treatment led to clear benefits since patients recover from ongoing infections with poor prognosis (disseminated infections) and live in a normal environment without evidence of increased susceptibility to infection.

lomatous disease (CGD) and WAS PID using lentiviral vectors (LVs) have started in the last

This chapter intend to illustrate the past, present and near future of GT for the treatment of

**2. Gamma-retroviral vector based gene therapy clinical trials for primary**

Gammaretrovirus, also named oncoretrovirus, are efficient, integrative, easy to manipulate and poorly immunogenic. Vector derived for these retroviruses are often named "retroviral vectors" and "oncoretroviral vectors". All the clinical data that will be presented in this section was obtained using a similar gammaretroviral backbone: **LTR---ψ-----transgene------LTR**. As consequence the therapeutic gene is expressed through the promoter and enhancer sequences present at the viral LTR. Another common aspect of all the GT strategies presented in this section is the modification of the patient´s hematopoietic stem cells (HSCs). However HSCs are quiescent or very slowly dividing cells and gammaretroviral-based vectors require active cell division for transduction [16]. Therefore HSCs transduction protocols require cytokine "pre-stimulation" to induce cell proliferation [17], a process that can modify the characteristics of the haematopoietic precursors [18]. However, since LTR-driven gammaretroviral vectors were the only integrative vectors available at the time, several clinical trials started on SCID-X1, ADA CGD and WAS. An overall conclusion of these clinical trials was that GT is as efficient and safe as haploidentical BMT. However it was also evident the necessity of improving the

SCID-X1 is a monogenic disease caused by mutations in the interleukin-2 receptor gamma chain gene (γc). Patients with SCID-X1 deficiency do not have T nor NK cells, consequently B-lymphocyte function is also intrinsically compromised [19]. SCID-X1 has been an attractive GT target because patient's cells expressing the transgene have a growth advantage over nonexpressing cells [20, 21]. Therefore, GT could, in theory, achieved complete immune reconsti‐ tution with a relatively low number of gene-corrected cells. The Fischer group at the "Unité d'Immunologie et d'Hématologie Pédiatriques, Hôpital Necker" in France achieved the first unequivocal success of gene therapy in the two patients treated [6]. The authors transduced

cDNA following pre-activation with stem cell factor (SCF), polyethylene glycol-megakaryo‐ cyte differentiation factor (PG-MDF), IL-3 and Flt3-L. The continuation of this work and other clinical trials in other countries enrolled a total of 20 SCID-X1 patients [7, 8, 22, 23]. Between 5 and 12 years after GT, 17 of the 20 treated patients are alive and display full or nearly full correction of the T cell deficiency [24, 25]. The GT treatment led to clear benefits since patients

) with a Murine Leukaemia Virus (MLV) based vector expressing the γc

vector system before GT of PID could be of general use in clinic.

**2.2. X-linked Severe Combined Immunodeficiency (SCID-X1)**

few years.

severe PIDs

**immunodeficiencies**

454 Gene Therapy - Tools and Potential Applications

patients HSCs (CD34+

**2.1. Gammaretrovirus-based vectors**

However, 5 of the 20 patients with SCID-X1 on GT trials developed leukaemia 3-6 years after treatment. Four patients were successfully treated with chemotherapy and they are alive and doing well. However the other patient died from chemotherapy-refractory leukemia [26]. This leukaemia-like disease was a result of vector-mediated up-regulation of host cellular onco‐ genes (i.e. LMO2) [8, 27]. Several studies have demonstrated that MLV-derived vectors integration favour transcriptionally active genes near transcription start sites (TSSs) [28-30]. Leukemogenesis could also be the result of insertional mutagenesis (activation of the LMO2 oncogene) combined with the acquisition of genetic abnormalities unrelated to vector inser‐ tion, such as the increase activity of NOTCH1 or the deletion of CDKN2A gene [8].

However, in spite of the secondary effects observed, the results obtained with GT using first generation MLV-based vectors are comparable to those obtained with HLA-identical HSC transplant (HSCT). It is expected that next generation vectors will certainly improve these results as it will discussed later.

#### **2.3. Adenosine Deaminase (ADA) Severe Combined Immunodeficiency (ADA-SCID)**

ADA-deficiency has been also considered an important target for GT. The *ADA* gene codify for an enzyme that is expressed in all tissues and catalyses the deamination of 2'-deoxyade‐ nosine and adenosine to 2'deoxyinosine and inosine. Its absence or malfunction cause the accumulation of purine metabolites that are toxic to the cells. Although the *ADA* gene is expressed in all tissues, the accumulation of purine metabolites in the immune cells is the main problem. As consequence, ADA patients suffer from lymphopenia, reduced (or absent) cellular and humoral immunity, failure to thrive and recurrent infections. Additionally, the accumu‐ lation of purine metabolites in other tissues also produces skeletal, hepatic, renal, lung, and neurologic abnormalities [31, 32]. Like for SCID-X1, bone marrow transplantation (BMT) is the best therapeutic alternative. However, contrary to SCID-X1, there are other treatment options that allow ADA patients to have near-normal lives: Enzyme replacement therapy (ERT) with polyethylene-glycol-conjugated bovine ADA (PEG-ADA). However, although ERT treatment is well tolerated and can partially restore immune function, its effect decline over time and, in addition, lifelong treatment is very expensive[33].

ADA deficiency has been successfully treated by GT using a similar approach to that for SCID-X1, but requiring mild bone-marrow chemoablation [34]. The authors showed immunological and metabolic reconstitution after transplantation of gene-modified CD34+ using ADAexpressing-MLV based vectors. The selective growth advantage of ADA-expressing lympho‐ cytes played an important role in the success of this trial. Similar findings have been reported by Gaspar *et. al.* [23] and again by Aiuti *et al*[10]. In total, over 40 patients with ADA have been treated in Italy, UK and USA. At present all patients are alive and 29 of them do not require ERT [9, 10, 23, 25, 34-36].

It is important to remark that no leukaemia-like disease have been observed in the ADA-SCID GT trial. The author propose that the differences between SCID-X1 and ADA might be related with SCID-X1 genetic background or the role of the therapeutic transgene (*ADA* is a house‐ keeping enzyme whereas γc is a potential oncogene growth factor receptor). However, in the last clinical trial some non-life threatening adverse effects have been reported such as neutro‐ penia (2 patients), treatment-related infections (2 patients), Epstein-Barr virus reactivations (1 patient) and autoimmune hepatitis (1 patient).

microtrombocytopenia, eczema and higher susceptibility to autoimmune diseases and

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457

As for other PID, HLA-identical sibling HSC donor transplantation is considered the treatment of choice (over 80% survival rate). Allogeneic HSCTs is offering nowadays good outcomes due to improvements in HLA-typing and new alternative donor sources and myeloablative conditioning regimens [46]. However, patients lacking a HLA-matched donor still require alternative therapeutic approaches. In this direction GT could be an alternative in the near future for these patients. In fact WAS is an attractive target for GT since expression of WASP

Dr Klein group (Hannover Medical School, Hannover, Germany) performed the first clinical trial for WAS GT [53]. 10 patients were enrolled in this trial and they received autologous

 cells transduced with LTR-driven gammaretroviral vectors expressing WASP. All patients received reduced intensity conditioning with Busulfan. Most of the patients treated gain WASP expression in multiple lineages. Platelet counts increased and clinical condition improved with resolution of eczema and bleeding disorder [54, 55]. However, as occurred in the SCID-X1 clinical trials, four out of 10 of the treated patients developed leukaemia [55, 56]. The presence of the strong LTR enhancer and the patient's predisposition to develop lympho‐

lymphoid malignancies [45].

**immunodeficiencies**

**transgene------- LTRΔU3**

CD34+

confer selective growth advantage [47-52].

mas could favour the high frequency of leukaemia in this trial.

**3. Lentiviral-vector based gene therapy clinical trials for primary**

As soon as the first cases of leukaemia appeared in the SCID-X1 GT trial, it was clear that LTRdriven gammaretroviral vectors were not the vector of choice to go further into clinic. Im‐ provements in the gammaretroviral vectors and the design of new integrative vectors became the main goal in the GT field. Several groups have dedicated considerable effort to understand the mechanism of leukomogenesis upon gammaretroviral transduction. The LMO2 oncogene was found in 4/5 cases in the SCID-X1 trial and it is now clear that retrovirus-mediated gene transfer can deregulate proto-oncogene expression through the LTR enhancer activity. With this in mind, Dr. Naldini's group have developed self-inactivated (LTR mutated) lentiviral vectors (based in HIV-1) which have one of the best efficiency/safety ratio [57-59]. LVs, contrary to gammaretroviral vectors are able to achieve efficient transduction of HSCs with minimal activation [60]. They are also safer than gammaretroviral vectors due to their less genotoxic integration site [61-63]. Several clinical trials for PID have started using HIV-1-based vectors and some promising results have already been shown on international meetings. In most cases, the general structure of the vectors is as follow: **LTRΔU3-- ψ ----human promoter ------**

There are at the moment two GT clinical trials on going for SCID-X1 using lentiviral vectors (http://www.wiley.com/legacy/wileychi/genmed/clinical/). One is designed for newly diag‐ nose children (St Jude Children's Research Hospital) and other is a Phase I/II non-randomized clinical trial designed to treat 13 patients with SCID-X1 who are between 2 and 30 years of age

#### **2.4. X-linked Chronic Granulomatous Disease (X-CGD)**

Chronic granulomatous disease (CGD) is a rare PID characterized by severe, life threatening bacterial and fungal infections. Patients with CGD have also defective degradation of inflam‐ matory mediators leading to granuloma formation. All of these defects are caused by muta‐ tions in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in phagocytic cells [37]. gp91phox mutations occur in up to 70% of the CGD cases and represent the X-linked form of this disorder (X-CGD). Neutrophils, monocytes, macrophages, and eosinophils from CGD patients cannot generate superoxide and other reactive oxygen intermediates to destroy invading bacteria and fungi.

Contrary to SCID-X1 and ADA, CGD is a difficult target for GT, since the expression of the correct form of the gene does not provide selective advantage to hematopoietic progenitors. In addition, myeloid cells have a short life span and therefore a large amount of HSC must be corrected to achieve clinical benefits. Myeloablative conditioning is therefore required to increase the amount of gene-modified cells that engraft into the patients. Several GT clinical trials for CGD have been conducted since 1997. Initial studies using retroviral vector to express p47-phox into CD34+ cells, resulted in low and short-term engraftment of CGD-corrected cells [38]. More recent GT clinical trials on X-CGD conducted in Franckfurt, Zurinch, London, USA and Seoul resulted in higher correction and clinical benefit in several patients. Dr Grez´s group showed the most dramatic effects in two children (5 and 8 years old) showing recovery from severe pulmonary and spinal aspergillosis. GT treatment also achieved recovery from paraparesis of both legs in one of the children [39]. However, the efficacy was only partial due to a progressive lost of gene-corrected cells over time [39-41]. The lost of transgene expression was, at least in part, due to inactivation of the vector promoter. However, there are other hypothesis that point to the potential toxicity of ectopic expression of gp91 gene on HSCs as a potential cause of the lost of gene-corrected cells [42]. In addition, three patients developed a myelodisplastic syndrome (MDS) due to transactivation of the MDS/EVI oncogene by the retroviral enhancer [40]. The MDS was fatal for two of the patients while the third was treated with HSCTs. These results revealed the importance of developing new, safer and more efficient vectors for GT in CGD.

#### **2.5. Wiskott-Aldrich Syndrome (WAS)**

Wiskott–Aldrich syndrome (WAS) is a X-linked PID caused by mutation in the WAS gene coding for the Wiskott-Aldrich syndrome protein (WASP), a hematopoietic-specific member of regulators of the actin cytoskeleton [43, 44]. The most severe form of WAS (where the mutation cause total absence of protein or function) is characterized by recurrent infections, microtrombocytopenia, eczema and higher susceptibility to autoimmune diseases and lymphoid malignancies [45].

with SCID-X1 genetic background or the role of the therapeutic transgene (*ADA* is a house‐ keeping enzyme whereas γc is a potential oncogene growth factor receptor). However, in the last clinical trial some non-life threatening adverse effects have been reported such as neutro‐ penia (2 patients), treatment-related infections (2 patients), Epstein-Barr virus reactivations (1

Chronic granulomatous disease (CGD) is a rare PID characterized by severe, life threatening bacterial and fungal infections. Patients with CGD have also defective degradation of inflam‐ matory mediators leading to granuloma formation. All of these defects are caused by muta‐ tions in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in phagocytic cells [37]. gp91phox mutations occur in up to 70% of the CGD cases and represent the X-linked form of this disorder (X-CGD). Neutrophils, monocytes, macrophages, and eosinophils from CGD patients cannot generate superoxide and other reactive oxygen

Contrary to SCID-X1 and ADA, CGD is a difficult target for GT, since the expression of the correct form of the gene does not provide selective advantage to hematopoietic progenitors. In addition, myeloid cells have a short life span and therefore a large amount of HSC must be corrected to achieve clinical benefits. Myeloablative conditioning is therefore required to increase the amount of gene-modified cells that engraft into the patients. Several GT clinical trials for CGD have been conducted since 1997. Initial studies using retroviral vector to express p47-phox into CD34+ cells, resulted in low and short-term engraftment of CGD-corrected cells [38]. More recent GT clinical trials on X-CGD conducted in Franckfurt, Zurinch, London, USA and Seoul resulted in higher correction and clinical benefit in several patients. Dr Grez´s group showed the most dramatic effects in two children (5 and 8 years old) showing recovery from severe pulmonary and spinal aspergillosis. GT treatment also achieved recovery from paraparesis of both legs in one of the children [39]. However, the efficacy was only partial due to a progressive lost of gene-corrected cells over time [39-41]. The lost of transgene expression was, at least in part, due to inactivation of the vector promoter. However, there are other hypothesis that point to the potential toxicity of ectopic expression of gp91 gene on HSCs as a potential cause of the lost of gene-corrected cells [42]. In addition, three patients developed a myelodisplastic syndrome (MDS) due to transactivation of the MDS/EVI oncogene by the retroviral enhancer [40]. The MDS was fatal for two of the patients while the third was treated with HSCTs. These results revealed the importance of developing new, safer and more efficient

Wiskott–Aldrich syndrome (WAS) is a X-linked PID caused by mutation in the WAS gene coding for the Wiskott-Aldrich syndrome protein (WASP), a hematopoietic-specific member of regulators of the actin cytoskeleton [43, 44]. The most severe form of WAS (where the mutation cause total absence of protein or function) is characterized by recurrent infections,

patient) and autoimmune hepatitis (1 patient).

456 Gene Therapy - Tools and Potential Applications

**2.4. X-linked Chronic Granulomatous Disease (X-CGD)**

intermediates to destroy invading bacteria and fungi.

vectors for GT in CGD.

**2.5. Wiskott-Aldrich Syndrome (WAS)**

As for other PID, HLA-identical sibling HSC donor transplantation is considered the treatment of choice (over 80% survival rate). Allogeneic HSCTs is offering nowadays good outcomes due to improvements in HLA-typing and new alternative donor sources and myeloablative conditioning regimens [46]. However, patients lacking a HLA-matched donor still require alternative therapeutic approaches. In this direction GT could be an alternative in the near future for these patients. In fact WAS is an attractive target for GT since expression of WASP confer selective growth advantage [47-52].

Dr Klein group (Hannover Medical School, Hannover, Germany) performed the first clinical trial for WAS GT [53]. 10 patients were enrolled in this trial and they received autologous CD34+ cells transduced with LTR-driven gammaretroviral vectors expressing WASP. All patients received reduced intensity conditioning with Busulfan. Most of the patients treated gain WASP expression in multiple lineages. Platelet counts increased and clinical condition improved with resolution of eczema and bleeding disorder [54, 55]. However, as occurred in the SCID-X1 clinical trials, four out of 10 of the treated patients developed leukaemia [55, 56]. The presence of the strong LTR enhancer and the patient's predisposition to develop lympho‐ mas could favour the high frequency of leukaemia in this trial.

#### **3. Lentiviral-vector based gene therapy clinical trials for primary immunodeficiencies**

As soon as the first cases of leukaemia appeared in the SCID-X1 GT trial, it was clear that LTRdriven gammaretroviral vectors were not the vector of choice to go further into clinic. Im‐ provements in the gammaretroviral vectors and the design of new integrative vectors became the main goal in the GT field. Several groups have dedicated considerable effort to understand the mechanism of leukomogenesis upon gammaretroviral transduction. The LMO2 oncogene was found in 4/5 cases in the SCID-X1 trial and it is now clear that retrovirus-mediated gene transfer can deregulate proto-oncogene expression through the LTR enhancer activity. With this in mind, Dr. Naldini's group have developed self-inactivated (LTR mutated) lentiviral vectors (based in HIV-1) which have one of the best efficiency/safety ratio [57-59]. LVs, contrary to gammaretroviral vectors are able to achieve efficient transduction of HSCs with minimal activation [60]. They are also safer than gammaretroviral vectors due to their less genotoxic integration site [61-63]. Several clinical trials for PID have started using HIV-1-based vectors and some promising results have already been shown on international meetings. In most cases, the general structure of the vectors is as follow: **LTRΔU3-- ψ ----human promoter ----- transgene------- LTRΔU3**

There are at the moment two GT clinical trials on going for SCID-X1 using lentiviral vectors (http://www.wiley.com/legacy/wileychi/genmed/clinical/). One is designed for newly diag‐ nose children (St Jude Children's Research Hospital) and other is a Phase I/II non-randomized clinical trial designed to treat 13 patients with SCID-X1 who are between 2 and 30 years of age and who have clinically significant impairment of immunity. Both cases are based on mice experiments showing a better profile of lentiviral vectors both in term of reconstitution and safety [64].

expression pattern. New, undesired side effects could appear in the future. New vectors must still consider improving two safety aspects: 1- genotoxicity (genomic alteration due to vector integrations) and 2- ectopic/unregulated expression of the transgene. Strategies to minimize or eliminate genotoxicity problems can be grouped in those based in improving retroviral vectors and those based in the development of non-viral technologies such as gene editing

Gene Therapy for Primary Immunodeficiencies

http://dx.doi.org/10.5772/55594

459

This work has been financed by Fondo de Investigaciones Sanitarias ISCIII (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union, through the research grant Nº PS09/00340, by the Consejería de Innovación Ciencia y Empresa (grants Nº P09- CTS-04532 and PAIDI-Bio-326) and Consejería de Salud (grant Nº PI0001/2009) from the Junta de Andalucía and FEDER/ Fondo de Cohesion Europeo (FSE) de Andalucía 2007-2013 to F.M.

, Alejandra Gutierrez-Guerrero and Karim Benabdellah

Gene and Cell Therapy Group. Human DNA variability department. GENYO. Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional

[1] Marodi L, Notarangelo LD. Immunological and genetic bases of new primary immu‐

[2] Pessach I, Walter J, Notarangelo LD. Recent advances in Primary Immunodeficien‐ cies: identification of novel genetic defects and unanticipated phenotypes. Pediatric

[3] Filipovich A. Hematopoietic cell transplantation for correction of primary immuno‐

[4] Neven B, Leroy S, Decaluwe H, Le Deist F, Picard C, Moshous D, et al. Long-term outcome after hematopoietic stem cell transplantation of a single-center cohort of 90 patients with severe combined immunodeficiency. Blood. 2009 Apr 23;113(17):

deficiencies. Bone Marrow Transplant. 2008 Aug;42 Suppl 1:S49-S52.

Government. Parque Tecnológico Ciencias de la Salud (PTCS), Granada, Spain

nodeficiencies. Nat Rev Immunol. 2007 Nov;7(11):851-61.

\*Address all correspondence to: francisco.martin@genyo.es

(revised in [14, 65]).

**Author details**

Francisco Martin\*

**References**

research. 2009 Jan 28.

4114-24.

**Acknowledgements**

Dr Gaspar and Dr Kohn have launched two other clinical trials using lentiviral vectors to treat ADA patients in UK and USA respectively. Both groups use EF1 promoter driven lentiviral vectors produced at the same site (Indiana University Vector Production Facility) through a Transatlantic Gene Therapy Consortium. The primary objective of the trial is to examine the safety of the protocol in 10 patients transplanted with LV gene-modified CD34+ cells. The protocol will involve non-myeloablative conditioning with busulfan and withholding of PEG-ADA ERT. As secondary objectives the trial will aim for the expression of ADA in peripheral blood leucocytes and immune reconstitution.

CGD is probably the PID where the necessity to improve vector efficiency and safety has been more obvious. The absence of the selective advantage of the gene-modified cells and the short life span of myeloid cells reduce the clinical benefits of gammaretroviral vectors but kept all the secondary effects. In addition, the potential toxicity of ectopic expression of gp91phox on HSCs required the use of physiologically regulated vectors [65] expressing the transgene specifically in granulocytes. Very encouraging results have been obtained in animal models using transcriptionally regulated LV [66, 67]. The first clinical trial for CGD using LV started on November 2011 directed by Adrian Thrasher at Great Ormond Street Hospital for Children (UK). The primary outcome measures will be overall survival but the trial will also study reduction in frequency of infections and long-term immune reconstitution (http://clinicaltri‐ als.gov/ct2/show/NCT01381003].

As SCID-X1 and CGD, GT for WAS has also good reasons to change the therapeutic vectors (see above). There are four clinical trials on going for WAS using LV (FR-0047, UK-0168 and US-1052: journal of gene medicine GT clinical trials data base; NCT01515462: Clincaltrial.gov). All trials will use a similar construct which drive the expression of the WASP cDNA through its own promoter. The WASp-promoter-driven LVs are haematopoietic-specific [47, 49, 68], physiological [49, 69] and avoid deleterious effects of over-expression in non-target cells[70]. Preliminary data presented at the 20th European Society of Gene and Cell Therapy by the Italian and French groups showed impressive results both, in terms of immune reconstitution and safety profile. It is important to note that integration site analysis in these patients did not show any preference for the proto-oncongens LMO2 or EVI1. In addition they didn't observe, at the time of analysis, any evidence of clonal dominance (usually indicative of proto-onco‐ genes activation).

#### **4. Future directions**

Based on the data shown, it does appear that new generation LVs driving the expression of the transgene through physiological promoters could be a big step toward GT clinical trans‐ lation. Exciting results are expected on the clinical trials undergoing at the moment. Still, LV integrates randomly at active sites in the cell genome and can therefore alter its normal expression pattern. New, undesired side effects could appear in the future. New vectors must still consider improving two safety aspects: 1- genotoxicity (genomic alteration due to vector integrations) and 2- ectopic/unregulated expression of the transgene. Strategies to minimize or eliminate genotoxicity problems can be grouped in those based in improving retroviral vectors and those based in the development of non-viral technologies such as gene editing (revised in [14, 65]).

#### **Acknowledgements**

and who have clinically significant impairment of immunity. Both cases are based on mice experiments showing a better profile of lentiviral vectors both in term of reconstitution and

Dr Gaspar and Dr Kohn have launched two other clinical trials using lentiviral vectors to treat ADA patients in UK and USA respectively. Both groups use EF1 promoter driven lentiviral vectors produced at the same site (Indiana University Vector Production Facility) through a Transatlantic Gene Therapy Consortium. The primary objective of the trial is to examine the

protocol will involve non-myeloablative conditioning with busulfan and withholding of PEG-ADA ERT. As secondary objectives the trial will aim for the expression of ADA in peripheral

CGD is probably the PID where the necessity to improve vector efficiency and safety has been more obvious. The absence of the selective advantage of the gene-modified cells and the short life span of myeloid cells reduce the clinical benefits of gammaretroviral vectors but kept all the secondary effects. In addition, the potential toxicity of ectopic expression of gp91phox on HSCs required the use of physiologically regulated vectors [65] expressing the transgene specifically in granulocytes. Very encouraging results have been obtained in animal models using transcriptionally regulated LV [66, 67]. The first clinical trial for CGD using LV started on November 2011 directed by Adrian Thrasher at Great Ormond Street Hospital for Children (UK). The primary outcome measures will be overall survival but the trial will also study reduction in frequency of infections and long-term immune reconstitution (http://clinicaltri‐

As SCID-X1 and CGD, GT for WAS has also good reasons to change the therapeutic vectors (see above). There are four clinical trials on going for WAS using LV (FR-0047, UK-0168 and US-1052: journal of gene medicine GT clinical trials data base; NCT01515462: Clincaltrial.gov). All trials will use a similar construct which drive the expression of the WASP cDNA through its own promoter. The WASp-promoter-driven LVs are haematopoietic-specific [47, 49, 68], physiological [49, 69] and avoid deleterious effects of over-expression in non-target cells[70]. Preliminary data presented at the 20th European Society of Gene and Cell Therapy by the Italian and French groups showed impressive results both, in terms of immune reconstitution and safety profile. It is important to note that integration site analysis in these patients did not show any preference for the proto-oncongens LMO2 or EVI1. In addition they didn't observe, at the time of analysis, any evidence of clonal dominance (usually indicative of proto-onco‐

Based on the data shown, it does appear that new generation LVs driving the expression of the transgene through physiological promoters could be a big step toward GT clinical trans‐ lation. Exciting results are expected on the clinical trials undergoing at the moment. Still, LV integrates randomly at active sites in the cell genome and can therefore alter its normal

cells. The

safety of the protocol in 10 patients transplanted with LV gene-modified CD34+

blood leucocytes and immune reconstitution.

als.gov/ct2/show/NCT01381003].

genes activation).

**4. Future directions**

safety [64].

458 Gene Therapy - Tools and Potential Applications

This work has been financed by Fondo de Investigaciones Sanitarias ISCIII (Spain) and Fondo Europeo de Desarrollo Regional (FEDER) from the European Union, through the research grant Nº PS09/00340, by the Consejería de Innovación Ciencia y Empresa (grants Nº P09- CTS-04532 and PAIDI-Bio-326) and Consejería de Salud (grant Nº PI0001/2009) from the Junta de Andalucía and FEDER/ Fondo de Cohesion Europeo (FSE) de Andalucía 2007-2013 to F.M.

#### **Author details**

Francisco Martin\* , Alejandra Gutierrez-Guerrero and Karim Benabdellah

\*Address all correspondence to: francisco.martin@genyo.es

Gene and Cell Therapy Group. Human DNA variability department. GENYO. Centre for Genomics and Oncological Research: Pfizer, University of Granada, Andalusian Regional Government. Parque Tecnológico Ciencias de la Salud (PTCS), Granada, Spain

#### **References**


[5] Kildebeck E, Checketts J, Porteus M. Gene therapy for primary immunodeficiencies. Curr Opin Pediatr. 2012 Dec;24(6):731-8.

[18] Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, et al. Side effects of retro‐ viral gene transfer into hematopoietic stem cells. Blood. 2003 Mar 15;101(6):2099-114.

Gene Therapy for Primary Immunodeficiencies

http://dx.doi.org/10.5772/55594

461

[19] Noguchi M, Yi H, Rosenblatt HM, Filipovich AH, Adelstein S, Modi WS, et al. Inter‐ leukin-2 receptor gamma chain mutation results in X-linked severe combined immu‐

[20] Stephan V, Wahn V, Le Deist F, Dirksen U, Broker B, Muller-Fleckenstein I, et al. Atypical X-linked severe combined immunodeficiency due to possible spontaneous reversion of the genetic defect in T cells. N Engl J Med. 1996 Nov 21;335(21):1563-7.

[21] Hacein-Bey-Abina S, Fischer A, Cavazzana-Calvo M. Gene therapy of X-linked se‐

[22] Hacein-Bey-Abina S, Le Deist F, Carlier F, Bouneaud C, Hue C, De Villartay JP, et al. Sustained correction of X-linked severe combined immunodeficiency by ex vivo gene

[23] Gaspar HB, Bjorkegren E, Parsley K, Gilmour KC, King D, Sinclair J, et al. Successful reconstitution of immunity in ADA-SCID by stem cell gene therapy following cessa‐ tion of PEG-ADA and use of mild preconditioning. Mol Ther. 2006 Oct;14(4):505-13.

[24] Hacein-Bey-Abina S, Hauer J, Lim A, Picard C, Wang GP, Berry CC, et al. Efficacy of gene therapy for X-linked severe combined immunodeficiency. N Engl J Med. 2010

[25] Gaspar HB, Cooray S, Gilmour KC, Parsley KL, Adams S, Howe SJ, et al. Long-term persistence of a polyclonal T cell repertoire after gene therapy for X-linked severe

[26] Hacein-Bey-Abina S, Garrigue A, Wang GP, Soulier J, Lim A, Morillon E, et al. Inser‐ tional oncogenesis in 4 patients after retrovirus-mediated gene therapy of SCID-X1. J

[27] Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al. A serious adverse event after successful gene therapy for X-linked severe com‐

[28] Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome are favored targets for MLV integration. Science. 2003 Jun 13;300(5626):1749-51.

[29] Laufs S, Nagy KZ, Giordano FA, Hotz-Wagenblatt A, Zeller WJ, Fruehauf S. Inser‐ tion of retroviral vectors in NOD/SCID repopulating human peripheral blood pro‐ genitor cells occurs preferentially in the vicinity of transcription start regions and in

[30] Bushman F, Lewinski M, Ciuffi A, Barr S, Leipzig J, Hannenhalli S, et al. Genomewide analysis of retroviral DNA integration. Nat Rev Microbiol. 2005 Nov;3(11):

combined immunodeficiency. Sci Transl Med. 2011 Aug 24;3(97):97ra79.

bined immunodeficiency. N Engl J Med. 2003 Jan 16;348(3):255-6.

vere combined immunodeficiency. Int J Hematol. 2002 Nov;76(4):295-8.

nodeficiency in humans. Cell. 1993 Apr 9;73(1):147-57.

therapy. N Engl J Med. 2002 Apr 18;346(16):1185-93.

Jul 22;363(4):355-64.

Clin Invest. 2008 Sep;118(9):3132-42.

introns. Mol Ther. 2004 Nov;10(5):874-81.

848-58.


[18] Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, et al. Side effects of retro‐ viral gene transfer into hematopoietic stem cells. Blood. 2003 Mar 15;101(6):2099-114.

[5] Kildebeck E, Checketts J, Porteus M. Gene therapy for primary immunodeficiencies.

[6] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

[7] Cavazzana-Calvo M, Lagresle C, Hacein-Bey-Abina S, Fischer A. Gene therapy for

[8] Howe SJ, Mansour MR, Schwarzwaelder K, Bartholomae C, Hubank M, Kempski H, et al. Insertional mutagenesis combined with acquired somatic mutations causes leu‐ kemogenesis following gene therapy of SCID-X1 patients. J Clin Invest. 2008 Sep

[9] Fischer A, Cavazzana-Calvo M. Gene therapy of inherited diseases. Lancet. 2008 Jun

[10] Aiuti A, Cattaneo F, Galimberti S, Benninghoff U, Cassani B, Callegaro L, et al. Gene therapy for immunodeficiency due to adenosine deaminase deficiency. N Engl J

[11] Ott MG, Schmidt M, Schwarzwaelder K, Stein S, Siler U, Koehl U, et al. Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med. 2006 Apr;12(4):401-9.

[12] Boztug K, Schmidt M, Schwarzer A, Banerjee PP, Diez IA, Dewey RA, et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med. 2010 Nov 11;363(20):

[13] Galy A, Thrasher AJ. Gene therapy for the Wiskott-Aldrich syndrome. Curr Opin Al‐

[14] Romero Z, Toscano MG, Unciti JD, Molina I, Martin F. Safer Vectors For Gene Thera‐

[15] Toscano MG, Romero Z, Munoz P, Cobo M, Benabdellah K, Martin F. Physiological and tissue-specific vectors for treatment of inherited diseases. Gene Ther. 2011 Feb;

[16] Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990 Aug;

[17] Demaison C, Brouns G, Blundell MP, Goldman JP, Levinsky RJ, Grez M, et al. A de‐ fined window for efficient gene marking of severe combined immunodeficient-repo‐ pulating cells using a gibbon ape leukemia virus-pseudotyped retroviral vector.

py Of Primary Immunodeficiencies. Curr Gene Ther. 2009 Aug 1.

severe combined immunodeficiency. Annu Rev Med. 2005;56:585-602.

Curr Opin Pediatr. 2012 Dec;24(6):731-8.

Science. 2000;288(5466):669-72.

2;118(9):3143-50.

460 Gene Therapy - Tools and Potential Applications

1918-27.

18(2):117-27.

10(8):4239-42.

14;371(9629):2044-7.

Med. 2009 Jan 29;360(5):447-58.

lergy Clin Immunol. 2011 Dec;11(6):545-50.

Hum Gene Ther. 2000;11(1):91-100.


[31] Ratech H, Hirschhorn R, Greco MA. Pathologic findings in adenosine deaminase de‐ ficient-severe combined immunodeficiency. II. Thymus, spleen, lymph node, and gastrointestinal tract lymphoid tissue alterations. Am J Pathol. 1989 Dec;135(6): 1145-56.

[44] Ochs HD, Thrasher AJ. The Wiskott-Aldrich syndrome. J Allergy Clin Immunol. 2006

Gene Therapy for Primary Immunodeficiencies

http://dx.doi.org/10.5772/55594

463

[45] Bosticardo M, Marangoni F, Aiuti A, Villa A, Roncarolo MG. Recent advances in un‐ derstanding the pathophysiology of Wiskott-Aldrich syndrome. Blood. 2009 Apr 7.

[46] Moratto D, Giliani S, Bonfim C, Mazzolari E, Fischer A, Ochs HD, et al. Long-term outcome and lineage-specific chimerism in 194 patients with Wiskott-Aldrich syn‐ drome treated by hematopoietic cell transplantation in the period 1980-2009: an inter‐

[47] Dupre L, Trifari S, Follenzi A, Marangoni F, Lain de Lera T, Bernad A, et al. Lentivi‐ ral vector-mediated gene transfer in T cells from Wiskott-Aldrich syndrome patients

[48] Konno A, Wada T, Schurman SH, Garabedian EK, Kirby M, Anderson SM, et al. Dif‐ ferential contribution of Wiskott-Aldrich syndrome protein to selective advantage in

[49] Martin F, Toscano MG, Blundell M, Frecha C, Srivastava GK, Santamaria M, et al. Lentiviral vectors transcriptionally targeted to hematopoietic cells by WASP gene

[50] Ariga T, Kondoh T, Yamaguchi K, Yamada M, Sasaki S, Nelson DL, et al. Spontane‐ ous in vivo reversion of an inherited mutation in the Wiskott-Aldrich syndrome. J

[51] Wada T, Konno A, Schurman SH, Garabedian EK, Anderson SM, Kirby M, et al. Sec‐ ond-site mutation in the Wiskott-Aldrich syndrome (WAS) protein gene causes so‐

[52] Wada T, Schurman SH, Otsu M, Garabedian EK, Ochs HD, Nelson DL, et al. Somatic mosaicism in Wiskott--Aldrich syndrome suggests in vivo reversion by a DNA slip‐

[53] Boztug K, Dewey RA, Klein C. Development of hematopoietic stem cell gene therapy

[54] Boztug K, Schmidt M, Schwarzer A, Banerjee PP, Diez IA, Dewey RA, et al. Stem-cell gene therapy for the Wiskott-Aldrich syndrome. N Engl J Med. 2010 Nov 11;363(20):

[55] Corrigan-Curay J, Cohen-Haguenauer O, O'Reilly M, Ross SR, Fan H, Rosenberg N, et al. Challenges in vector and trial design using retroviral vectors for long-term gene correction in hematopoietic stem cell gene therapy. Mol Ther. 2012 Jun;20(6):1084-94.

[56] Avedillo Diez I, Zychlinski D, Coci EG, Galla M, Modlich U, Dewey RA, et al. Devel‐ opment of novel efficient SIN vectors with improved safety features for Wiskott-Al‐ drich syndrome stem cell based gene therapy. Mol Pharm. 2011 Oct 3;8(5):1525-37.

matic mosaicism in two WAS siblings. J Clin Invest. 2003 May;111(9):1389-97.

page mechanism. Proc Natl Acad Sci U S A. 2001 Jul 17;98(15):8697-702.

for Wiskott-Aldrich syndrome. Curr Opin Mol Ther. 2006 Oct;8(5):390-5.

national collaborative study. Blood. 2011 Aug 11;118(6):1675-84.

leads to functional correction. Mol Ther. 2004 Nov;10(5):903-15.

proximal promoter sequences. Gene Ther. 2005 Apr;12(8):715-23.

T- and B-cell lineages. Blood. 2004 Jan 15;103(2):676-8.

Immunol. 2001 Apr 15;166(8):5245-9.

1918-27.

Apr;117(4):725-38; quiz 39.


[44] Ochs HD, Thrasher AJ. The Wiskott-Aldrich syndrome. J Allergy Clin Immunol. 2006 Apr;117(4):725-38; quiz 39.

[31] Ratech H, Hirschhorn R, Greco MA. Pathologic findings in adenosine deaminase de‐ ficient-severe combined immunodeficiency. II. Thymus, spleen, lymph node, and gastrointestinal tract lymphoid tissue alterations. Am J Pathol. 1989 Dec;135(6):

[32] Rogers MH, Lwin R, Fairbanks L, Gerritsen B, Gaspar HB. Cognitive and behavioral abnormalities in adenosine deaminase deficient severe combined immunodeficiency.

[33] Gaspar HB, Aiuti A, Porta F, Candotti F, Hershfield MS, Notarangelo LD. How I

[34] Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A, et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Sci‐

[35] Sakiyama Y, Ariga T, Ohtsu M. [Gene therapy for adenosine deaminase deficiency].

[36] Aiuti A, Cassani B, Andolfi G, Mirolo M, Biasco L, Recchia A, et al. Multilineage hematopoietic reconstitution without clonal selection in ADA-SCID patients treated

[37] Seger RA. Modern management of chronic granulomatous disease. Br J Haematol.

[38] Malech HL, Maples PB, Whiting-Theobald N, Linton GF, Sekhsaria S, Vowells SJ, et al. Prolonged production of NADPH oxidase-corrected granulocytes after gene ther‐ apy of chronic granulomatous disease. Proc Natl Acad Sci U S A. 1997 Oct 28;94(22):

[39] Grez M, Reichenbach J, Schwable J, Seger R, Dinauer MC, Thrasher AJ. Gene therapy of chronic granulomatous disease: the engraftment dilemma. Mol Ther. 2011 Jan;

[40] Stein S, Ott MG, Schultze-Strasser S, Jauch A, Burwinkel B, Kinner A, et al. Genomic instability and myelodysplasia with monosomy 7 consequent to EVI1 activation after gene therapy for chronic granulomatous disease. Nat Med. 2010 Feb;16(2):198-204.

[41] Kuhns DB, Alvord WG, Heller T, Feld JJ, Pike KM, Marciano BE, et al. Residual NADPH oxidase and survival in chronic granulomatous disease. N Engl J Med. 2010

[42] Bedard K, Krause KH. The NOX family of ROS-generating NADPH oxidases: physi‐

[43] Gallego MD, Santamaria M, Pena J, Molina IJ. Defective actin reorganization and pol‐ ymerization of Wiskott-Aldrich T cells in response to CD3-mediated stimulation.

ology and pathophysiology. Physiol Rev. 2007 Jan;87(1):245-313.

with stem cell gene therapy. J Clin Invest. 2007 Aug;117(8):2233-40.

treat ADA deficiency. Blood. 2009 Oct 22;114(17):3524-32.

1145-56.

462 Gene Therapy - Tools and Potential Applications

J Pediatr. 2001 Jul;139(1):44-50.

ence. 2002 Jun 28;296(5577):2410-3.

2008 Feb;140(3):255-66.

Dec 30;363(27):2600-10.

Blood. 1997;90(8):3089-97.

12133-8.

19(1):28-35.

Nippon Rinsho. 2005 Mar;63(3):448-52.


[57] Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C, et al. Hema‐ topoietic stem cell gene transfer in a tumor-prone mouse model uncovers low geno‐ toxicity of lentiviral vector integration. NatBiotechnol. 2006;24(6):687-96.

[69] Charrier S, Dupre L, Scaramuzza S, Jeanson-Leh L, Blundell MP, Danos O, et al. Len‐ tiviral vectors targeting WASp expression to hematopoietic cells, efficiently trans‐

Gene Therapy for Primary Immunodeficiencies

http://dx.doi.org/10.5772/55594

465

[70] Toscano MG, Frecha C, Benabdellah K, Cobo M, Blundell M, Thrasher AJ, et al. Hem‐ atopoietic-specific lentiviral vectors circumvent cellular toxicity due to ectopic ex‐ pression of Wiskott-Aldrich syndrome protein. Hum Gene Ther. 2008 Feb;19(2):

duce and correct cells from WAS patients. Gene Ther. 2007 Mar;14(5):415-28.

179-97.


[69] Charrier S, Dupre L, Scaramuzza S, Jeanson-Leh L, Blundell MP, Danos O, et al. Len‐ tiviral vectors targeting WASp expression to hematopoietic cells, efficiently trans‐ duce and correct cells from WAS patients. Gene Ther. 2007 Mar;14(5):415-28.

[57] Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C, et al. Hema‐ topoietic stem cell gene transfer in a tumor-prone mouse model uncovers low geno‐

[58] Zufferey R, Dull T, Mandel RJ, Bukovsky A, Quiroz D, Naldini L, et al. Self-inactivat‐ ing lentivirus vector for safe and efficient in vivo gene delivery. J Virol. 1998;72(12):

[59] Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, et al. In vivo gene deliv‐ ery and stable transduction of nondividing cells by a lentiviral vector. Science.

[60] Case SS, Price MA, Jordan CT, Yu XJ, Wang L, Bauer G, et al. Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vec‐

[61] Montini E, Cesana D, Schmidt M, Sanvito F, Ponzoni M, Bartholomae C, et al. Hema‐ topoietic stem cell gene transfer in a tumor-prone mouse model uncovers low geno‐

[62] Gonzalez-Murillo A, Lozano ML, Montini E, Bueren JA, Guenechea G. Unaltered re‐ population properties of mouse hematopoietic stem cells transduced with lentiviral

[63] Montini E, Cesana D, Schmidt M, Sanvito F, Bartholomae CC, Ranzani M, et al. The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy. J Clin Invest. 2009

[64] Zhou S, Mody D, DeRavin SS, Hauer J, Lu T, Ma Z, et al. A self-inactivating lentiviral vector for SCID-X1 gene therapy that does not activate LMO2 expression in human T

[65] Toscano MG, Romero Z, Munoz P, Cobo M, Benabdellah K, Martin F. Physiological and tissue-specific vectors for treatment of inherited diseases. Gene Ther. 2011 Feb;

[66] Santilli G, Almarza E, Brendel C, Choi U, Beilin C, Blundell MP, et al. Biochemical correction of X-CGD by a novel chimeric promoter regulating high levels of trans‐

[67] Barde I, Laurenti E, Verp S, Wiznerowicz M, Offner S, Viornery A, et al. Lineage- and stage-restricted lentiviral vectors for the gene therapy of chronic granulomatous dis‐

[68] Frecha C, Toscano MG, Costa C, Saez-Lara MJ, Cosset FL, Verhoeyen E, et al. Im‐ proved lentiviral vectors for Wiskott-Aldrich syndrome gene therapy mimic endoge‐ nous expression profiles throughout haematopoiesis. Gene Ther. 2008 Jun;15(12):

gene expression in myeloid cells. Mol Ther. 2011 Jan;19(1):122-32.

toxicity of lentiviral vector integration. Nat Biotechnol. 2006 Jun;24(6):687-96.

tors. Proc Natl Acad Sci U S A. 1999 Mar 16;96(6):2988-93.

toxicity of lentiviral vector integration. NatBiotechnol. 2006;24(6):687-96.

9873-80.

1996;272(5259):263-7.

464 Gene Therapy - Tools and Potential Applications

vectors. Blood. 2008 Aug 6.

cells. Blood. 2010 Aug 12;116(6):900-8.

ease. Gene Ther. 2011 Nov;18(11):1087-97.

Apr;119(4):964-75.

18(2):117-27.

930-41.

[70] Toscano MG, Frecha C, Benabdellah K, Cobo M, Blundell M, Thrasher AJ, et al. Hem‐ atopoietic-specific lentiviral vectors circumvent cellular toxicity due to ectopic ex‐ pression of Wiskott-Aldrich syndrome protein. Hum Gene Ther. 2008 Feb;19(2): 179-97.

**Chapter 19**

**Gene Therapy for Diabetic Retinopathy – Targeting the**

**Renin-Angiotensin System**

Yiguo Qiu, Takahiko Nakagawa,

http://dx.doi.org/10.5772/52702

**1. Introduction**

after 20 years [5-7].

Qiuhong Li, Amrisha Verma, Ping Zhu, Bo Lei,

Mohan K Raizada and William W Hauswirth

Additional information is available at the end of the chapter

**1.1. Diabetic retinopathy clinical features and current treatment options**

The prevalence of diabetes has been continuously increasing for the last few decades and it is being recognized as a worldwide epidemic [1]. Diabetic retinopathy (DR) is the most com‐ mon diabetic microvascular complication, and despite recent advances in therapeutics and management, DR remains the leading cause of severe vision loss in people under age of six‐ ty [2-4]. The prevalence of DR increases with duration of diabetes, and nearly all individuals with type 1 diabetes and more than 60% of those with type 2 have some form of retinopathy

Diabetic retinopathy (DR) is characterized by the development of progressive pathologi‐ cal changes in the retinal neuro-glial cells and microvasculature. The earlier hallmarks of diabetic retinopathy include breakdown of the blood-retinal barrier (BRB), loss of peri‐ cytes, thickening of basement membrane, and the formation of microaneuryms, which are outpouchings of capillaries [8]. BRB breakdown results in increased vascular permeability and leakage of fluid into the macula causing macular edema, another significant cause of vision loss in those with diabetes. With the progression of diabetic retinopathy, hemor‐ rhage, macular edema, cotton wool spots, all signs of retinal ischemia, and hard exu‐ dates, the result of precipitation of lipoproteins and other circulating proteins through abnormally leaky retinal vessels become increasingly apparent. More severe and later stages of diabetic retinopathy, known as proliferative diabetic retinopathy (PDR), is char‐

> © 2013 Li et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

## **Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System**

Qiuhong Li, Amrisha Verma, Ping Zhu, Bo Lei, Yiguo Qiu, Takahiko Nakagawa, Mohan K Raizada and William W Hauswirth

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52702

#### **1. Introduction**

#### **1.1. Diabetic retinopathy clinical features and current treatment options**

The prevalence of diabetes has been continuously increasing for the last few decades and it is being recognized as a worldwide epidemic [1]. Diabetic retinopathy (DR) is the most com‐ mon diabetic microvascular complication, and despite recent advances in therapeutics and management, DR remains the leading cause of severe vision loss in people under age of six‐ ty [2-4]. The prevalence of DR increases with duration of diabetes, and nearly all individuals with type 1 diabetes and more than 60% of those with type 2 have some form of retinopathy after 20 years [5-7].

Diabetic retinopathy (DR) is characterized by the development of progressive pathologi‐ cal changes in the retinal neuro-glial cells and microvasculature. The earlier hallmarks of diabetic retinopathy include breakdown of the blood-retinal barrier (BRB), loss of peri‐ cytes, thickening of basement membrane, and the formation of microaneuryms, which are outpouchings of capillaries [8]. BRB breakdown results in increased vascular permeability and leakage of fluid into the macula causing macular edema, another significant cause of vision loss in those with diabetes. With the progression of diabetic retinopathy, hemor‐ rhage, macular edema, cotton wool spots, all signs of retinal ischemia, and hard exu‐ dates, the result of precipitation of lipoproteins and other circulating proteins through abnormally leaky retinal vessels become increasingly apparent. More severe and later stages of diabetic retinopathy, known as proliferative diabetic retinopathy (PDR), is char‐

© 2013 Li et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

acterized by pathological neovascularization. Vision loss can occur from vitreous hemor‐ rhage or from tractional retinal detachment [8, 9].

tions as a carboxymonopeptidase, cleaving a single C-terminal residue from peptide sub‐ strates, thus ACE2 is able to cleave Ang II to form Ang (1-7). Ang (1-7), a biologically active component of the RAS [28-30] binds to a G-protein coupled receptor, Mas receptor [31], and plays a counter-regulatory role in the RAS by opposing the vascular and proliferative effects of Ang II [32]. A current view of RAS consists of at least two axis with counteracting biologic

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

469

**Figure 1.** Schematic diagram depicting the key components of the Renin Angiotensin System. Angiotensinogen is cleaved by renin to form angiotensin I (Ang I). Angiotensin converting enzyme (ACE) converts Ang I into Angiotensin II (Ang II) the main effector peptide of the RAS. Ang II elicits is cellular effects by activating the main receptor, Angioten‐ sin II receptor 1 (AT1R), as well as other receptors (not shown). Angiotensin II-converting enzyme 2 (ACE2), a recently discovered component of RAS, cleaves Ang II to form Angiotensin (1-7) (Ang 1-7), which activate Mas receptor to pro‐ duce counteracting effects mediated by Ang II. All these components are expressed locally in various cell types in the eye, regulating metabolism, cell survival, and other local neuronal-vascular and immune-modulating functions in the

This vasoprotective axis of RAS counteracts the traditional proliferative, fibrotic, proinflam‐ matory and hypertrophic effects of the ACE/Ang II/AT1R axis of the RAS [24]. The impor‐ tance of the vasodeleterious axis of the RAS [ACE/angiotensin II (Ang II)/ AT1R] in cardiovascular disease, as well as in diabetes and diabetic complications, is well established since ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are leading therapeu‐ tic strategies [20, 33-35]. However, the impact of the vasoprotective axis of the RAS remains poorly understood [24, 36-38]. The concept that shifting the balance of the RAS towards the vasodilatory axis by activation of ACE2 or its product, Ang-(1-7) is beneficial has been sup‐ ported by many studies in cardiac, pulmonary, and vascular fibrosis [24, 39-43]. Indeed, ACE2/Ang-(1-7) activation is now considered to be a critical part of the beneficial actions of

effects (Figure 1).

retina.

ACEi and ARB drugs [24, 36].

Despite recent developments in the pharmacotherapy of DR, treatment options for patients with DR are still limited. Laser photocoagulation, the primary treatment option for patients with PDR, is still considered gold standard therapy for the treatment of PDR. Although this treatment slows the loss of vision in those with PDR, it does not represent a cure, and is in itself a cell destructive therapy. Corticosteroids and anti-VEGF agents have shown promis‐ ing results with regard to prevention of neovascularization, but remain limited in use due to their short-duration effects. More importantly, none of these agents have been able to substi‐ tute for the durability and effectiveness of laser mediated panretinal photocoagulation in preventing vision loss in the late stages of DR.

#### **1.2. RAS and diabetic complications**

The renin-angiotensin system (RAS) plays a vital role in the cardiovascular homeostasis by regulating vascular tone, fluid and electrolyte balance, and in the sympathetic nerve system. Angiotensin II (Ang II), a peptide hormone of RAS, has been known to regulate a variety of hemodynamic physiological responses, including fluid homeostasis, renal function, and contraction of vascular smooth muscle [10]. In addition, Ang II is capable of inducing a mul‐ titude of non-hemodynamic effects, such as the induction of reactive oxygen species (ROS), cytokines, and the stimulation of collagen synthesis [11-14]. Most of the pathophysiological actions of Ang II are mediated via activation of Ang II type 1 receptors (AT1R), G protein– coupled receptors (GPCRs) that couple to many signaling molecules, including small G pro‐ teins, phospholipases, mitogen-activated protein (MAP) kinases, phosphatases, tyrosine kin‐ ases, NADPH oxidase, and transcription factors to stimulate vascular smooth muscle cell growth, inflammation, and fibrosis [11, 15, 16]. Dysregulation of RAS has been implicated in a number of major cardiovascular and metabolic diseases, including endothelial dysfunc‐ tion, atherosclerosis, hypertension, renal disease, diabetic complications, stroke, myocardial infarction and congestive heart failure [17, 18]. RAS blockade produces beneficial cardiovas‐ cular and renal effects in numerous clinical trials [19-21].

#### **1.3. Recent advances in RAS research**

Recent discoveries have revealed that the RAS hormonal signaling cascade is more com‐ plex than initially conceived with multiple enzymes, effector molecules, and receptors that coordinately regulate the effects of the RAS. Recent studies have identified additional peptides with important physiological and pathological roles, new enzymatic cascades that generate these peptides and more receptors and signaling pathways that mediate their function [22, 23].

Discovery of angiotensin-converting enzyme 2 (ACE2) has resulted in the establishment of a novel axis of the RAS involving ACE2/Ang-(1-7)/Mas [24-27]. ACE2, like ACE, is a zinc-met‐ allopeptidase, exhibiting approximately 42% amino acid identity with ACE in its catalytic domain. However, unlike somatic ACE, ACE2 only contains a single catalytic site and func‐ tions as a carboxymonopeptidase, cleaving a single C-terminal residue from peptide sub‐ strates, thus ACE2 is able to cleave Ang II to form Ang (1-7). Ang (1-7), a biologically active component of the RAS [28-30] binds to a G-protein coupled receptor, Mas receptor [31], and plays a counter-regulatory role in the RAS by opposing the vascular and proliferative effects of Ang II [32]. A current view of RAS consists of at least two axis with counteracting biologic effects (Figure 1).

acterized by pathological neovascularization. Vision loss can occur from vitreous hemor‐

Despite recent developments in the pharmacotherapy of DR, treatment options for patients with DR are still limited. Laser photocoagulation, the primary treatment option for patients with PDR, is still considered gold standard therapy for the treatment of PDR. Although this treatment slows the loss of vision in those with PDR, it does not represent a cure, and is in itself a cell destructive therapy. Corticosteroids and anti-VEGF agents have shown promis‐ ing results with regard to prevention of neovascularization, but remain limited in use due to their short-duration effects. More importantly, none of these agents have been able to substi‐ tute for the durability and effectiveness of laser mediated panretinal photocoagulation in

The renin-angiotensin system (RAS) plays a vital role in the cardiovascular homeostasis by regulating vascular tone, fluid and electrolyte balance, and in the sympathetic nerve system. Angiotensin II (Ang II), a peptide hormone of RAS, has been known to regulate a variety of hemodynamic physiological responses, including fluid homeostasis, renal function, and contraction of vascular smooth muscle [10]. In addition, Ang II is capable of inducing a mul‐ titude of non-hemodynamic effects, such as the induction of reactive oxygen species (ROS), cytokines, and the stimulation of collagen synthesis [11-14]. Most of the pathophysiological actions of Ang II are mediated via activation of Ang II type 1 receptors (AT1R), G protein– coupled receptors (GPCRs) that couple to many signaling molecules, including small G pro‐ teins, phospholipases, mitogen-activated protein (MAP) kinases, phosphatases, tyrosine kin‐ ases, NADPH oxidase, and transcription factors to stimulate vascular smooth muscle cell growth, inflammation, and fibrosis [11, 15, 16]. Dysregulation of RAS has been implicated in a number of major cardiovascular and metabolic diseases, including endothelial dysfunc‐ tion, atherosclerosis, hypertension, renal disease, diabetic complications, stroke, myocardial infarction and congestive heart failure [17, 18]. RAS blockade produces beneficial cardiovas‐

Recent discoveries have revealed that the RAS hormonal signaling cascade is more com‐ plex than initially conceived with multiple enzymes, effector molecules, and receptors that coordinately regulate the effects of the RAS. Recent studies have identified additional peptides with important physiological and pathological roles, new enzymatic cascades that generate these peptides and more receptors and signaling pathways that mediate

Discovery of angiotensin-converting enzyme 2 (ACE2) has resulted in the establishment of a novel axis of the RAS involving ACE2/Ang-(1-7)/Mas [24-27]. ACE2, like ACE, is a zinc-met‐ allopeptidase, exhibiting approximately 42% amino acid identity with ACE in its catalytic domain. However, unlike somatic ACE, ACE2 only contains a single catalytic site and func‐

rhage or from tractional retinal detachment [8, 9].

468 Gene Therapy - Tools and Potential Applications

preventing vision loss in the late stages of DR.

cular and renal effects in numerous clinical trials [19-21].

**1.3. Recent advances in RAS research**

their function [22, 23].

**1.2. RAS and diabetic complications**

**Figure 1.** Schematic diagram depicting the key components of the Renin Angiotensin System. Angiotensinogen is cleaved by renin to form angiotensin I (Ang I). Angiotensin converting enzyme (ACE) converts Ang I into Angiotensin II (Ang II) the main effector peptide of the RAS. Ang II elicits is cellular effects by activating the main receptor, Angioten‐ sin II receptor 1 (AT1R), as well as other receptors (not shown). Angiotensin II-converting enzyme 2 (ACE2), a recently discovered component of RAS, cleaves Ang II to form Angiotensin (1-7) (Ang 1-7), which activate Mas receptor to pro‐ duce counteracting effects mediated by Ang II. All these components are expressed locally in various cell types in the eye, regulating metabolism, cell survival, and other local neuronal-vascular and immune-modulating functions in the retina.

This vasoprotective axis of RAS counteracts the traditional proliferative, fibrotic, proinflam‐ matory and hypertrophic effects of the ACE/Ang II/AT1R axis of the RAS [24]. The impor‐ tance of the vasodeleterious axis of the RAS [ACE/angiotensin II (Ang II)/ AT1R] in cardiovascular disease, as well as in diabetes and diabetic complications, is well established since ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are leading therapeu‐ tic strategies [20, 33-35]. However, the impact of the vasoprotective axis of the RAS remains poorly understood [24, 36-38]. The concept that shifting the balance of the RAS towards the vasodilatory axis by activation of ACE2 or its product, Ang-(1-7) is beneficial has been sup‐ ported by many studies in cardiac, pulmonary, and vascular fibrosis [24, 39-43]. Indeed, ACE2/Ang-(1-7) activation is now considered to be a critical part of the beneficial actions of ACEi and ARB drugs [24, 36].

#### **1.4. Tissue RAS in end-organ damage**

The classical (endocrine) RAS has been traditionally regarded as systemic hormonal sys‐ tem. Ang II is formed from liver-synthesized angiotensinogen via a series of proteolytic cleavage events. Circulating Ang II activates AT1 and AT2 receptors in various tissues, such as the brain, adrenal and vascular tissues to modulate cardiovascular and hydro‐ mineral homeostasis.

Hyperglycemia has been shown to directly stimulate angiotensin gene expression via the hex‐ ominase pathway, thus contributing to increased Ang II synthesis [62]. Elevated levels of re‐ nin, prorenin, and Ang II have been found in patients with DR. In fact, ACE inhibitors and angiotensin receptor blockers (ARBs) have been shown to improve diabetes-induced vascular, neuronal, and glial dysfunction [61, 63-66]. Recent clinical studies have also clearly demon‐ strated the beneficial effects of RAS inhibition in both type 1 and type 2 diabetic patients with retinopathy [67-71]. Despite these positive outcomes, RAS blockers are not completely retino‐ protective and retinopathy still progresses to more advanced stages. This could be attributed to the existence of local Ang II formation and that current therapeutic agents are unable to cross the blood-retina barrier (BRB) in a concentration sufficient to influence the local RAS in the eye. In addition, increasing evidence suggests that Ang II can be generated via multiple pathways, many of which may not be blocked by classic inhibitors of ACE [72-75]. Furthermore, addition‐ al components of RAS that contribute to end-organ damage, such as receptors for renin and prorenin (PRR), have been recently identified [76]. Activation of prorenin/PRR signaling path‐

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

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Ang II may contribute to development and progression of DR by several mechanisms. First, Ang II has been shown to increase VEGF expression directly via activation of AT1R signaling and indirectly by PCK activation [77] to enhance the role of VEGF induced vascular permeabil‐ ity and angiogenesis. Treatment with ACE inhibitors reduces vitreous levels of VEGF and at‐ tenuates VEGF-mediated BRB breakdown [78, 79]. Second, Ang II, mediated via AT1R, also contributes to diabetes-induced retinal inflammation by activation of nuclear factor-κβ signal‐ ing pathway within retinal endothelial cells [80, 81] leading to the release of inflammatory cy‐ tokines which perpetuates the inflammatory cycle. Pro-inflammatory cytokines, chemokines and other inflammatory mediators play an important role in the pathogenesis of DR [82, 83]. These lead to persistent low-grade inflammation, the adhesion of leukocytes to the retinal vas‐ culature (leukostasis), breakdown of BRB and neovascularization with subsequent sub-retinal fibrosis or disciform scarring [84-88]. Third, Ang II may contribute to increased oxidative stress in diabetic retina. Ang II induces reactive oxygen species (ROS) production by activation of NADPH oxidases [89], which has been implicated in diabetic complications [90, 91]. Ang II al‐ so induces mitochondrial ROS production, which further stimulate of NADPH oxidases lead‐

Fourth, Ang II may also contribute to neuronal dysfunction induced by diabetes [94]. Recep‐ tors for Ang II are also expressed in the inner retinal neurons (Table 1). Ang II induced AT1R signaling may cause neuronal dysfunction by reducing the synaptophysin protein in

The discovery of ACE2- mediated degradation of Ang II into the protective peptide Ang 1-7 thereby negatively regulating the classic RAS, has instigated stimulated interest regarding the potential of ACE2 as a therapeutic target [88, 89], and strategies aimed at enhancing

**3. Protective role of the ACE2/Ang1-7-Mas axis of RAS in diabetic**

way can initiate the RAS cascade independent of Ang II [76].

ing to vicious cycle and contributing tissue damage [92, 93].

the synaptic vesicles [94].

**complications**

However, most components of RAS have also been identified in essentially every organ in‐ cluding kidney, heart, liver, brain, adipose tissue, reproductive tissue, hematopoietic tissue, immune cells and eye, and increasing evidence supports the existence of tissue- specific RAS that exerts diverse physiological effects locally and independently of circulating Ang II [44-46]. These tissue- specific paracrine, intracrine andautocrine actions of RAS may contrib‐ ute to end-organ damage in many pathological conditions including diabetic complications and maybe the basis for the reported limited beneficial effects of RAS blockade.

#### **2. Ocular RAS in pathogenesis of diabetic retinopathy**

Increasing evidence continues to implicate the involvement of the local renin-angiotensinsystem (RAS) in retinal vascular dysfunctions. Various components of RAS have been de‐ tected in the different cell types of the eye (Table 1).


**Table 1.** All components of RAS are expressed locally in the eye.

Hyperglycemia has been shown to directly stimulate angiotensin gene expression via the hex‐ ominase pathway, thus contributing to increased Ang II synthesis [62]. Elevated levels of re‐ nin, prorenin, and Ang II have been found in patients with DR. In fact, ACE inhibitors and angiotensin receptor blockers (ARBs) have been shown to improve diabetes-induced vascular, neuronal, and glial dysfunction [61, 63-66]. Recent clinical studies have also clearly demon‐ strated the beneficial effects of RAS inhibition in both type 1 and type 2 diabetic patients with retinopathy [67-71]. Despite these positive outcomes, RAS blockers are not completely retino‐ protective and retinopathy still progresses to more advanced stages. This could be attributed to the existence of local Ang II formation and that current therapeutic agents are unable to cross the blood-retina barrier (BRB) in a concentration sufficient to influence the local RAS in the eye. In addition, increasing evidence suggests that Ang II can be generated via multiple pathways, many of which may not be blocked by classic inhibitors of ACE [72-75]. Furthermore, addition‐ al components of RAS that contribute to end-organ damage, such as receptors for renin and prorenin (PRR), have been recently identified [76]. Activation of prorenin/PRR signaling path‐ way can initiate the RAS cascade independent of Ang II [76].

**1.4. Tissue RAS in end-organ damage**

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mineral homeostasis.

The classical (endocrine) RAS has been traditionally regarded as systemic hormonal sys‐ tem. Ang II is formed from liver-synthesized angiotensinogen via a series of proteolytic cleavage events. Circulating Ang II activates AT1 and AT2 receptors in various tissues, such as the brain, adrenal and vascular tissues to modulate cardiovascular and hydro‐

However, most components of RAS have also been identified in essentially every organ in‐ cluding kidney, heart, liver, brain, adipose tissue, reproductive tissue, hematopoietic tissue, immune cells and eye, and increasing evidence supports the existence of tissue- specific RAS that exerts diverse physiological effects locally and independently of circulating Ang II [44-46]. These tissue- specific paracrine, intracrine andautocrine actions of RAS may contrib‐ ute to end-organ damage in many pathological conditions including diabetic complications

Increasing evidence continues to implicate the involvement of the local renin-angiotensinsystem (RAS) in retinal vascular dysfunctions. Various components of RAS have been de‐

**RAS components Retinal Localization Reference** Angiotensinogen Retinal microvasculature, RGCs, RPE [47, 48] Angiotensin I Aqueous, vitreous, and subretinal fluid [49]

photoreceptors

Renin Muller cells and vitreous fluid [52, 53] Renin receptor Retinal microvasculature, microglia, astrocytes, RGCs, RPE [54-58]

ACE Muller cells, RGCs, retinal endothelial cells, photoreceptors, and vitreous [51, 59-61] ACE2 Retina [50] AT1R Muller cells, retinal blood vessels, photoreceptors and RGCs [50, 51] AT2R Muller cells, nuclei of some inner, nuclear layer neurons, and ganglion cells [50] Mas receptor RGCs, retinal microvasculature, microglia, subset of astrocytes unpublished results

Angiotensin 1-7 Muller cells [50]

[49-51]

Angiotensin II Aqueous, vitreous, and subretinal fluid, RGCs, retinal endothelial cells and

and maybe the basis for the reported limited beneficial effects of RAS blockade.

**2. Ocular RAS in pathogenesis of diabetic retinopathy**

tected in the different cell types of the eye (Table 1).

GC: retinal ganglion cells; RPE: retinal pigment epithelium.

**Table 1.** All components of RAS are expressed locally in the eye.

Ang II may contribute to development and progression of DR by several mechanisms. First, Ang II has been shown to increase VEGF expression directly via activation of AT1R signaling and indirectly by PCK activation [77] to enhance the role of VEGF induced vascular permeabil‐ ity and angiogenesis. Treatment with ACE inhibitors reduces vitreous levels of VEGF and at‐ tenuates VEGF-mediated BRB breakdown [78, 79]. Second, Ang II, mediated via AT1R, also contributes to diabetes-induced retinal inflammation by activation of nuclear factor-κβ signal‐ ing pathway within retinal endothelial cells [80, 81] leading to the release of inflammatory cy‐ tokines which perpetuates the inflammatory cycle. Pro-inflammatory cytokines, chemokines and other inflammatory mediators play an important role in the pathogenesis of DR [82, 83]. These lead to persistent low-grade inflammation, the adhesion of leukocytes to the retinal vas‐ culature (leukostasis), breakdown of BRB and neovascularization with subsequent sub-retinal fibrosis or disciform scarring [84-88]. Third, Ang II may contribute to increased oxidative stress in diabetic retina. Ang II induces reactive oxygen species (ROS) production by activation of NADPH oxidases [89], which has been implicated in diabetic complications [90, 91]. Ang II al‐ so induces mitochondrial ROS production, which further stimulate of NADPH oxidases lead‐ ing to vicious cycle and contributing tissue damage [92, 93].

Fourth, Ang II may also contribute to neuronal dysfunction induced by diabetes [94]. Recep‐ tors for Ang II are also expressed in the inner retinal neurons (Table 1). Ang II induced AT1R signaling may cause neuronal dysfunction by reducing the synaptophysin protein in the synaptic vesicles [94].

#### **3. Protective role of the ACE2/Ang1-7-Mas axis of RAS in diabetic complications**

The discovery of ACE2- mediated degradation of Ang II into the protective peptide Ang 1-7 thereby negatively regulating the classic RAS, has instigated stimulated interest regarding the potential of ACE2 as a therapeutic target [88, 89], and strategies aimed at enhancing ACE2 action may have important therapeutic potential for cardiovascular disorders as well as for diabetic complications [40, 95-99]. Ang (1-7) has been shown to prevent diabetes-in‐ duced cardiovascular dysfunction [100] and nephropathy [101]. The protective effect of Ang 1-7 signaling is at least in part mediated by direct inhibition of diabetes-induced ROS pro‐ duction due to elevated NADPH oxidase activity [101, 102] and reduction in PPAR-gamma and catalase activities [102]. Adenovirus mediated gene delivery of human ACE2 in pan‐ creas improved fasting blood glucose, beta-cell dysfunction and apoptosis occurring in type 2 diabetes mouse model [103]. The importance of ACE2 as a negative regulator of RAS in diabetic complications is supported by the facts that ACE2 deficiency exacerbates diabetic complications [104, 105] and enhancing ACE2 action counteracts the deleterious effects of Ang II and produces protective effects [96-99, 106].

in samples isolated from un-transfected cells, or cells transfected with the control plasmid ex‐ pressing only the cytoplasmic GFP protein (data not shown). Intravitreal administration of AAV-Ang-(1-7) resulted in a robust transduction of retinal cells primarily within the inner reti‐ nal layer (Figure 3C-F). This was associated with an increase in both cellular and secreted Ang- (1-7) (Figure 3G-H). Similarly, ACE2 protein level was increased in the retina following

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**Figure 2.** Real-time RT-PCR analysis of retinal mRNA for renin-angiotensin system genes. Values represent fold differ‐ ence compared to age matched non-diabetic retinal samples for each gene at each time point (14 day and 1 month after induced diabetes). DM: diabetic. NDM: non-diabetic. At least 4 eyes were analyzed at each time point. \*p<0.01

(versus NDM group). (From [108] with permission of Mol. Therapy).

transduction with AAV-ACE2 (Figure 3G).

#### **3.1. Diabetes induced changes in the expression of the retinal RAS genes in the mouse retina during the progression of diabetes**

We have previously shown that diabetes induced by STZ treatment in eNOS-/- mice results in more severe, accelerated retinopathy than diabetes in untreated eNOS+/+ animals [107]. Thus it became critical to compare retinal mRNA levels of the RAS genes in control and dia‐ betic animals during the progression of diabetes. We observed significant (3-10 fold) increas‐ es in the mRNA levels of the vasodeleterious axis of the RAS (angiotensinogen, renin, pro/ renin receptor, ACE and AT1 receptor subtypes) following STZ treatment (Figure 2) [108]. In contrast, there was ~ 30% reduction in ACE2 mRNA following an initial stimulatory re‐ sponse. As a result the ACE/ACE2 mRNA ratio was increased by 10-fold, while AT1R/Mas ratio was increased by 3-fold following one month of diabetes (Figure 2). These observations were our initial indication that DR is associated with a shifting balance of the retinal RAS towards vasodeleterious axis.

#### **3.2. Enhancing ACE2/Ang1-7-Mas axis by AAV-mediated gene delivery**

#### *3.2.1. Characterization of AAV vectors expressing ACE2 and Ang-(1-7)*

AAV vector expressing the secreted form of human ACE2 was constructed under the control of the chicken-beta-actin (CBA) promoter (Figure 3A). This secreted form of ACE2 has been pre‐ viously characterized and shown to be active enzymatically [109]. Since Ang-(1-7) peptide con‐ tains only 7 amino acids and small peptides are usually difficult to express in mammalian cells, we designed an expression construct in which the Ang-(1-7) peptide is expressed as part of the secreted fusion GFP protein, and is subsequently cleaved upon secretion into the active pep‐ tide. Expression of the fusion sGFP-FC-Ang-(1-7) is under the control of the CBA promoter in the AAV vector (Figure 3A) and was confirmed by tranfecting HEK293 cells using this plas‐ mid DNA (Figure 3B). To ensure that the fusion protein was indeed secreted, proteins isolated from the culture supernatants as well as cell lysates from transfected, sham-transfected or un‐ transfected cells were analysized by western blotting (Figure 3B). Mass spectrometry analysis of Ang (1-7) peptide in supernatant samples of HEK293 cells transfected with the sGFP-FC-Ang-(1-7) plasmid DNA was also performed. The Ang-(1-7) peptide is detectable in superna‐ tant isolated from cells transfected with sGFP-FC-Ang-(1-7) plasmid DNA, but not detectable in samples isolated from un-transfected cells, or cells transfected with the control plasmid ex‐ pressing only the cytoplasmic GFP protein (data not shown). Intravitreal administration of AAV-Ang-(1-7) resulted in a robust transduction of retinal cells primarily within the inner reti‐ nal layer (Figure 3C-F). This was associated with an increase in both cellular and secreted Ang- (1-7) (Figure 3G-H). Similarly, ACE2 protein level was increased in the retina following transduction with AAV-ACE2 (Figure 3G).

ACE2 action may have important therapeutic potential for cardiovascular disorders as well as for diabetic complications [40, 95-99]. Ang (1-7) has been shown to prevent diabetes-in‐ duced cardiovascular dysfunction [100] and nephropathy [101]. The protective effect of Ang 1-7 signaling is at least in part mediated by direct inhibition of diabetes-induced ROS pro‐ duction due to elevated NADPH oxidase activity [101, 102] and reduction in PPAR-gamma and catalase activities [102]. Adenovirus mediated gene delivery of human ACE2 in pan‐ creas improved fasting blood glucose, beta-cell dysfunction and apoptosis occurring in type 2 diabetes mouse model [103]. The importance of ACE2 as a negative regulator of RAS in diabetic complications is supported by the facts that ACE2 deficiency exacerbates diabetic complications [104, 105] and enhancing ACE2 action counteracts the deleterious effects of

**3.1. Diabetes induced changes in the expression of the retinal RAS genes in the mouse**

**3.2. Enhancing ACE2/Ang1-7-Mas axis by AAV-mediated gene delivery**

*3.2.1. Characterization of AAV vectors expressing ACE2 and Ang-(1-7)*

We have previously shown that diabetes induced by STZ treatment in eNOS-/- mice results in more severe, accelerated retinopathy than diabetes in untreated eNOS+/+ animals [107]. Thus it became critical to compare retinal mRNA levels of the RAS genes in control and dia‐ betic animals during the progression of diabetes. We observed significant (3-10 fold) increas‐ es in the mRNA levels of the vasodeleterious axis of the RAS (angiotensinogen, renin, pro/ renin receptor, ACE and AT1 receptor subtypes) following STZ treatment (Figure 2) [108]. In contrast, there was ~ 30% reduction in ACE2 mRNA following an initial stimulatory re‐ sponse. As a result the ACE/ACE2 mRNA ratio was increased by 10-fold, while AT1R/Mas ratio was increased by 3-fold following one month of diabetes (Figure 2). These observations were our initial indication that DR is associated with a shifting balance of the retinal RAS

AAV vector expressing the secreted form of human ACE2 was constructed under the control of the chicken-beta-actin (CBA) promoter (Figure 3A). This secreted form of ACE2 has been pre‐ viously characterized and shown to be active enzymatically [109]. Since Ang-(1-7) peptide con‐ tains only 7 amino acids and small peptides are usually difficult to express in mammalian cells, we designed an expression construct in which the Ang-(1-7) peptide is expressed as part of the secreted fusion GFP protein, and is subsequently cleaved upon secretion into the active pep‐ tide. Expression of the fusion sGFP-FC-Ang-(1-7) is under the control of the CBA promoter in the AAV vector (Figure 3A) and was confirmed by tranfecting HEK293 cells using this plas‐ mid DNA (Figure 3B). To ensure that the fusion protein was indeed secreted, proteins isolated from the culture supernatants as well as cell lysates from transfected, sham-transfected or un‐ transfected cells were analysized by western blotting (Figure 3B). Mass spectrometry analysis of Ang (1-7) peptide in supernatant samples of HEK293 cells transfected with the sGFP-FC-Ang-(1-7) plasmid DNA was also performed. The Ang-(1-7) peptide is detectable in superna‐ tant isolated from cells transfected with sGFP-FC-Ang-(1-7) plasmid DNA, but not detectable

Ang II and produces protective effects [96-99, 106].

**retina during the progression of diabetes**

472 Gene Therapy - Tools and Potential Applications

towards vasodeleterious axis.

**Figure 2.** Real-time RT-PCR analysis of retinal mRNA for renin-angiotensin system genes. Values represent fold differ‐ ence compared to age matched non-diabetic retinal samples for each gene at each time point (14 day and 1 month after induced diabetes). DM: diabetic. NDM: non-diabetic. At least 4 eyes were analyzed at each time point. \*p<0.01 (versus NDM group). (From [108] with permission of Mol. Therapy).

and AAV2-sGFP-FC-Ang-(1-7) (bottom) compared to a molecular weight standard (right lane). H: Ang-(1-7) peptide levels in the retina with and without AAV-sGFP-FC-Ang-(1-7) injection. There was more than a 10-fold increase in Ang- (1-7) peptide level detected by using an Ang-(1-7) specific EIA kit (Bachem, San Carlos, CA) in retinas receiving injec‐ tion of AAV-sGFP-FC Ang-(1-7). PR: photoreceptor; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner

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*3.2.2. Ocular gene delivery of ACE2/Ang-(1-7) via the AAV vector in the retina results increased*

Diabetes induced more than a 5-fold increase in ACE activity in the retinas of eNOS-/- mice, whereas ACE2 activity was relatively unchanged (Figure 4A). AAV2-ACE2 injected retinas show more than a two-fold increase in ACE2 enzymatic activity (Figure 4A) and this is asso‐ ciated with a reduced level of Ang II and increased Ang-(1-7) peptide level (Figure 4B), but has only a marginal effect on ACE activity (Figure 4A). Injection of AAV2-Ang-(1-7) has no

We also determined Ang II and Ang-(1-7) peptide levels using a commercial EIA kit (Bach‐ em, San Carlos, CA). STZ induced diabetes resulted in more than a 2-fold increase in Ang II levels whereas the Ang-(1-7) level was unchanged in the retinas of eNOS-/- mice (Figure 4B). This increase of Ang II was completely normalized in retinas injected with AAV-ACE2 but

**Figure 4.** ACE, ACE2 activities and angiotensin peptide levels in the mouse retina.A: ACE and ACE2 enzymatic activities and ACE/ACE2 ratios in non-diabetic (NDM), 1 month diabetic (1M DM), and 1 month diabeticeNOS-/- mouse retinas treated with AAV-ACE2/Ang-(1-7). Values are expressed as fold differences compared with age-matched non-diabetic group. \*p<0.01 (versus untreated DM group, N=6/group). B: Ang II and Ang-(1-7) peptide levels in non-diabetic (NDM), 1 month diabetic (1M DM), and 1 month diabetic eNOS-/- retinas treated with AAV-ACE2/Ang-(1-7), meas‐ ured by ELISA using a commercial kit. \*p<0.01 (versus untreated DM group). Values represent fold difference com‐ pared with age-matched non-diabetic group. Three retinas were pooled for each measurement, each measurement was done in duplicates, and three separate pools were averaged for each group. (From [108] with permission of Mol.

plexiform layer; RGC: retinal ganglion cells. (From [108] with permission of Mol. Therapy).

effect on ACE2 activity, but significantly decreased ACE activity (Figure 4A).

was unchanged in retinas injected with AAV-Ang-(1-7) vector (Figure 4B).

*ACE2 activities and Ang-(1-7) peptide levels*

Therapy).

**Figure 3.** Construction and characterization of AAV vectors expressing ACE2 and Ang-(1-7).A: Maps of the AAV vector expressing the human ACE2 gene (hACE2) and the AAV vector expressing Ang-(1-7) gene. The Ang-(1-7) peptide is expressed as part of fusion protein, and cleaved in vivo upon secretion at the furin cleavage (FC) site. ITR: inverted terminal repeat; CBA: CMV- chicken-β-actin promoter. A control vector contains the coding region for the secreted GFP without the Ang-(1-7) peptide coding sequence. B: Expression and cleavage of the fusion protein. In cultured HEK293 cells transfected with the plasmid sGFP-FC-Ang-(1-7), or infected with AAV-sGFP-FC-Ang-(1-7), there was ro‐ bust expression of GFP as expected. Proteins isolated from cell lysates contained a single protein band with molecular weight ~30 kd, as predicted for the precursor (fusion protein), but culture supernatants contained two protein bands (30kd and a 27kd), indicating that the secreted protein is cleaved at the furin cleavage site as predicted. C-F: Transduc‐ tion of mouse retina with AAV vector expressing sGFP-FC-Ang-(1-7) and hACE2. A single intravitreal injection of 1μl AAV vector (109 vg/eye) resulted in efficient transduction of inner retinal cells, primarily retinal ganglion cells. C. Low magnification of cross section of a mouse eye that received AAV2-sGFP-FC-Ang-(1-7) injection. D. Higher magnifica‐ tion of the same eye. E. A retinal whole mount showing GFP expression. F. Higher magnification of the same retinal whole mount. G: Western blot of proteins isolated from an uninjected eye and an eye injected with AAV2-ACE2 (top)

and AAV2-sGFP-FC-Ang-(1-7) (bottom) compared to a molecular weight standard (right lane). H: Ang-(1-7) peptide levels in the retina with and without AAV-sGFP-FC-Ang-(1-7) injection. There was more than a 10-fold increase in Ang- (1-7) peptide level detected by using an Ang-(1-7) specific EIA kit (Bachem, San Carlos, CA) in retinas receiving injec‐ tion of AAV-sGFP-FC Ang-(1-7). PR: photoreceptor; OPL: outer plexiform layer; INL: inner nuclear layer; IPL: inner plexiform layer; RGC: retinal ganglion cells. (From [108] with permission of Mol. Therapy).

#### *3.2.2. Ocular gene delivery of ACE2/Ang-(1-7) via the AAV vector in the retina results increased ACE2 activities and Ang-(1-7) peptide levels*

Diabetes induced more than a 5-fold increase in ACE activity in the retinas of eNOS-/- mice, whereas ACE2 activity was relatively unchanged (Figure 4A). AAV2-ACE2 injected retinas show more than a two-fold increase in ACE2 enzymatic activity (Figure 4A) and this is asso‐ ciated with a reduced level of Ang II and increased Ang-(1-7) peptide level (Figure 4B), but has only a marginal effect on ACE activity (Figure 4A). Injection of AAV2-Ang-(1-7) has no effect on ACE2 activity, but significantly decreased ACE activity (Figure 4A).

We also determined Ang II and Ang-(1-7) peptide levels using a commercial EIA kit (Bach‐ em, San Carlos, CA). STZ induced diabetes resulted in more than a 2-fold increase in Ang II levels whereas the Ang-(1-7) level was unchanged in the retinas of eNOS-/- mice (Figure 4B). This increase of Ang II was completely normalized in retinas injected with AAV-ACE2 but was unchanged in retinas injected with AAV-Ang-(1-7) vector (Figure 4B).

**Figure 3.** Construction and characterization of AAV vectors expressing ACE2 and Ang-(1-7).A: Maps of the AAV vector expressing the human ACE2 gene (hACE2) and the AAV vector expressing Ang-(1-7) gene. The Ang-(1-7) peptide is expressed as part of fusion protein, and cleaved in vivo upon secretion at the furin cleavage (FC) site. ITR: inverted terminal repeat; CBA: CMV- chicken-β-actin promoter. A control vector contains the coding region for the secreted GFP without the Ang-(1-7) peptide coding sequence. B: Expression and cleavage of the fusion protein. In cultured HEK293 cells transfected with the plasmid sGFP-FC-Ang-(1-7), or infected with AAV-sGFP-FC-Ang-(1-7), there was ro‐ bust expression of GFP as expected. Proteins isolated from cell lysates contained a single protein band with molecular weight ~30 kd, as predicted for the precursor (fusion protein), but culture supernatants contained two protein bands (30kd and a 27kd), indicating that the secreted protein is cleaved at the furin cleavage site as predicted. C-F: Transduc‐ tion of mouse retina with AAV vector expressing sGFP-FC-Ang-(1-7) and hACE2. A single intravitreal injection of 1μl AAV vector (109 vg/eye) resulted in efficient transduction of inner retinal cells, primarily retinal ganglion cells. C. Low magnification of cross section of a mouse eye that received AAV2-sGFP-FC-Ang-(1-7) injection. D. Higher magnifica‐ tion of the same eye. E. A retinal whole mount showing GFP expression. F. Higher magnification of the same retinal whole mount. G: Western blot of proteins isolated from an uninjected eye and an eye injected with AAV2-ACE2 (top)

474 Gene Therapy - Tools and Potential Applications

**Figure 4.** ACE, ACE2 activities and angiotensin peptide levels in the mouse retina.A: ACE and ACE2 enzymatic activities and ACE/ACE2 ratios in non-diabetic (NDM), 1 month diabetic (1M DM), and 1 month diabeticeNOS-/- mouse retinas treated with AAV-ACE2/Ang-(1-7). Values are expressed as fold differences compared with age-matched non-diabetic group. \*p<0.01 (versus untreated DM group, N=6/group). B: Ang II and Ang-(1-7) peptide levels in non-diabetic (NDM), 1 month diabetic (1M DM), and 1 month diabetic eNOS-/- retinas treated with AAV-ACE2/Ang-(1-7), meas‐ ured by ELISA using a commercial kit. \*p<0.01 (versus untreated DM group). Values represent fold difference com‐ pared with age-matched non-diabetic group. Three retinas were pooled for each measurement, each measurement was done in duplicates, and three separate pools were averaged for each group. (From [108] with permission of Mol. Therapy).

#### **3.3. Protective role of ACE2/Ang (1-7) AAV gene delivery in mouse model of DR**

#### *3.3.1. Enhanced ACE2/Ang1-7 expression in the retina reduced diabetes-induced retinal vascular leakage*

We investigated if elevated expression of retinal ACE2 or Ang-(1-7) would overcome the vasodeleterious effect of the ACE/AT1R axis and prevent the development of diabetes-in‐ duced retinopathy. Effects of increased ACE2 and Ang-(1-7) expression on retinal vascular permeability were evaluated by FITC-labeled albumin extravasations and quantified by measuring its fluorescence intensity in serial sections from non-diabetic, untreated, ACE2 treated diabetic eNOS-/- mice and Ang 1-7 treated diabetic eNOS-/- mice. Induction of diabe‐ tes for 2 month in eNOS-/- mice resulted in a 2-fold increase in vascular permeability. This pathophysiology was significantly reduced in diabetic retinas which received ACE2/Ang- (1-7) vector treatments (Figure 5), but not in the retinas receiving control vector containing the coding sequence for secreted GFP without Ang-(1-7) or ACE2 (data not shown).

**Figure 6.** Intravitreal administration of ACE2 or Ang-(1-7)-AAV reduces diabetes-induced ocular inflammation. A. Quan‐ tification of CD45positive inflammatory cells in the retinas from untreated non-diabetic, ACE2 treated and Ang-(1-7) treated diabetic eNOS-/- mouse retinas at 1 month after induced diabetes or the equivalent age in untreated controls. B. Quantification of CD11b positive inflammatory cells in the retinas from untreated non-diabetic, ACE2 treated and Ang- (1-7) treated diabetic eNOS-/- mouse retinas at 1 month after induced diabetes or the equivalent age in untreated con‐ trols. N=4 for each group. \*p<0.01 (versus untreated DM group). (From [108] with permission of Mol. Therapy).

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**Figure 7.** Evaluation of acellular capillary formation in untreated and AAV-ACE2/Ang-(1-7) treated retinas of diabetic mice. Treatments with ACE2 and Ang 1-7 vectors in the diabetic eNOS-/- mouse retinas reduced acellular capillaries. A: Representative images of trypsin-digested retinal vascular preparations from untreated non-diabetic eNOS-/-, ACE2 and Ang-(1-7) treated diabetic eNOS-/- mouse retinas (2 months after induced diabetes or the equivalent age in un‐ treated controls. Arrows indicate the acellular capillaries. B. Quantitative measurements of acellular capillaries. The val‐ ues on Y-axis represent the number of acellular capillaries per mm2 retina. NDM: non-diabetes; DM: diabetes. N=6.

\*p<0.01 (versus untreated DM group). (From [108] with permission of Mol. Therapy).

**Figure 5.** Effects of ocular treatments with ACE2 and Ang-(1-7)-AAV2 on retinal vascular permeability in diabetic eNOS-/- mice. Retinal vascular permeability was evaluated by FITC-labeled albumin extravasations and quantified by measuring the fluorescence intensity in serial sections from eNOS-/- mice at 1 month after induced diabetes. Data are presented as mean ± SD from 6 eyes in each group. \*p<0.01 (versus untreated DM group). NDM: non-diabetes; DM: diabetes. (From [108] with permission of Mol. Therapy).

#### *3.3.2. Increased expression of ACE2 and Ang1-7 resulted in reduced ocular inflammation in diabetic retina*

Diabetes-induced ocular inflammation, as demonstrated by increased infiltrating CD45 posi‐ tive macrophages and activation of CD11b positive microglial cells, was significantly re‐ duced in eyes treated with ACE2 and Ang-(1-7) expression vectors (Figure 6).

#### *3.3.3. Increased ACE2/Ang1-7 expression reduced the number of acellular capillaries in the diabetic retina*

Induction of diabetes for 2 month in eNOS-/- mice resulted in a >10-fold increase in the for‐ mation of acellular capillaries that was significantly reduced in diabetic retinas which re‐ ceived ACE2/Ang-(1-7) vector treatments (Figure 7). Furthermore, increasing the level of ACE2 also prevented basement membrane thickening in diabetic eNOS-/- retina (Figure 8).

**3.3. Protective role of ACE2/Ang (1-7) AAV gene delivery in mouse model of DR**

the coding sequence for secreted GFP without Ang-(1-7) or ACE2 (data not shown).

**Figure 5.** Effects of ocular treatments with ACE2 and Ang-(1-7)-AAV2 on retinal vascular permeability in diabetic eNOS-/- mice. Retinal vascular permeability was evaluated by FITC-labeled albumin extravasations and quantified by measuring the fluorescence intensity in serial sections from eNOS-/- mice at 1 month after induced diabetes. Data are presented as mean ± SD from 6 eyes in each group. \*p<0.01 (versus untreated DM group). NDM: non-diabetes; DM:

*3.3.2. Increased expression of ACE2 and Ang1-7 resulted in reduced ocular inflammation in diabetic*

Diabetes-induced ocular inflammation, as demonstrated by increased infiltrating CD45 posi‐ tive macrophages and activation of CD11b positive microglial cells, was significantly re‐

*3.3.3. Increased ACE2/Ang1-7 expression reduced the number of acellular capillaries in the diabetic*

Induction of diabetes for 2 month in eNOS-/- mice resulted in a >10-fold increase in the for‐ mation of acellular capillaries that was significantly reduced in diabetic retinas which re‐ ceived ACE2/Ang-(1-7) vector treatments (Figure 7). Furthermore, increasing the level of ACE2 also prevented basement membrane thickening in diabetic eNOS-/- retina (Figure 8).

duced in eyes treated with ACE2 and Ang-(1-7) expression vectors (Figure 6).

diabetes. (From [108] with permission of Mol. Therapy).

*leakage*

476 Gene Therapy - Tools and Potential Applications

*retina*

*retina*

*3.3.1. Enhanced ACE2/Ang1-7 expression in the retina reduced diabetes-induced retinal vascular*

We investigated if elevated expression of retinal ACE2 or Ang-(1-7) would overcome the vasodeleterious effect of the ACE/AT1R axis and prevent the development of diabetes-in‐ duced retinopathy. Effects of increased ACE2 and Ang-(1-7) expression on retinal vascular permeability were evaluated by FITC-labeled albumin extravasations and quantified by measuring its fluorescence intensity in serial sections from non-diabetic, untreated, ACE2 treated diabetic eNOS-/- mice and Ang 1-7 treated diabetic eNOS-/- mice. Induction of diabe‐ tes for 2 month in eNOS-/- mice resulted in a 2-fold increase in vascular permeability. This pathophysiology was significantly reduced in diabetic retinas which received ACE2/Ang- (1-7) vector treatments (Figure 5), but not in the retinas receiving control vector containing

**Figure 6.** Intravitreal administration of ACE2 or Ang-(1-7)-AAV reduces diabetes-induced ocular inflammation. A. Quan‐ tification of CD45positive inflammatory cells in the retinas from untreated non-diabetic, ACE2 treated and Ang-(1-7) treated diabetic eNOS-/- mouse retinas at 1 month after induced diabetes or the equivalent age in untreated controls. B. Quantification of CD11b positive inflammatory cells in the retinas from untreated non-diabetic, ACE2 treated and Ang- (1-7) treated diabetic eNOS-/- mouse retinas at 1 month after induced diabetes or the equivalent age in untreated con‐ trols. N=4 for each group. \*p<0.01 (versus untreated DM group). (From [108] with permission of Mol. Therapy).

**Figure 7.** Evaluation of acellular capillary formation in untreated and AAV-ACE2/Ang-(1-7) treated retinas of diabetic mice. Treatments with ACE2 and Ang 1-7 vectors in the diabetic eNOS-/- mouse retinas reduced acellular capillaries. A: Representative images of trypsin-digested retinal vascular preparations from untreated non-diabetic eNOS-/-, ACE2 and Ang-(1-7) treated diabetic eNOS-/- mouse retinas (2 months after induced diabetes or the equivalent age in un‐ treated controls. Arrows indicate the acellular capillaries. B. Quantitative measurements of acellular capillaries. The val‐ ues on Y-axis represent the number of acellular capillaries per mm2 retina. NDM: non-diabetes; DM: diabetes. N=6. \*p<0.01 (versus untreated DM group). (From [108] with permission of Mol. Therapy).

**Figure 8.** Transmission electron micrographs of retinal capillaries from a untreated 2 month diabetic eNOS-/- mouse eye (A), and an eye that received AAV-ACE2 treatment 2 weeks before STZ-induction of diabetes (B). CL: capillary lu‐ men; En: endothelial cell; P: pericyte; \* indicates the capillary basement membrane. Scale bar = 500nm. We have previ‐ ously shown that the basement membranes of retinal capillaries from the diabetic eNOS-/- animals at two months after STZ induction of diabetes was significantly thicker than those from age-matched, non-diabetic animals [107]. The thickening of the basement membrane was prevented in the AAV-ACE2 treated eyes (73.81+17nm, versus 95.72+20 nm in untreated DM eye).

**Figure 9.** Evaluation of acellular capillary formation in untreated and ACE2/Ang-(1-7) AAV2 vector treated retinas of diabetic SD rats. (A) Representative images of trypsin-digested retinal vascular preparations from non-diabetic SD rat, untreated, ACE2 and Ang-(1-7) treated diabetic SD rat retinas (14 months after induced diabetes). (B) Quantitative measurements of acellular capillaries. Values on Y-axis represent the number of acellular capillaries per mm2of retina. NDM: non-diabetes; DM: diabetes. N=6. \*p<0.01(versus untreated DM group). (From [108] with permission of Mol.

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

479

**Figure 10.** TBARs levels in eNOS-/- mouse retinas (A) and SD rat retinas (B). Diabetes resulted in increased TBARs levels in both eNOS-/- mouse retinas at1 month of diabetes and SD rat retinas at 4 months of diabetes. These increases were prevented by AAV-ACE2/Ang-(1-7) treatments. NDM: non-diabetes; DM: diabetes. N=6/group. \*p<0.01(vs untreated

Therapy).

DM). (From [108] with permission of Mol. Therapy).

#### **3.4. Protective role of ACE2/Ang (1-7) AAV gene delivery in a rat model of DR**

#### *3.4.1. Increased ACE2/Ang1-7 expression reduced the number of acellular capillaries in the diabetic rat retina*

We also used STZ-induced diabetic SD rats as an additional animal model of diabetes to provide conceptual validation. We observed more than a 5-fold increase in the number of acellular capillaries in STZ-induced diabetic rat retinas at 14 month of diabetes. This increase was almost completely prevented by gene delivery of either ACE2 or Ang-(1-7) (Figure 9).

#### *3.4.2. Increased expression of ACE2/Ang-(1-7) reduces oxidative damage in diabetic retina*

Diabetes and its complications are associated with increased oxidative stress. We assessed oxidative damage measuring the levels of thiobarbituric acid-reactive substances (TBARs, is a marker for oxidative damage [110]) in the retina). Diabetes induced a significant increase in TBARs (Figure 10A) in eNOS-/- mouse retinas (Figure 10A). This increase is completely prevented by AAV-ACE2 or Ang-(1-7) treatment. Similar results were also obtained in SD rat retinas (Figure 10B).

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System http://dx.doi.org/10.5772/52702 479

**Figure 9.** Evaluation of acellular capillary formation in untreated and ACE2/Ang-(1-7) AAV2 vector treated retinas of diabetic SD rats. (A) Representative images of trypsin-digested retinal vascular preparations from non-diabetic SD rat, untreated, ACE2 and Ang-(1-7) treated diabetic SD rat retinas (14 months after induced diabetes). (B) Quantitative measurements of acellular capillaries. Values on Y-axis represent the number of acellular capillaries per mm2of retina. NDM: non-diabetes; DM: diabetes. N=6. \*p<0.01(versus untreated DM group). (From [108] with permission of Mol. Therapy).

**Figure 8.** Transmission electron micrographs of retinal capillaries from a untreated 2 month diabetic eNOS-/- mouse eye (A), and an eye that received AAV-ACE2 treatment 2 weeks before STZ-induction of diabetes (B). CL: capillary lu‐ men; En: endothelial cell; P: pericyte; \* indicates the capillary basement membrane. Scale bar = 500nm. We have previ‐ ously shown that the basement membranes of retinal capillaries from the diabetic eNOS-/- animals at two months after STZ induction of diabetes was significantly thicker than those from age-matched, non-diabetic animals [107]. The thickening of the basement membrane was prevented in the AAV-ACE2 treated eyes (73.81+17nm, versus

*3.4.1. Increased ACE2/Ang1-7 expression reduced the number of acellular capillaries in the diabetic*

We also used STZ-induced diabetic SD rats as an additional animal model of diabetes to provide conceptual validation. We observed more than a 5-fold increase in the number of acellular capillaries in STZ-induced diabetic rat retinas at 14 month of diabetes. This increase was almost completely prevented by gene delivery of either ACE2 or Ang-(1-7) (Figure 9).

Diabetes and its complications are associated with increased oxidative stress. We assessed oxidative damage measuring the levels of thiobarbituric acid-reactive substances (TBARs, is a marker for oxidative damage [110]) in the retina). Diabetes induced a significant increase in TBARs (Figure 10A) in eNOS-/- mouse retinas (Figure 10A). This increase is completely prevented by AAV-ACE2 or Ang-(1-7) treatment. Similar results were also obtained in SD

**3.4. Protective role of ACE2/Ang (1-7) AAV gene delivery in a rat model of DR**

*3.4.2. Increased expression of ACE2/Ang-(1-7) reduces oxidative damage in diabetic retina*

95.72+20 nm in untreated DM eye).

478 Gene Therapy - Tools and Potential Applications

rat retinas (Figure 10B).

*rat retina*

**Figure 10.** TBARs levels in eNOS-/- mouse retinas (A) and SD rat retinas (B). Diabetes resulted in increased TBARs levels in both eNOS-/- mouse retinas at1 month of diabetes and SD rat retinas at 4 months of diabetes. These increases were prevented by AAV-ACE2/Ang-(1-7) treatments. NDM: non-diabetes; DM: diabetes. N=6/group. \*p<0.01(vs untreated DM). (From [108] with permission of Mol. Therapy).

#### **3.5. Possible mechanisms of protective action of ACE2/Ang (1-7) in diabetic retina**

We demonstrate that all the genes within the RAS are expressed in the retina, consistent with various previous reports (reviewed in [111] and references therein), and the expression levels of genes in the vasoconstrictive arm of RAS (renin, ACE, AT1R) are highly elevated in diabetic retinas, whereas there is initial increase in the expression of genes in the vascodila‐ tive axis (ACE2 and MAS) earlier in diabetes that attenuate over time with the progression of diabetes, thus tipping the balance towards more vasoconstrictive, proinflammatory, hy‐ pertrophic effects of RAS mediated by ACE/Ang II/AT1R axis. This is associated with in‐ creased ACE activity and Ang II levels in diabetic retinas, whereas ACE2 activity and Ang- (1-7) levels are not significantly changed, while the mRNA levels for ACE2 and Mas receptor are reduced under these conditions.

show that increased expression of either ACE2 or Ang-(1-7) is protective in both eNOS-/ mouse and rat models of diabetic retinopathy. However the action of ACE2 and Ang-(1-7) may be different. The protective effect of ACE2 may result from reduced Ang II, by catalyz‐ ing its conversion to Ang-(1-7), thus increasing the level of Ang-(1-7), or combination of both. Indeed, in the AAV-ACE2 treated retina diabetes-induced elevation of Ang II is re‐ duced and this is associated with an increased level of Ang-(1-7). On other hand, the fact that increased Ang-(1-7) expressed from AAV vector in the retina is also protective and that the Ang II level remained high in AAV-Ang-(1-7) treated retinas suggest that Ang-(1-7) can produce physiological responses that direct counteract these of Ang II, consistent with well-

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

481

It is interesting to note that ACE2 over-expression resulted in reduced Ang II and increased Ang-(1-7) levels as expected, but has no effect on ACE activity. However, over-expression of Ang-(1-7) had no effect on endogenous ACE2 activity, but significantly reduced ACE activi‐ ty. Paradoxically, despite reduced ACE activity in AAV-Ang-(1-7) treated retinas, Ang II levels remained high. It is possible that other enzymes/pathways may be involved in Ang II formation in addition to ACE. One such candidate is chymase, which has been detected in vascular systems and other tissues including eye [115]. Another candidate is the receptor for prorenin and renin (pro/renin). It has been recently demonstrated that binding of pro/renin to its receptor, pro/renin receptor (PRR), causes its prosegment to unfold, thereby activating prorenin so that it is able to generate angiotensin peptides that stimulate the Ang II-depend‐ ent pathway [76]. Considering the fact that retina contains high level of prorenin, and its lev‐ el is further increased in patients with diabetic retinopathy [52], this pathway likely contributes to increased Ang II level under diabetic conditions. The existence of multiple pathways for Ang II formation at the tissue level may explain the limited beneficial effects of classic RAS blockers, and may also lend support for thtoe notion that enhancing the protec‐ tive axis of RAS (ACE2/Ang-(1-7)/Mas) may represent a more effective strategy for treat‐

AAV vector mediated gene therapy for ocular diseases has been studied in animal models for more than a decade. Reports focusing on retinal therapy include a wide variety of retinal degenerative animal models of corresponding human retinopathies, as well as the therapeu‐ tic effects of AAV-vector mediated expression of neuroprotective, anti-apoptotic, and antiangiogenic agents in the retina [116]. In view of recent clinical trials in which AAV delivered RPE65 gene led to restoration of vision in human patients and other reports on successful trials on treatment of ocular diseases and inherited immune deficiencies (reviewed in [117] and references therein), gene therapy has emerged as promising approach and may become a standard treatment option for a wide range of diseases in the future. In particular, when considering that the diabetic individual experience this serious ocular complication for deca‐ des, a therapeutic strategy that is long-lasting and does not require patient compliance is particularly desirable. Thus, the delivery of ACE2 and/or Ang-(1-7) could serve as a novel gene therapeutic target for DR in combination with existing strategies to control hyperglyce‐

established effects of Ang-(1-7) [114].

mic and insulin resistance states.

ment of diabetic retinopathy and other diabetic complications.

Furthermore, we show that enhanced expression of either ACE2 or Ang-(1-7) via AAV vec‐ tor mediated gene delivery in the retina prevents diabetes-induced retinal vascular permea‐ bility, thickening of basement membrane, retinal inflammation, formation of acellular capillaries, and oxidative damage in both mouse and rat models of diabetic retinopathy. More importantly, these beneficial effects occur in the absence of systemic control of glu‐ cose, blood pressure, which is elevated in eNOS-/- mice [107], and other diabetic complica‐ tions [112], suggesting that local RAS activation plays a significant role of pathogenesis of diabetic retinopathy, and can be modulated locally to restore the balance between the two counter-acting arms by enhancing the ACE2/Ang-(1-7)/MAS axis. These observations pro‐ vide conceptual support that enhancing ACE2/ Ang-(1-7) axis maybe an effective strategy for the treatment of DR.

Although various components of RAS have been detected in retina, our study is the first to examine the expression levels of all known RAS genes during the progression of diabetes in the eNOS-/- mice, which exhibit accelerated retinopathy [107]. We show that increased ex‐ pression of genes in the vasoconstrictive, proinflammatory axis of RAS (ACE, AT1R, renin, renin receptor) occur early, 14 days after STZ-induced diabetes. We have previously shown that increased retinal vascular permeability and gliosis are already detectable at this time point in diabetic eNOS-/- mouse retina, suggesting that local hyperactivity of the deleterious axis (ACE/Ang II/AT1R) may contribute to these pathological changes. We also measured ACE and ACE2 activities in diabetic eNOS-/- mouse retina. In contrast to a previous report which showed that ACE enzyme activity was decreased, whereas ACE2 enzyme activity was increased in diabetic rat retinas [113], we found that ACE activity is highly increased in diabetic retinas, whereas ACE2 activity remains unchanged. This discrepancy may be due to the difference in animal models or the time points at which these assays were performed.

The importance of the vasodeleterious axis of the RAS (ACE/ Ang II/ AT1R) in cardiovascu‐ lar disease, as well as in diabetes and diabetic complications, is well established since ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are leading therapeutic strat‐ egies [20, 33-35]. However, the impact of the vasoprotective axis of the RAS remains poorly understood, particularly in the eye. The concept that shifting the balance of the RAS towards the vasodilatory axis by activation of ACE2 or its product, Ang-(1-7) is beneficial has been supported by many studies in cardiac, pulmonary, and vascular fibrosis [24, 36-38]. We show that increased expression of either ACE2 or Ang-(1-7) is protective in both eNOS-/ mouse and rat models of diabetic retinopathy. However the action of ACE2 and Ang-(1-7) may be different. The protective effect of ACE2 may result from reduced Ang II, by catalyz‐ ing its conversion to Ang-(1-7), thus increasing the level of Ang-(1-7), or combination of both. Indeed, in the AAV-ACE2 treated retina diabetes-induced elevation of Ang II is re‐ duced and this is associated with an increased level of Ang-(1-7). On other hand, the fact that increased Ang-(1-7) expressed from AAV vector in the retina is also protective and that the Ang II level remained high in AAV-Ang-(1-7) treated retinas suggest that Ang-(1-7) can produce physiological responses that direct counteract these of Ang II, consistent with wellestablished effects of Ang-(1-7) [114].

**3.5. Possible mechanisms of protective action of ACE2/Ang (1-7) in diabetic retina**

receptor are reduced under these conditions.

480 Gene Therapy - Tools and Potential Applications

for the treatment of DR.

We demonstrate that all the genes within the RAS are expressed in the retina, consistent with various previous reports (reviewed in [111] and references therein), and the expression levels of genes in the vasoconstrictive arm of RAS (renin, ACE, AT1R) are highly elevated in diabetic retinas, whereas there is initial increase in the expression of genes in the vascodila‐ tive axis (ACE2 and MAS) earlier in diabetes that attenuate over time with the progression of diabetes, thus tipping the balance towards more vasoconstrictive, proinflammatory, hy‐ pertrophic effects of RAS mediated by ACE/Ang II/AT1R axis. This is associated with in‐ creased ACE activity and Ang II levels in diabetic retinas, whereas ACE2 activity and Ang- (1-7) levels are not significantly changed, while the mRNA levels for ACE2 and Mas

Furthermore, we show that enhanced expression of either ACE2 or Ang-(1-7) via AAV vec‐ tor mediated gene delivery in the retina prevents diabetes-induced retinal vascular permea‐ bility, thickening of basement membrane, retinal inflammation, formation of acellular capillaries, and oxidative damage in both mouse and rat models of diabetic retinopathy. More importantly, these beneficial effects occur in the absence of systemic control of glu‐ cose, blood pressure, which is elevated in eNOS-/- mice [107], and other diabetic complica‐ tions [112], suggesting that local RAS activation plays a significant role of pathogenesis of diabetic retinopathy, and can be modulated locally to restore the balance between the two counter-acting arms by enhancing the ACE2/Ang-(1-7)/MAS axis. These observations pro‐ vide conceptual support that enhancing ACE2/ Ang-(1-7) axis maybe an effective strategy

Although various components of RAS have been detected in retina, our study is the first to examine the expression levels of all known RAS genes during the progression of diabetes in the eNOS-/- mice, which exhibit accelerated retinopathy [107]. We show that increased ex‐ pression of genes in the vasoconstrictive, proinflammatory axis of RAS (ACE, AT1R, renin, renin receptor) occur early, 14 days after STZ-induced diabetes. We have previously shown that increased retinal vascular permeability and gliosis are already detectable at this time point in diabetic eNOS-/- mouse retina, suggesting that local hyperactivity of the deleterious axis (ACE/Ang II/AT1R) may contribute to these pathological changes. We also measured ACE and ACE2 activities in diabetic eNOS-/- mouse retina. In contrast to a previous report which showed that ACE enzyme activity was decreased, whereas ACE2 enzyme activity was increased in diabetic rat retinas [113], we found that ACE activity is highly increased in diabetic retinas, whereas ACE2 activity remains unchanged. This discrepancy may be due to the difference in animal models or the time points at which these assays were performed. The importance of the vasodeleterious axis of the RAS (ACE/ Ang II/ AT1R) in cardiovascu‐ lar disease, as well as in diabetes and diabetic complications, is well established since ACE inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are leading therapeutic strat‐ egies [20, 33-35]. However, the impact of the vasoprotective axis of the RAS remains poorly understood, particularly in the eye. The concept that shifting the balance of the RAS towards the vasodilatory axis by activation of ACE2 or its product, Ang-(1-7) is beneficial has been supported by many studies in cardiac, pulmonary, and vascular fibrosis [24, 36-38]. We

It is interesting to note that ACE2 over-expression resulted in reduced Ang II and increased Ang-(1-7) levels as expected, but has no effect on ACE activity. However, over-expression of Ang-(1-7) had no effect on endogenous ACE2 activity, but significantly reduced ACE activi‐ ty. Paradoxically, despite reduced ACE activity in AAV-Ang-(1-7) treated retinas, Ang II levels remained high. It is possible that other enzymes/pathways may be involved in Ang II formation in addition to ACE. One such candidate is chymase, which has been detected in vascular systems and other tissues including eye [115]. Another candidate is the receptor for prorenin and renin (pro/renin). It has been recently demonstrated that binding of pro/renin to its receptor, pro/renin receptor (PRR), causes its prosegment to unfold, thereby activating prorenin so that it is able to generate angiotensin peptides that stimulate the Ang II-depend‐ ent pathway [76]. Considering the fact that retina contains high level of prorenin, and its lev‐ el is further increased in patients with diabetic retinopathy [52], this pathway likely contributes to increased Ang II level under diabetic conditions. The existence of multiple pathways for Ang II formation at the tissue level may explain the limited beneficial effects of classic RAS blockers, and may also lend support for thtoe notion that enhancing the protec‐ tive axis of RAS (ACE2/Ang-(1-7)/Mas) may represent a more effective strategy for treat‐ ment of diabetic retinopathy and other diabetic complications.

AAV vector mediated gene therapy for ocular diseases has been studied in animal models for more than a decade. Reports focusing on retinal therapy include a wide variety of retinal degenerative animal models of corresponding human retinopathies, as well as the therapeu‐ tic effects of AAV-vector mediated expression of neuroprotective, anti-apoptotic, and antiangiogenic agents in the retina [116]. In view of recent clinical trials in which AAV delivered RPE65 gene led to restoration of vision in human patients and other reports on successful trials on treatment of ocular diseases and inherited immune deficiencies (reviewed in [117] and references therein), gene therapy has emerged as promising approach and may become a standard treatment option for a wide range of diseases in the future. In particular, when considering that the diabetic individual experience this serious ocular complication for deca‐ des, a therapeutic strategy that is long-lasting and does not require patient compliance is particularly desirable. Thus, the delivery of ACE2 and/or Ang-(1-7) could serve as a novel gene therapeutic target for DR in combination with existing strategies to control hyperglyce‐ mic and insulin resistance states.

#### **4. Summary**

All genes of the RAS are locally expressed in the retina, establishing the existence of an in‐ trinsic retinal RAS. It is clear that the expression of genes of the vasoconstrictive/pro-inflam‐ matory/ proliferative/fibrotic (i.e., vasodeleterious) axis (ACE/Ang II/AT1R) is highly elevated, while the vasoprotective axis [ACE2/Ang-(1-7)/Mas] is decreased in the diabetic retina. We have demonstrated that increased expression of ACE2 or Ang-(1-7), two key members of the vasoprotective axis, via AAV-mediated gene delivery to the retina attenu‐ ates diabetes-induced retinal vascular pathology. Moreover, these beneficial effects of gene transfer occur without influencing the systemic hyperglycemic status. Thus, strategies en‐ hancing the protective ACE2/Ang-(1-7) axis of RAS could serve as a novel therapeutic target for DR.

ance states may represent a better strategy for preventing and treating diabetic

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

Supported in part by grants from American Diabetes Association, American Heart Associa‐

, Bo Lei1

2 The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laborato‐

3 Division of Renal Disease and Hypertension, University of Colorado Denver, Aurora, CO,

4 Department of Physiology & Functional Genomics, University of Florida, Gainesville, FL,

[1] Shaw, J.E., R.A. Sicree, and P.Z. Zimmet, Global estimates of the prevalence of diabe‐

[2] Cheung, N., P. Mitchell, and T.Y. Wong, Diabetic retinopathy. Lancet, 2010.

[3] Klein, B.E., Overview of epidemiologic studies of diabetic retinopathy. Ophthalmic

[4] Yau, J.W., et al., Global prevalence and major risk factors of diabetic retinopathy.

[5] Fong, D.S., et al., Retinopathy in diabetes. Diabetes Care, 2004. 27 Suppl 1: p. S84-7.

[6] Fong, D.S., et al., Diabetic retinopathy. Diabetes Care, 2004. 27(10): p. 2540-53.

tes for 2010 and 2030. Diabetes Res ClinPract, 2010. 87(1): p. 4-14.

, Yiguo Qiu1,2, Takahiko Nakagawa3

,

http://dx.doi.org/10.5772/52702

483

tion, Research to Prevent Blindness, NIH grants EY021752 and EY021721.

, Ping Zhu1

1 Departments of Ophthalmology, University of Florida, Gainesville, FL, USA

and William W Hauswirth1

complications such as diabetic retinopathy.

, Amrisha Verma1

\*Address all correspondence to: qli@ufl.edu

ry of Ophthalmology, Chongqing Eye Institute, China

**Acknowledgments**

**Author details**

Mohan K Raizada4

Qiuhong Li1

USA

USA

**References**

376(9735): p. 124-36.

Epidemiol, 2007. 14(4): p. 179-83.

Diabetes Care, 2012. 35(3): p. 556-64.

#### **5. Implications and future challenges**

Hyperactivity of RAS, resulting in elevated concentrations of the principal effector peptide Ang II, is central to pathways leading to increased vascular inflammation, oxidative stress, endothelial dysfunction and tissue remodeling in variety of conditions including heart fail‐ ure, stroke, renal failure, diabetes and its associated complications including DR. As a result, RAS inhibitors are one of the first-line therapeutic agents for treating patients with cardio‐ vascular diseases, metabolic syndrome, diabetes and diabetic complications. Ang II block‐ ade has shown to be antiangiogenic [66, 118, 119], anti-inflammatory [120] and improves retinal function [65], and indeed Ang II blockade therapy for retinopathy is in several clini‐ cal trials [67, 68, 121]. Despite the clear beneficial effects of RAS blockers (ACE inhibitors [ACEi] and angiotensin receptor blockers [ARBs]) [70, 71, 122], end-organ damage still en‐ sue in patients with diabetes. Overwhelming evidence now supports the notion thatactiva‐ tion of RAS at tissue levels contributes to the development and progression of diabetic complications including DR, independent of circulating RAS regulation. However the pre‐ cise molecular and cellular mechanisms as to how retinal RAS contributes to the develop‐ ment and progression of DR remain to be elucidated. Recent studies have also revealed the evolving complexity of RAS with a myriad cellular and intracellular pathways leading to formation of Ang II, as well as Ang II- independent signaling pathways resulting in hyper‐ activity of tissue RAS. The physiological implications of many of these components are still not well understood and new antagonists/agonists specific to these new components remain to be discovered. Nevertheless, our results clearly demonstrate that enhancing the protective axis of RAS (ACE2/Ang1-7/Mas) locally may be a better strategy for counteracting the effects of the pathological RAS activation than present systemic approaches. Furthermore, since AAV vector mediated gene delivery has been shown to be safe, and improve vision for ex‐ tended periods of time after a single administration in several clinical trials, enhancing the endogenous protective axis of RAS (ACE2/Ang1-7/Mas) by local gene delivery, in combina‐ tion with combination with existing strategies to control hyperglycemic and insulin resist‐ ance states may represent a better strategy for preventing and treating diabetic complications such as diabetic retinopathy.

#### **Acknowledgments**

**4. Summary**

482 Gene Therapy - Tools and Potential Applications

for DR.

**5. Implications and future challenges**

All genes of the RAS are locally expressed in the retina, establishing the existence of an in‐ trinsic retinal RAS. It is clear that the expression of genes of the vasoconstrictive/pro-inflam‐ matory/ proliferative/fibrotic (i.e., vasodeleterious) axis (ACE/Ang II/AT1R) is highly elevated, while the vasoprotective axis [ACE2/Ang-(1-7)/Mas] is decreased in the diabetic retina. We have demonstrated that increased expression of ACE2 or Ang-(1-7), two key members of the vasoprotective axis, via AAV-mediated gene delivery to the retina attenu‐ ates diabetes-induced retinal vascular pathology. Moreover, these beneficial effects of gene transfer occur without influencing the systemic hyperglycemic status. Thus, strategies en‐ hancing the protective ACE2/Ang-(1-7) axis of RAS could serve as a novel therapeutic target

Hyperactivity of RAS, resulting in elevated concentrations of the principal effector peptide Ang II, is central to pathways leading to increased vascular inflammation, oxidative stress, endothelial dysfunction and tissue remodeling in variety of conditions including heart fail‐ ure, stroke, renal failure, diabetes and its associated complications including DR. As a result, RAS inhibitors are one of the first-line therapeutic agents for treating patients with cardio‐ vascular diseases, metabolic syndrome, diabetes and diabetic complications. Ang II block‐ ade has shown to be antiangiogenic [66, 118, 119], anti-inflammatory [120] and improves retinal function [65], and indeed Ang II blockade therapy for retinopathy is in several clini‐ cal trials [67, 68, 121]. Despite the clear beneficial effects of RAS blockers (ACE inhibitors [ACEi] and angiotensin receptor blockers [ARBs]) [70, 71, 122], end-organ damage still en‐ sue in patients with diabetes. Overwhelming evidence now supports the notion thatactiva‐ tion of RAS at tissue levels contributes to the development and progression of diabetic complications including DR, independent of circulating RAS regulation. However the pre‐ cise molecular and cellular mechanisms as to how retinal RAS contributes to the develop‐ ment and progression of DR remain to be elucidated. Recent studies have also revealed the evolving complexity of RAS with a myriad cellular and intracellular pathways leading to formation of Ang II, as well as Ang II- independent signaling pathways resulting in hyper‐ activity of tissue RAS. The physiological implications of many of these components are still not well understood and new antagonists/agonists specific to these new components remain to be discovered. Nevertheless, our results clearly demonstrate that enhancing the protective axis of RAS (ACE2/Ang1-7/Mas) locally may be a better strategy for counteracting the effects of the pathological RAS activation than present systemic approaches. Furthermore, since AAV vector mediated gene delivery has been shown to be safe, and improve vision for ex‐ tended periods of time after a single administration in several clinical trials, enhancing the endogenous protective axis of RAS (ACE2/Ang1-7/Mas) by local gene delivery, in combina‐ tion with combination with existing strategies to control hyperglycemic and insulin resist‐

Supported in part by grants from American Diabetes Association, American Heart Associa‐ tion, Research to Prevent Blindness, NIH grants EY021752 and EY021721.

#### **Author details**

Qiuhong Li1 , Amrisha Verma1 , Ping Zhu1 , Bo Lei1 , Yiguo Qiu1,2, Takahiko Nakagawa3 , Mohan K Raizada4 and William W Hauswirth1

\*Address all correspondence to: qli@ufl.edu

1 Departments of Ophthalmology, University of Florida, Gainesville, FL, USA

2 The First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laborato‐ ry of Ophthalmology, Chongqing Eye Institute, China

3 Division of Renal Disease and Hypertension, University of Colorado Denver, Aurora, CO, USA

4 Department of Physiology & Functional Genomics, University of Florida, Gainesville, FL, USA

#### **References**


[7] Williams, R., et al., Epidemiology of diabetic retinopathy and macular oedema: a sys‐ tematic review. Eye, 2004. 18(10): p. 963-83.

[23] Nguyen Dinh Cat, A. and R.M. Touyz, A new look at the renin-angiotensin system--

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

485

[24] Ferreira, A.J., et al., Therapeutic implications of the vasoprotective axis of the reninangiotensin system in cardiovascular diseases. Hypertension, 2010. 55(2): p. 207-13.

[25] Ferrario, C.M., A.J. Trask, and J.A. Jessup, Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of car‐ diovascular function. Am J Physiol Heart CircPhysiol, 2005.289(6): p. H2281-90.

[26] Donoghue, M., et al., A novel angiotensin-converting enzyme-related carboxypepti‐ dase (ACE2) converts angiotensin I to angiotensin 1-9.Circ Res, 2000. 87(5): p. E1-9.

[27] Tipnis, S.R., et al., A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. J BiolChem,

[28] Ferreira, A.J. and R.A. Santos, Cardiovascular actions of angiotensin-(1-7).Braz J Med

[30] Mercure, C., et al., Angiotensin(1-7) blunts hypertensive cardiac remodeling by a di‐

[31] Santos, R.A., et al., Angiotensin-(1-7) is an endogenous ligand for the G protein-cou‐

[32] Santos, R.A., A.J. Ferreira, and E.S.A.C. Simoes, Recent advances in the angiotensinconverting enzyme 2-angiotensin(1-7)-Mas axis.ExpPhysiol, 2008. 93(5): p. 519-27.

[33] Sica, D.A., The practical aspects of combination therapy with angiotensin receptor blockers and angiotensin-converting enzyme inhibitors. J Renin Angiotensin Aldos‐

[34] Ribeiro-Oliveira, A., Jr., et al., The renin-angiotensin system and diabetes: an up‐

[35] Perkins, J.M. and S.N. Davis, The renin-angiotensin-aldosterone system: a pivotal role in insulin sensitivity and glycemic control.CurrOpinEndocrinol Diabetes Obes,

[36] Keidar, S., M. Kaplan, and A. Gamliel-Lazarovich, ACE2 of the heart: From angioten‐

[37] Iwai, M. and M. Horiuchi, Devil and angel in the renin-angiotensin system: ACE-an‐ giotensin II-AT1 receptor axis vs. ACE2-angiotensin-(1-7)-Mas receptor axis.Hyper‐

[38] Der Sarkissian, S., et al., ACE2: A novel therapeutic target for cardiovascular diseas‐

[29] Varagic, J., et al., New angiotensins. J Mol Med, 2008. 86(6): p. 663-71.

pled receptor Mas.ProcNatlAcadSci U S A, 2003. 100(14): p. 8258-63.

rect effect on the heart.Circ Res, 2008. 103(11): p. 1319-26.

date.Vasc Health Risk Manag, 2008. 4(4): p. 787-803.

es.ProgBiophysMolBiol, 2006. 91(1-2): p. 163-98.

sin I to angiotensin (1-7).Cardiovasc Res, 2007. 73(3): p. 463-9.

focusing on the vascular system. Peptides, 2011. 32(10): p. 2141-50.

2000. 275(43): p. 33238-43.

Biol Res, 2005. 38(4): p. 499-507.

terone Syst, 2002. 3(2): p. 66-71.

2008. 15(2): p. 147-52.

tens Res, 2009. 32(7): p. 533-6.


[23] Nguyen Dinh Cat, A. and R.M. Touyz, A new look at the renin-angiotensin system- focusing on the vascular system. Peptides, 2011. 32(10): p. 2141-50.

[7] Williams, R., et al., Epidemiology of diabetic retinopathy and macular oedema: a sys‐

[10] Hunyady, L. and K.J. Catt, Pleiotropic AT1 receptor signaling pathways mediating physiological and pathogenic actions of angiotensin II.MolEndocrinol, 2006. 20(5): p.

[11] Mehta, P.K. and K.K. Griendling, Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system. Am J Physiol Cell Physiol,

[12] Marchesi, C., P. Paradis, and E.L. Schiffrin, Role of the renin-angiotensin system in

[13] de Cavanagh, E.M., et al., From mitochondria to disease: role of the renin-angiotensin

[14] de Cavanagh, E.M., et al., Angiotensin II, mitochondria, cytoskeletal, and extracellu‐ lar matrix connections: an integrating viewpoint. Am J Physiol Heart CircPhysiol,

[15] Touyz, R.M. and E.L. Schiffrin, Signal transduction mechanisms mediating the phys‐ iological and pathophysiological actions of angiotensin II in vascular smooth muscle

[16] Nguyen Dinh Cat, A. and R.M. Touyz, Cell signaling of angiotensin II on vascular

[17] Carey, R.M. and H.M. Siragy, Newly recognized components of the renin-angioten‐ sin system: potential roles in cardiovascular and renal regulation.Endocr Rev, 2003.

[18] Putnam, K., et al., The renin-angiotensin system: a target of and contributor to dysli‐ pidemias, altered glucose homeostasis, and hypertension of the metabolic syndrome.

[19] Nakao, Y.M., et al., Effects of renin-angiotensin system blockades on cardiovascular outcomes in patients with diabetes mellitus: A systematic review and meta-analysis.

[20] Perret-Guillaume, C., et al., Benefits of the RAS blockade: clinical evidence before the

[21] Ostergren, J., Renin-angiotensin-system blockade in the prevention of diabetes. Dia‐

[22] Crowley, S.D. and T.M. Coffman, Recent advances involving the renin-angiotensin

tone: novel mechanisms.CurrHypertens Rep, 2011. 13(2): p. 122-8.

Am J Physiol Heart CircPhysiol, 2012.302(6): p. H1219-30.

ONTARGET study. J Hypertens, 2009. 27 Suppl 2: p. S3-7.

vascular inflammation. Trends PharmacolSci, 2008. 29(7): p. 367-74.

[8] Frank, R.N., Diabetic retinopathy. N Engl J Med, 2004. 350(1): p. 48-58.

tematic review. Eye, 2004. 18(10): p. 963-83.

system. Am J Nephrol, 2007.27(6): p. 545-53.

cells.Pharmacol Rev, 2000. 52(4): p. 639-72.

Diabetes Res ClinPract, 2012. 96(1): p. 68-75.

betes Res ClinPract, 2007. 76 Suppl 1: p. S13-21.

system.Exp Cell Res, 2012. 318(9): p. 1049-56.

953-70.

2007.292(1): p. C82-97.

484 Gene Therapy - Tools and Potential Applications

2009.296(3): p. H550-8.

24(3): p. 261-71.

[9] Watkins, P.J., Retinopathy. BMJ, 2003. 326(7395): p. 924-6.


[39] Huentelman, M.J., et al., Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats.ExpPhysiol, 2005. 90(5): p. 783-90.

[54] Alcazar, O., et al., (Pro)renin receptor is expressed in human retinal pigment epitheli‐ um and participates in extracellular matrix remodeling.Exp Eye Res, 2009. 89(5): p.

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

487

[55] Satofuka, S., et al., Role of nonproteolytically activated prorenin in pathologic, but not physiologic, retinal neovascularization. Invest Ophthalmol Vis Sci, 2007. 48(1): p.

[56] Satofuka, S., et al., (Pro)renin receptor promotes choroidal neovascularization by acti‐ vating its signal transduction and tissue renin-angiotensin system. Am J Pathol,

[57] Satofuka, S., et al., (Pro)renin receptor-mediated signal transduction and tissue reninangiotensin system contribute to diabetes-induced retinal inflammation. Diabetes,

[58] Wilkinson-Berka, J.L., et al., RILLKKMPSV influences the vasculature, neurons and glia, and (pro)renin receptor expression in the retina. Hypertension, 2010. 55(6): p.

[59] Maruichi, M., et al., Measurement of activities in two different angiotensin II generat‐ ing systems, chymase and angiotensin-converting enzyme, in the vitreous fluid of vitreoretinal diseases: a possible involvement of chymase in the pathogenesis of mac‐

[60] Kida, T., et al., Renin-angiotensin system in proliferative diabetic retinopathy and its gene expression in cultured human muller cells.Jpn J Ophthalmol, 2003. 47(1): p.

[61] Zhang, J.Z., et al., Captopril inhibits glucose accumulation in retinal cells in diabetes.

[62] Hsieh, T.J., et al., High glucose stimulates angiotensinogen gene expression and cell hypertrophy via activation of the hexosamine biosynthesis pathway in rat kidney

[63] Zhang, J.Z., et al., Captopril inhibits capillary degeneration in the early stages of dia‐

[64] Moravski, C.J., et al., The renin-angiotensin system influences ocular endothelial cell proliferation in diabetes: transgenic and interventional studies. Am J Pathol,

[65] Phipps, J.A., J.L. Wilkinson-Berka, and E.L. Fletcher, Retinal dysfunction in diabetic ren-2 rats is ameliorated by treatment with valsartan but not atenolol. Invest Oph‐

[66] Wilkinson-Berka, J.L., et al., Valsartan but not atenolol improves vascular pathology

in diabetic Ren-2 rat retina. Am J Hypertens, 2007.20(4): p. 423-30.

proximal tubular cells. Endocrinology, 2003. 144(10): p. 4338-49.

ular hole patients.Curr Eye Res, 2004. 29(4-5): p. 321-5.

Invest Ophthalmol Vis Sci, 2003. 44(9): p. 4001-5.

betic retinopathy.Curr Eye Res, 2007. 32(10): p. 883-9.

638-47.

422-9.

1454-60.

36-41.

2008.173(6): p. 1911-8.

2009. 58(7): p. 1625-33.

2003.162(1): p. 151-60.

thalmol Vis Sci, 2007. 48(2): p. 927-34.


[54] Alcazar, O., et al., (Pro)renin receptor is expressed in human retinal pigment epitheli‐ um and participates in extracellular matrix remodeling.Exp Eye Res, 2009. 89(5): p. 638-47.

[39] Huentelman, M.J., et al., Protection from angiotensin II-induced cardiac hypertrophy and fibrosis by systemic lentiviral delivery of ACE2 in rats.ExpPhysiol, 2005. 90(5): p.

[40] Hernandez Prada, J.A., et al., Structure-based identification of small-molecule angio‐ tensin-converting enzyme 2 activators as novel antihypertensive agents. Hyperten‐

[41] Ferreira, A.J., et al., Evidence for angiotensin-converting enzyme 2 as a therapeutic target for the prevention of pulmonary hypertension.Am J RespirCrit Care Med,

[42] Fraga-Silva, R.A., et al., ACE2 activation promotes antithrombotic activity.Mol Med,

[43] Der Sarkissian, S., et al., Cardiac overexpression of angiotensin converting enzyme 2 protects the heart from ischemia-induced pathophysiology. Hypertension, 2008.

[44] Paul, M., A. PoyanMehr, and R. Kreutz, Physiology of local renin-angiotensin sys‐

[45] Bader, M., et al., Tissue renin-angiotensin systems: new insights from experimental animal models in hypertension research. J Mol Med, 2001. 79(2-3): p. 76-102.

[46] Baltatu, O.C., L.A. Campos, and M. Bader, Local renin-angiotensin system and the

[47] Wagner, J., et al., Demonstration of renin mRNA, angiotensinogen mRNA, and an‐ giotensin converting enzyme mRNA expression in the human eye: evidence for an

[48] Sarlos, S. and J.L. Wilkinson-Berka, The renin-angiotensin system and the developing

[49] Danser, A.H., et al., Angiotensin levels in the eye. Invest Ophthalmol Vis Sci, 1994.

[50] Senanayake, P., et al., Angiotensin II and Its Receptor Subtypes in the Human Retina.

[51] Savaskan, E., et al., Immunohistochemical localization of angiotensin-converting en‐ zyme, angiotensin II and AT1 receptor in human ocular tissues. Ophthalmic Res,

[52] Danser, A.H., et al., Renin, prorenin, and immunoreactive renin in vitreous fluid from eyes with and without diabetic retinopathy. J ClinEndocrinolMetab, 1989. 68(1):

[53] Berka, J.L., et al., Renin-containing Muller cells of the retina display endocrine fea‐

intraocular renin-angiotensin system. Br J Ophthalmol, 1996. 80(2): p. 159-63.

retinal vasculature. Invest Ophthalmol Vis Sci, 2005. 46(3): p. 1069-77.

Invest Ophthalmol Vis Sci, 2007. 48(7): p. 3301-11.

tures. Invest Ophthalmol Vis Sci, 1995. 36(7): p. 1450-8.

brain--a continuous quest for knowledge. Peptides, 2011. 32(5): p. 1083-6.

783-90.

486 Gene Therapy - Tools and Potential Applications

sion, 2008. 51(5): p. 1312-7.

2009. 179(11): p. 1048-54.

2010. 16(5-6): p. 210-5.

tems.Physiol Rev, 2006. 86(3): p. 747-803.

51(3): p. 712-8.

35(3): p. 1008-18.

2004. 36(6): p. 312-20.

p. 160-7.


[67] Chaturvedi, N., et al., Effect of candesartan on prevention (DIRECT-Prevent 1) and progression (DIRECT-Protect 1) of retinopathy in type 1 diabetes: randomised, place‐ bo-controlled trials. Lancet, 2008. 372(9647): p. 1394-402.

[82] Adamis, A.P., Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol,

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

489

[83] Joussen, A.M., et al., Nonsteroidal anti-inflammatory drugs prevent early diabetic

[84] Tang, J. and T.S. Kern, Inflammation in diabetic retinopathy.ProgRetin Eye Res, 2012.

[85] Joussen, A.M., et al., A central role for inflammation in the pathogenesis of diabetic

[86] Adamis, A.P. and A.J. Berman, Immunological mechanisms in the pathogenesis of

[87] Kern, T.S., Contributions of inflammatory processes to the development of the early

[88] Kurihara, T., et al., Renin-Angiotensin system hyperactivation can induce inflamma‐

[89] Lavoie, J.L. and C.D. Sigmund, Minireview: overview of the renin-angiotensin sys‐ tem--an endocrine and paracrine system. Endocrinology, 2003. 144(6): p. 2179-83.

[90] Sedeek, M., et al., Oxidative stress, nox isoforms and complications of diabetes-po‐ tential targets for novel therapies. J CardiovascTransl Res, 2012. 5(4): p. 509-18.

[91] Gao, L. and G.E. Mann, Vascular NAD(P)H oxidase activation in diabetes: a double-

[92] Dikalov, S., Cross talk between mitochondria and NADPH oxidases. Free RadicBiol

[93] Dikalov, S.I. and R.R. Nazarewicz, Angiotensin II-Induced Production of Mitochon‐ drial Reactive Oxygen Species: Potential Mechanisms and Relevance for Cardiovas‐

[94] Kurihara, T., et al., Angiotensin II type 1 receptor signaling contributes to synapto‐ physin degradation and neuronal dysfunction in the diabetic retina. Diabetes, 2008.

[95] Katovich, M.J., et al., Angiotensin-converting enzyme 2 as a novel target for gene

[96] Oudit, G.Y., et al., Human recombinant ACE2 reduces the progression of diabetic

[97] Oudit, G.Y. and J.M. Penninger, Recombinant human angiotensin-converting en‐ zyme 2 as a new renin-angiotensin system peptidase for heart failure therapy.Curr

therapy for hypertension.ExpPhysiol, 2005. 90(3): p. 299-305.

retinopathy via TNF-alpha suppression. FASEB J, 2002. 16(3): p. 438-40.

diabetic retinopathy.SeminImmunopathol, 2008. 30(2): p. 65-84.

stages of diabetic retinopathy.Exp Diabetes Res, 2007. 2007: p. 95103.

tion and retinal neural dysfunction.Int J Inflam, 2012. 2012: p. 581695.

edged sword in redox signalling.Cardiovasc Res, 2009. 82(1): p. 9-20.

2002. 86(4): p. 363-5.

30(5): p. 343-58.

retinopathy. FASEB J, 2004. 18(12): p. 1450-2.

Med, 2011. 51(7): p. 1289-301.

57(8): p. 2191-8.

cular Disease.Antioxid Redox Signal, 2012.

nephropathy. Diabetes, 2010. 59(2): p. 529-38.

Heart Fail Rep, 2011. 8(3): p. 176-83.


[82] Adamis, A.P., Is diabetic retinopathy an inflammatory disease? Br J Ophthalmol, 2002. 86(4): p. 363-5.

[67] Chaturvedi, N., et al., Effect of candesartan on prevention (DIRECT-Prevent 1) and progression (DIRECT-Protect 1) of retinopathy in type 1 diabetes: randomised, place‐

[68] Sjolie, A.K., et al., Effect of candesartan on progression and regression of retinopathy in type 2 diabetes (DIRECT-Protect 2): a randomised placebo-controlled trial. Lancet,

[69] Mauer, M., et al., Renal and retinal effects of enalapril and losartan in type 1 diabetes.

[70] Ghattas, A., P.L. Lip, and G.Y. Lip, Renin-angiotensin blockade in diabetic retinop‐

[71] Wright, A.D. and P.M. Dodson, Diabetic retinopathy and blockade of the renin-an‐ giotensin system: new data from the DIRECT study programme. Eye (Lond), 2010.

[72] Miyazaki, M. and S. Takai, Tissue angiotensin II generating system by angiotensin-

[73] Cristovam, P.C., et al., ACE-dependent and chymase-dependent angiotensin II gen‐ eration in normal and glucose-stimulated human mesangial cells.ExpBiol Med (May‐

[74] Kumar, R. and M.A. Boim, Diversity of pathways for intracellular angiotensin II syn‐

[75] Shiota, N., et al., Angiotensin II-generating system in dog and monkey ocular tis‐

[76] Nguyen, G., et al., Pivotal role of the renin/prorenin receptor in angiotensin II pro‐ duction and cellular responses to renin. J Clin Invest, 2002. 109(11): p. 1417-27. [77] Malhotra, A., et al., Angiotensin II promotes glucose-induced activation of cardiac protein kinase C isozymes and phosphorylation of troponin I. Diabetes, 2001. 50(8):

[78] Kim, H.W., et al., Enalapril alters expression of key growth factors in experimental

[79] Kim, J.H., et al., Blockade of angiotensin II attenuates VEGF-mediated blood-retinal barrier breakdown in diabetic retinopathy. J Cereb Blood Flow Metab, 2008.

[80] Li, X.C. and J.L. Zhuo, Nuclear factor-kappaB as a hormonal intracellular signaling molecule: focus on angiotensin II-induced cardiovascular and renal injury.CurrOpin‐

[81] Brasier, A.R., et al., Angiotensin II induces gene transcription through cell-type-de‐ pendent effects on the nuclear factor-kappaB (NF-kappaB) transcription factor.Mol

converting enzyme and chymase. J PharmacolSci, 2006. 100(5): p. 391-7.

thesis.CurrOpinNephrolHypertens, 2009. 18(1): p. 33-9.

sues.ClinExpPharmacolPhysiol, 1997. 24(3-4): p. 243-8.

diabetic retinopathy.Curr Eye Res, 2009. 34(11): p. 976-87.

NephrolHypertens, 2008. 17(1): p. 37-43.

Cell Biochem, 2000. 212(1-2): p. 155-69.

bo-controlled trials. Lancet, 2008. 372(9647): p. 1394-402.

2008. 372(9647): p. 1385-93.

488 Gene Therapy - Tools and Potential Applications

24(1): p. 1-6.

p. 1918-26.

N Engl J Med, 2009. 361(1): p. 40-51.

wood), 2008. 233(8): p. 1035-43.

athy.Int J ClinPract, 2011. 65(2): p. 113-6.


[98] Zhong, J., et al., Angiotensin-converting enzyme 2 suppresses pathological hypertro‐ phy, myocardial fibrosis, and cardiac dysfunction. Circulation, 2010. 122(7): p. 717-28, 18 p following 728.

[113] Tikellis, C., et al., Identification of angiotensin converting enzyme 2 in the rodent ret‐

Gene Therapy for Diabetic Retinopathy – Targeting the Renin-Angiotensin System

http://dx.doi.org/10.5772/52702

491

[114] Ferrario, C.M., et al., Counterregulatory actions of angiotensin-(1-7). Hypertension,

[115] Lorenz, J.N., Chymase: the other ACE? Am J Physiol Renal Physiol, 2010.298(1): p.

[116] Hauswirth, W.W., et al., Range of retinal diseases potentially treatable by AAV-vec‐ tored gene therapy. Novartis Found Symp, 2004. 255: p. 179-88; discussion 188-94.

[117] Herzog, R.W., O. Cao, and A. Srivastava, Two decades of clinical gene therapy--suc‐

[118] Sarlos, S., et al., Retinal angiogenesis is mediated by an interaction between the an‐ giotensin type 2 receptor, VEGF, and angiopoietin. Am J Pathol, 2003.163(3): p.

[119] Moravski, C.J., et al., Retinal neovascularization is prevented by blockade of the re‐

[120] Nagai, N., et al., Suppression of diabetes-induced retinal inflammation by blocking the angiotensin II type 1 receptor or its downstream nuclear factor-kappaB pathway.

[121] Chaturvedi, N., et al., Effect of lisinopril on progression of retinopathy in normoten‐ sive people with type 1 diabetes. The EUCLID Study Group. EURODIAB Controlled Trial of Lisinopril in Insulin-Dependent Diabetes Mellitus. Lancet, 1998. 351(9095): p.

[122] Sjolie, A.K., P. Dodson, and F.R. Hobbs, Does renin-angiotensin system blockade have a role in preventing diabetic retinopathy? A clinical review.Int J ClinPract, 2011.

cess is finally mounting.Discov Med, 2010. 9(45): p. 105-11.

nin-angiotensin system. Hypertension, 2000. 36(6): p. 1099-104.

Invest Ophthalmol Vis Sci, 2007. 48(9): p. 4342-50.

ina.Curr Eye Res, 2004. 29(6): p. 419-27.

1997. 30(3 Pt 2): p. 535-41.

F35-6.

879-87.

28-31.

65(2): p. 148-53.


[113] Tikellis, C., et al., Identification of angiotensin converting enzyme 2 in the rodent ret‐ ina.Curr Eye Res, 2004. 29(6): p. 419-27.

[98] Zhong, J., et al., Angiotensin-converting enzyme 2 suppresses pathological hypertro‐ phy, myocardial fibrosis, and cardiac dysfunction. Circulation, 2010. 122(7): p.

[99] Zhong, J., et al., Prevention of angiotensin II-mediated renal oxidative stress, inflam‐ mation, and fibrosis by angiotensin-converting enzyme 2. Hypertension, 2011. 57(2):

[100] Benter, I.F., et al., Angiotensin-(1-7) prevents diabetes-induced cardiovascular dys‐

[101] Moon, J.Y., et al., Attenuating effect of angiotensin-(1-7) on angiotensin II-mediated NAD(P)H oxidase activation in type 2 diabetic nephropathy of KK-Ay/Ta mice. Am J

[102] Dhaunsi, G.S., et al., Angiotensin-(1-7) prevents diabetes-induced attenuation in PPAR-gamma and catalase activities.Eur J Pharmacol, 2010. 638(1-3): p. 108-14.

[103] Bindom, S.M., et al., Angiotensin I-converting enzyme type 2 (ACE2) gene therapy improves glycemic control in diabetic mice. Diabetes, 2010. 59(10): p. 2540-8.

[104] Patel, V.B., et al., Loss of angiotensin-converting enzyme-2 exacerbates diabetic cardi‐ ovascular complications and leads to systolic and vascular dysfunction: a critical role

[105] Wong, D.W., et al., Loss of angiotensin-converting enzyme-2 (Ace2) accelerates dia‐

[106] Lo, J., et al., Angiotensin Converting Enzyme 2 antagonizes Ang II-induced pressor response and NADPH oxidase activation in WKY rats and in SHR model.ExpPhy‐

[107] Li, Q., et al., Diabetic eNOS-knockout mice develop accelerated retinopathy. Invest

[108] Verma, A., et al., ACE2 and Ang-(1-7) confer protection against development of dia‐

[109] Huentelman, M.J., et al., Cloning and characterization of a secreted form of angioten‐

[110] Dawn-Linsley, M., et al., Monitoring thiobarbituric acid-reactive substances (TBARs) as an assay for oxidative damage in neuronal cultures and central nervous system. J

[111] Fletcher, E.L., et al., The renin-angiotensin system in retinal health and disease: Its in‐ fluence on neurons, glia and the vasculature.ProgRetin Eye Res, 2010: p. 1-28.

[112] Nakagawa, T., et al., Diabetic endothelial nitric oxide synthase knockout mice devel‐ op advanced diabetic nephropathy. J Am SocNephrol, 2007. 18(2): p. 539-50.

of the angiotensin II/AT1 receptor axis.Circ Res, 2012. 110(10): p. 1322-35.

betic kidney injury. Am J Pathol, 2007.171(2): p. 438-51.

Ophthalmol Vis Sci, 2010. 51(10): p. 5240-6.

Neurosci Methods, 2005. 141(2): p. 219-22.

betic retinopathy.MolTher, 2012. 20(1): p. 28-36.

sin-converting enzyme 2.RegulPept, 2004. 122(2): p. 61-7.

function. Am J Physiol Heart CircPhysiol, 2007.292(1): p. H666-72.

717-28, 18 p following 728.

490 Gene Therapy - Tools and Potential Applications

Physiol Renal Physiol, 2011.

p. 314-22.

siol, 2012.


**Chapter 20**

**Gene Therapy for Retinitis Pigmentosa**

The retina comprises diverse differentiated neurons that have specific functions. Photoreceptor cells, the first-order neurons in the retina, have photopigments (rhodopsin and opsin) that absorb photons. Signals produced by the photoreceptor cells are transmitted to second-order neurons. Finally, visual signals are transmitted to the brain from the third-order neurons, the retinal ganglion cells (RGCs). Major diseases that cause blindness in advanced countries include glaucoma, diabetic retinopathy, retinitis pigmentosa (RP), and age-related retinop‐ athy. Loss of vision due to these diseases is irreversible. However, with regard to glaucoma, eye drops that have the effect of reducing intraocular pressure have been developed. In diabetic retinopathy, effective surgical treatments such as vitrectomy and photocoagulation have been established. Blindness due to glaucoma and diabetic retinopathy can be prevented by admin‐ istering these treatments in the early phase. On the other hand, in diseases caused by gene mutations, such as RP, effective treatments for delaying photoreceptor degeneration have not yet been established. Degeneration of photoreceptor cells results in loss of vision, even if other

RP is a disease that causes blindness due to photoreceptor degeneration. Symptoms include night blindness and loss of peripheral and central vision. Approximately 1 in 4,000 people are affectedbythisdisease[4].In1990,Dryjaetal.[5]firstidentifiedapointmutationintherhodopsin gene from RP patients. A number of gene mutations responsible for RP has subsequently been identified.Most ofthese genes are associated with thephototransductionpathway in the retina. Insome cases,themutatedgene existsnot only inphotoreceptor cells but also inretinalpigment epithelial cells. To date, 53 causative genes and 7 loci of RP have been identified (http:// www.sph.uth.tmc.edu/Retnet/).Leber'scongenitalamaurosis(LCA)isanotherretinaldegener‐ ativediseasepredictedto affect approximately 1/81000 individuals [6].MostLCApatients have

> © 2013 Tomita et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

Hiroshi Tomita, Eriko Sugano, Hitomi Isago,

Additional information is available at the end of the chapter

Namie Murayama and Makoto Tamai

http://dx.doi.org/10.5772/52987

retinal neurons are intact [1-3].

**1. Introduction**

### **Gene Therapy for Retinitis Pigmentosa**

Hiroshi Tomita, Eriko Sugano, Hitomi Isago, Namie Murayama and Makoto Tamai

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52987

#### **1. Introduction**

The retina comprises diverse differentiated neurons that have specific functions. Photoreceptor cells, the first-order neurons in the retina, have photopigments (rhodopsin and opsin) that absorb photons. Signals produced by the photoreceptor cells are transmitted to second-order neurons. Finally, visual signals are transmitted to the brain from the third-order neurons, the retinal ganglion cells (RGCs). Major diseases that cause blindness in advanced countries include glaucoma, diabetic retinopathy, retinitis pigmentosa (RP), and age-related retinop‐ athy. Loss of vision due to these diseases is irreversible. However, with regard to glaucoma, eye drops that have the effect of reducing intraocular pressure have been developed. In diabetic retinopathy, effective surgical treatments such as vitrectomy and photocoagulation have been established. Blindness due to glaucoma and diabetic retinopathy can be prevented by admin‐ istering these treatments in the early phase. On the other hand, in diseases caused by gene mutations, such as RP, effective treatments for delaying photoreceptor degeneration have not yet been established. Degeneration of photoreceptor cells results in loss of vision, even if other retinal neurons are intact [1-3].

RP is a disease that causes blindness due to photoreceptor degeneration. Symptoms include night blindness and loss of peripheral and central vision. Approximately 1 in 4,000 people are affectedbythisdisease[4].In1990,Dryjaetal.[5]firstidentifiedapointmutationintherhodopsin gene from RP patients. A number of gene mutations responsible for RP has subsequently been identified.Most ofthese genes are associated with thephototransductionpathway in the retina. Insome cases,themutatedgene existsnot only inphotoreceptor cells but also inretinalpigment epithelial cells. To date, 53 causative genes and 7 loci of RP have been identified (http:// www.sph.uth.tmc.edu/Retnet/).Leber'scongenitalamaurosis (LCA)isanotherretinaldegener‐ ativediseasepredictedto affect approximately 1/81000 individuals [6].MostLCApatients have

© 2013 Tomita et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

severe visual defects in childhood. Histological analysis of the retinas of LCA patients shows markedretinal atrophy in the outerretinal layer, vascularthickening andsclerosis, andatrophy of the retinal pigment epithelium (RPE) [7]. Leber classified the disease as a type of RP on the basis of these characteristics. Later, Franceschetti and Dieterle differentiated it from retinal dystrophy based on the features of electroretinograms (ERGs) in these patients. Many gene mutations involved in LCA have been identified and the disease has been classified into 15 subtypes based on the affected gene [8-13]. Among these, LCA2, accounting for 10% of LCA cases [14], is due to a mutation in the RPE65 gene, which encodes all-*trans* retinyl ester isomer‐ ase. Deficiency in RPE65, leads to severe loss of visual function. Thus, in the case of LCA2, the cause of the disease is clearly identified as the biochemical blockade of the visual cycle caused byRPE65deficiency[11,12].ReplacementtherapyusingtheRPE65geneisacandidatetherapeu‐ tic strategy for LCA2. Indeed, successful results have been reported in RPE65 replacement therapy with the LCA2 animal model, Briard dogs [15]. After proof-of-principle studies [16], phaseItrialsusingadeno-associatedvirusvectortype2wereconductedin3independentgroups [17].The results showednoadverse effects suchas systemicdisseminationofvectororimmuno‐ logical responses to the vector or transgene. Importantly, improvement of visual function as evaluated by microperimetry was observed in 1 subject [18,19]. Two other groups also report‐ ed improvement in visual function [20,21]. Continuous follow-ups for 1.5 years [22] have confirmed the safety and tolerability of replacement gene therapy [23]. The various hereditary forms of RP are as follows: autosomal dominant, recessive, and X-linked recessive. The Pro23 - > His gene mutation in the rhodopsin gene [24,25] occurs in 20–30% of all RP patients in Europe and the U.S. In contrast, the occurrence in Japan is only a few percent. Thus, in addition to the diversity of the gene mutations, their frequencies vary characteristically among different races. Differencesintheprogression,clinicalfindings,anddevelopmentofthediseasearealsoobserved among different patients, even in those with the same mutation. A common feature of photore‐ ceptor celldeathcausedbyvariousgenemutations iseventualapoptosisviaacommonpathway [26]. Based on this rationale, various kinds of methods to prevent apoptosis, such as chemical treatment [27,28] and gene therapy, including gene replacement and neurotrophic factor supplementation [29-31], have been investigated. However, these strategies have not been successful in the complete prevention of cell death, although they have been shown to delay degeneration. The diversity of clinical features and gene mutations makes it difficult to develop effective treatments for RP.

**2. Materials and methods**

models is shown in Fig. 1.

**Genetically blind rats**

**Thy-I ChR2 transgenic rats**

**Animals**

suffering of animals used in the following experiments.

All the experiments performed for this report were approved by the Tohoku University Animal Care Committee, which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Every effort was made to minimize the number and

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 495

We used 2 types of photoreceptor degeneration models: a genetically blind rat model and a light-induced photoreceptor degeneration model. The experimental design for each of these

**Figure 1.** Experimental design. Two types of photoreceptor degeneration models were used in this study. The photo‐ receptor cells of RCS rats degenerate by 3 months after birth due to the Mertk gene mutation. On the other hand, Thy-TG rats have native photoreceptors. Therefore, we subjected TG rats to continuous light exposure to induce pho‐ toreceptor degeneration. To confirm photoreceptor degeneration, ERGs were recorded before performing behavioral

Royal College of Surgeons (RCS; rdy/rdy) rats [43,44] were used as model animals for photorecep‐ tor degeneration in our experiments. The RCS rat, an animal model of recessively inherited retinal degeneration, is widely used in the study of photoreceptor degeneration. The gene responsible is the receptor tyrosine kinase gene Mertk [45], and mutations in MERTK, the human ortholog of the RCS rat retinal dystrophy gene, cause RP [46]. Photoreceptor degeneration is almost complete by 3 months after birth. We intravitreously injected the AAV-ChR2V vector into 6-month- or 10-

We established transgenic (TG) rats harboring the ChR2 gene regulated by the Thy-1.2 promoter to investigate contrast sensitivity at each spatial frequency [47]. The rat Thy-1.2 antigen has been found to be abundant in the brain and thymus [48,49]. In the retina, the Thy-1.2 antigen is recognized as a marker specific to RGCs [50,51]. It is necessary to induce the degeneration of native photoreceptor cells in order to investigate the visual function conferred by ChR2-expressing RGCs, because the Thy-I TG rat has native photoreceptor cells. For this purpose, Thy-I TG rats were subjected to light-induced photoreceptor degeneration. Briefly, Thy-I TG rats were kept in cyclic light (12 hours ON/OFF: 5–10 lux/dark) for at least 2 weeks

month-old RCS rats. The rats were obtained from CLEA Japan, Inc. (Tokyo, Japan).

assessments. Finally, the eyes from all animals were subjected to histological examination.

A retinal prosthesis, comprising electrodes, an image processor, and a camera, is the only method to restore vision that has been studied [32-36]. Recently, a new strategy involving gene therapy for restoring vision has been developed using bacteriorhodopsin family genes [37,38]. The channelrhodopsin-2 (ChR2) gene derived from the green alga *Chlamydomonas* functions as a photoreceptor and cation-selective channel [39]. After the absorption of photons by photopigments, photon acquisition is completed by a chain reaction involving certain photo‐ receptor-specific proteins. Thus, the phototransduction pathway in photoreceptor cells requires not only photopigments but also certain photoreceptor-specific proteins, which complicates the reaction. Due to the inherent characteristics of ChR2, photosensitive neurons can be produced by the transfer of the ChR2 gene into neurons [40-42]. Here, we introduce new strategies for restoring vision by using channelrhodopsins.

#### **2. Materials and methods**

All the experiments performed for this report were approved by the Tohoku University Animal Care Committee, which is accredited by the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Every effort was made to minimize the number and suffering of animals used in the following experiments.

#### **Animals**

severe visual defects in childhood. Histological analysis of the retinas of LCA patients shows markedretinal atrophy in the outerretinal layer, vascularthickening andsclerosis, andatrophy of the retinal pigment epithelium (RPE) [7]. Leber classified the disease as a type of RP on the basis of these characteristics. Later, Franceschetti and Dieterle differentiated it from retinal dystrophy based on the features of electroretinograms (ERGs) in these patients. Many gene mutations involved in LCA have been identified and the disease has been classified into 15 subtypes based on the affected gene [8-13]. Among these, LCA2, accounting for 10% of LCA cases [14], is due to a mutation in the RPE65 gene, which encodes all-*trans* retinyl ester isomer‐ ase. Deficiency in RPE65, leads to severe loss of visual function. Thus, in the case of LCA2, the cause of the disease is clearly identified as the biochemical blockade of the visual cycle caused byRPE65deficiency[11,12].ReplacementtherapyusingtheRPE65geneisacandidatetherapeu‐ tic strategy for LCA2. Indeed, successful results have been reported in RPE65 replacement therapy with the LCA2 animal model, Briard dogs [15]. After proof-of-principle studies [16], phaseItrialsusingadeno-associatedvirusvectortype2wereconductedin3independentgroups [17].The results showednoadverse effects suchas systemicdisseminationofvectororimmuno‐ logical responses to the vector or transgene. Importantly, improvement of visual function as evaluated by microperimetry was observed in 1 subject [18,19]. Two other groups also report‐ ed improvement in visual function [20,21]. Continuous follow-ups for 1.5 years [22] have confirmed the safety and tolerability of replacement gene therapy [23]. The various hereditary forms of RP are as follows: autosomal dominant, recessive, and X-linked recessive. The Pro23 - > His gene mutation in the rhodopsin gene [24,25] occurs in 20–30% of all RP patients in Europe and the U.S. In contrast, the occurrence in Japan is only a few percent. Thus, in addition to the diversity of the gene mutations, their frequencies vary characteristically among different races. Differencesintheprogression,clinicalfindings,anddevelopmentofthediseasearealsoobserved among different patients, even in those with the same mutation. A common feature of photore‐ ceptor celldeathcausedbyvariousgenemutations iseventualapoptosisviaacommonpathway [26]. Based on this rationale, various kinds of methods to prevent apoptosis, such as chemical treatment [27,28] and gene therapy, including gene replacement and neurotrophic factor supplementation [29-31], have been investigated. However, these strategies have not been successful in the complete prevention of cell death, although they have been shown to delay degeneration. The diversity of clinical features and gene mutations makes it difficult to develop

A retinal prosthesis, comprising electrodes, an image processor, and a camera, is the only method to restore vision that has been studied [32-36]. Recently, a new strategy involving gene therapy for restoring vision has been developed using bacteriorhodopsin family genes [37,38]. The channelrhodopsin-2 (ChR2) gene derived from the green alga *Chlamydomonas* functions as a photoreceptor and cation-selective channel [39]. After the absorption of photons by photopigments, photon acquisition is completed by a chain reaction involving certain photo‐ receptor-specific proteins. Thus, the phototransduction pathway in photoreceptor cells requires not only photopigments but also certain photoreceptor-specific proteins, which complicates the reaction. Due to the inherent characteristics of ChR2, photosensitive neurons can be produced by the transfer of the ChR2 gene into neurons [40-42]. Here, we introduce

new strategies for restoring vision by using channelrhodopsins.

effective treatments for RP.

494 Gene Therapy - Tools and Potential Applications

We used 2 types of photoreceptor degeneration models: a genetically blind rat model and a light-induced photoreceptor degeneration model. The experimental design for each of these models is shown in Fig. 1.

**Figure 1.** Experimental design. Two types of photoreceptor degeneration models were used in this study. The photo‐ receptor cells of RCS rats degenerate by 3 months after birth due to the Mertk gene mutation. On the other hand, Thy-TG rats have native photoreceptors. Therefore, we subjected TG rats to continuous light exposure to induce pho‐ toreceptor degeneration. To confirm photoreceptor degeneration, ERGs were recorded before performing behavioral assessments. Finally, the eyes from all animals were subjected to histological examination.

#### **Genetically blind rats**

Royal College of Surgeons (RCS; rdy/rdy) rats [43,44] were used as model animals for photorecep‐ tor degeneration in our experiments. The RCS rat, an animal model of recessively inherited retinal degeneration, is widely used in the study of photoreceptor degeneration. The gene responsible is the receptor tyrosine kinase gene Mertk [45], and mutations in MERTK, the human ortholog of the RCS rat retinal dystrophy gene, cause RP [46]. Photoreceptor degeneration is almost complete by 3 months after birth. We intravitreously injected the AAV-ChR2V vector into 6-month- or 10 month-old RCS rats. The rats were obtained from CLEA Japan, Inc. (Tokyo, Japan).

#### **Thy-I ChR2 transgenic rats**

We established transgenic (TG) rats harboring the ChR2 gene regulated by the Thy-1.2 promoter to investigate contrast sensitivity at each spatial frequency [47]. The rat Thy-1.2 antigen has been found to be abundant in the brain and thymus [48,49]. In the retina, the Thy-1.2 antigen is recognized as a marker specific to RGCs [50,51]. It is necessary to induce the degeneration of native photoreceptor cells in order to investigate the visual function conferred by ChR2-expressing RGCs, because the Thy-I TG rat has native photoreceptor cells. For this purpose, Thy-I TG rats were subjected to light-induced photoreceptor degeneration. Briefly, Thy-I TG rats were kept in cyclic light (12 hours ON/OFF: 5–10 lux/dark) for at least 2 weeks prior to light exposure. The rats were then exposed to a 3000-lux intensity of fluorescent light for 7 days [28]. We used a light exposure box (NK Systems, Tokyo, Japan) to control the timing and light intensity for the induction of photoreceptor degeneration. After induction, we recorded ERGs to confirm photoreceptor degeneration.

computer monitors (ProLite E1902WS; Iiyama, Tokyo, Japan) arranged in a square around a platform. The software controlled the speed of virtual optomotor rotation, which was set at 12 degrees per second (2 rpm) in all experiments. The spatial frequency and the contrast of the

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 497

The animal was allowed to move freely on the platform in the virtual optomotor system. The grating session was started at a low spatial frequency (0.06 cycles/degree) with maximal contrast. An experimenter assessed whether the animals tracked the rotation, by monitoring the head movement and the presented rotating stimulus simultaneously on another display connected to the video camera. If head movement simultaneous with the rotation was evident, the experimenter judged that the animal could discriminate the grating, and proceeded to the next grating session. If the movement was ambiguous, the same grating session was presented again. All behavioral tests were double-blinded and performed during the first few hours of

VEP measurements in 6- or 10-month-old RCS rats are expected to be abolished due to loss of photoreceptor cells. Generally, in RCS rats, photoreceptor degeneration is almost complete by 3 months after birth. Indeed, VEP measurements were not evoked even by the maximal LED flash in any of the aged RCS (rdy/rdy) rats (Fig. 2A). On the other hand, robust VEPs were evoked by the blue LED flash in RCS rats injected with the AAV-ChR2V vector (Fig. 2A). Initially, small VEP responses were observed at 2 weeks after AAV injection (data not shown), and the maximum amplitudes of VEP were observed 8 weeks later [58]. There were notable differences in sample waveforms from 6- and 10-month-old rats injected with AAV-ChR2V. Amplitudes and latencies of VEPs from 6-month-old rats were larger and shorter, respectively,

The expression of the ChR2 gene was evaluated by measuring Venus fluorescence in RCS rat retinas (Fig. 3A). The number of positive cells in rats injected at 10 months of age was signifi‐ cantly less than that injected at 6 months of age (Fig. 3B). The number of RGCs decreased linearly with age, following photoreceptor degeneration in the RCS rats (Fig. 3C). We have previously shown [56] that the ChR2 gene is mainly expressed in RGCs upon intravitreous injection of the AAV-ChR2V vector. Therefore, the observed decrease in the number of RGCs

There were 11–12 rows of photoreceptor nuclei in the outer nuclear layer (ONL) of the Thy-1 TG rats; this is a number usually observed in rodents without retinal degeneration [59].

with age suggests that the transduction efficiencies at both ages are very similar.

grating pattern were varied but the average brightness was kept constant.

the animals' light cycle (light on at 8 AM).

**4.1. Recording of VEP measurements in RCS rats**

than those from 10-month-old rats (Fig. 2B).

**4.3. Photoreceptor degeneration in Thy-I TG rats**

**4.2. Transduction efficiencies of ChR2 in retinas of RCS rats**

**4. Results**

Preparation of the adeno-associated virus vector

The adeno-associated virus (AAV) vector with the ChR2 gene was constructed as described previously [38]. Following this, the AAV Helper-Free System (Stratagene, La Jolla, CA) was used to produce infectious AAV-Venus (control) and AAV-ChR2V virions, which were purified by a single-step column purification method as previously described [52].

Recording of ERGs and visual electrophysiology (VEP)

ERGs and VEP readings were recorded using a Neuropack (MEB-9102; Nihon Kohden, Tokyo, Japan) according to methods previously described [38,53]. Briefly, rats were dark-adapted overnight, and the pupils were dilated with 1% atropine and 2.5% phenylephrine hydrochlor‐ ide. Small contact lenses with gold wire loops were placed on both corneas, and a silver wire reference electrode was inserted subcutaneously between the eyes. Eyes were stimulated with flash light stimuli of 10-ms duration using a blue LED. Full-field scotopic ERGs were recorded, band-pass filtered at 0.3–500 Hz, and averaged for 5 responses at each light intensity. For VEP recordings, recording electrodes (silver-silver chloride) were placed epidurally on each side, 7 mm behind the bregma and 3 mm lateral of the midline, and a reference electrode was placed epidurally on the midline 12 mm behind the bregma, at least 7 days before the experiments [54,55]. Under ketamine-xylazine anesthesia, the pupils were dilated with 1% atropine and 2.5% phenylephrine hydrochloride. The ground electrode clip was placed on the tail. Photic stimuli of 20-ms duration were generated under various intensities by pulse activation of a blue LED. The high- and low-pass filters were set to 50 kHz and 0.05 kHz, respectively. One hundred consecutive response waveforms were averaged for each VEP measurement.

Determination of transduction efficiency

At the end of the experiment, RCS and Thy-TG rats were sacrificed, and their eyes were resected and fixed in 4% paraformaldehyde and 0.1 M phosphate buffer, pH 7.4 [56]. The eye of each rat was flat-mounted on a slide and covered with Vectashield medium (Vector Laboratories, Burlingame, CA) to prevent the degradation of fluorescence. Then, the number of positive cells was counted.

#### **3. Behavioral assessment**

The spatial vision of each animal was quantified by its optomotor response. We used a virtual optomotor system to evaluate the contrast sensitivities of each spatial frequency. The original virtual optomotor system described by Prusky et al. [57] was modified for rats [47]. When a drum is rotated around an animal with printed visual stimuli on the inside wall, the animal tracks the stimulus by turning its head. A light-dark grating pattern was displayed on computer monitors (ProLite E1902WS; Iiyama, Tokyo, Japan) arranged in a square around a platform. The software controlled the speed of virtual optomotor rotation, which was set at 12 degrees per second (2 rpm) in all experiments. The spatial frequency and the contrast of the grating pattern were varied but the average brightness was kept constant.

The animal was allowed to move freely on the platform in the virtual optomotor system. The grating session was started at a low spatial frequency (0.06 cycles/degree) with maximal contrast. An experimenter assessed whether the animals tracked the rotation, by monitoring the head movement and the presented rotating stimulus simultaneously on another display connected to the video camera. If head movement simultaneous with the rotation was evident, the experimenter judged that the animal could discriminate the grating, and proceeded to the next grating session. If the movement was ambiguous, the same grating session was presented again. All behavioral tests were double-blinded and performed during the first few hours of the animals' light cycle (light on at 8 AM).

#### **4. Results**

prior to light exposure. The rats were then exposed to a 3000-lux intensity of fluorescent light for 7 days [28]. We used a light exposure box (NK Systems, Tokyo, Japan) to control the timing and light intensity for the induction of photoreceptor degeneration. After induction, we

The adeno-associated virus (AAV) vector with the ChR2 gene was constructed as described previously [38]. Following this, the AAV Helper-Free System (Stratagene, La Jolla, CA) was used to produce infectious AAV-Venus (control) and AAV-ChR2V virions, which were

ERGs and VEP readings were recorded using a Neuropack (MEB-9102; Nihon Kohden, Tokyo, Japan) according to methods previously described [38,53]. Briefly, rats were dark-adapted overnight, and the pupils were dilated with 1% atropine and 2.5% phenylephrine hydrochlor‐ ide. Small contact lenses with gold wire loops were placed on both corneas, and a silver wire reference electrode was inserted subcutaneously between the eyes. Eyes were stimulated with flash light stimuli of 10-ms duration using a blue LED. Full-field scotopic ERGs were recorded, band-pass filtered at 0.3–500 Hz, and averaged for 5 responses at each light intensity. For VEP recordings, recording electrodes (silver-silver chloride) were placed epidurally on each side, 7 mm behind the bregma and 3 mm lateral of the midline, and a reference electrode was placed epidurally on the midline 12 mm behind the bregma, at least 7 days before the experiments [54,55]. Under ketamine-xylazine anesthesia, the pupils were dilated with 1% atropine and 2.5% phenylephrine hydrochloride. The ground electrode clip was placed on the tail. Photic stimuli of 20-ms duration were generated under various intensities by pulse activation of a blue LED. The high- and low-pass filters were set to 50 kHz and 0.05 kHz, respectively. One hundred consecutive response waveforms were averaged for each VEP measurement.

At the end of the experiment, RCS and Thy-TG rats were sacrificed, and their eyes were resected and fixed in 4% paraformaldehyde and 0.1 M phosphate buffer, pH 7.4 [56]. The eye of each rat was flat-mounted on a slide and covered with Vectashield medium (Vector Laboratories, Burlingame, CA) to prevent the degradation of fluorescence. Then, the number

The spatial vision of each animal was quantified by its optomotor response. We used a virtual optomotor system to evaluate the contrast sensitivities of each spatial frequency. The original virtual optomotor system described by Prusky et al. [57] was modified for rats [47]. When a drum is rotated around an animal with printed visual stimuli on the inside wall, the animal tracks the stimulus by turning its head. A light-dark grating pattern was displayed on

purified by a single-step column purification method as previously described [52].

recorded ERGs to confirm photoreceptor degeneration.

Recording of ERGs and visual electrophysiology (VEP)

Determination of transduction efficiency

of positive cells was counted.

**3. Behavioral assessment**

Preparation of the adeno-associated virus vector

496 Gene Therapy - Tools and Potential Applications

#### **4.1. Recording of VEP measurements in RCS rats**

VEP measurements in 6- or 10-month-old RCS rats are expected to be abolished due to loss of photoreceptor cells. Generally, in RCS rats, photoreceptor degeneration is almost complete by 3 months after birth. Indeed, VEP measurements were not evoked even by the maximal LED flash in any of the aged RCS (rdy/rdy) rats (Fig. 2A). On the other hand, robust VEPs were evoked by the blue LED flash in RCS rats injected with the AAV-ChR2V vector (Fig. 2A). Initially, small VEP responses were observed at 2 weeks after AAV injection (data not shown), and the maximum amplitudes of VEP were observed 8 weeks later [58]. There were notable differences in sample waveforms from 6- and 10-month-old rats injected with AAV-ChR2V. Amplitudes and latencies of VEPs from 6-month-old rats were larger and shorter, respectively, than those from 10-month-old rats (Fig. 2B).

#### **4.2. Transduction efficiencies of ChR2 in retinas of RCS rats**

The expression of the ChR2 gene was evaluated by measuring Venus fluorescence in RCS rat retinas (Fig. 3A). The number of positive cells in rats injected at 10 months of age was signifi‐ cantly less than that injected at 6 months of age (Fig. 3B). The number of RGCs decreased linearly with age, following photoreceptor degeneration in the RCS rats (Fig. 3C). We have previously shown [56] that the ChR2 gene is mainly expressed in RGCs upon intravitreous injection of the AAV-ChR2V vector. Therefore, the observed decrease in the number of RGCs with age suggests that the transduction efficiencies at both ages are very similar.

#### **4.3. Photoreceptor degeneration in Thy-I TG rats**

There were 11–12 rows of photoreceptor nuclei in the outer nuclear layer (ONL) of the Thy-1 TG rats; this is a number usually observed in rodents without retinal degeneration [59].

However, robust VEP measurements could be recorded, even though the photoreceptor cells had completely degenerated (Fig. 4B). Intense expression of the ChR2 gene was observed in

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 499

**Figure 4.** Electrophysiological response of Thy-I TG rats after photoreceptor degeneration. (A) Hematoxylin-eosin staining of the retina showed the degeneration of the native photoreceptor cells after continuous light exposure. (B) Extensive expression of the ChR2 gene was observed throughout the retina. (C) The ERG response was completely abolished following continuous light exposure, indicating that native photoreceptor cells had degenerated through‐

In our virtual optomotor system, a stimulus of blue stripes over a black background was produced according to a sine wave function with variable amplitude and frequency (Fig. 5A). All the photoreceptor-degenerated Thy-I TG and wild-type (normal) rats tracked the virtual rotating blue/black gratings (Fig. 5B). However, tracking stopped when the contrast was reduced below a specific threshold. We observed that contrast sensitivity was small at the minimal spatial frequency of 0.06 cycles per degree (CPD), increased with an increase in spatial frequency, and was negligible at spatial frequencies over 0.52 CPD. Therefore, the relationship followed an inverted U-shaped curve, as noted in previous reports [57]. In photoreceptordegenerated Thy-I TG rats, no reduction of contrast sensitivity was observed at any spatial frequency. Unexpectedly, the contrast sensitivity was instead somewhat enhanced at low

The photo-acquisition system of mammalian photoreceptor cells, which mediates various photoreceptor-specific proteins, is very complicated. In contrast, the corresponding system in green algae such as *Chlamydomonas* and *Volvox* is simpler. ChR2 contains a 13-*cis* retinal that absorbs a photon, inducing a conformational change. The ChR2 functions as a cation-selective ion channel. For this reason, the transfer of a single gene, ChR2, to RGCs allows the generation

out the retina. VEP measurements could still be recorded after photoreceptor degeneration.

**4.4. Behavioral assessment in photoreceptor degenerated-Thy-I TG rat**

spatial frequencies such as 0.09 or 0.18 CPD (Fig. 5C).

**5. Discussion**

the entire retina, with about 45% of RGCs positive for ChR2 (Fig. 4C).

**Figure 2.** VEP recordings before and after the injection of AAV-ChR2V. (A) VEP recordings from both 6-month- and 10-month-old RCS rats showed no responses. However, VEPs responses were clearly elicited 8 weeks after injection. (B) The amplitudes and latencies from rats injected with AAV-ChR2 at 6 months of age (n = 8) were significantly larger and shorter than those injected at 10 months of age (n = 4).

**Figure 3.** Transduction efficiencies of ChR2 in retinas of RCS rats. (A) Retinal whole-mount specimens obtained from rats injected with AAV-ChR2 at 6 and 10 months of age. (B) Venus-positive cells expressing the ChR2 gene were ob‐ served in whole-mount specimens. (C) The number of RGCs decreased with age.

Following continuous light exposure, photoreceptor cells disappeared (Fig. 4A). ERGs showed no response, indicating that the photoreceptor cells degenerated in the whole retina (Fig. 4B). However, robust VEP measurements could be recorded, even though the photoreceptor cells had completely degenerated (Fig. 4B). Intense expression of the ChR2 gene was observed in the entire retina, with about 45% of RGCs positive for ChR2 (Fig. 4C).

**Figure 4.** Electrophysiological response of Thy-I TG rats after photoreceptor degeneration. (A) Hematoxylin-eosin staining of the retina showed the degeneration of the native photoreceptor cells after continuous light exposure. (B) Extensive expression of the ChR2 gene was observed throughout the retina. (C) The ERG response was completely abolished following continuous light exposure, indicating that native photoreceptor cells had degenerated through‐ out the retina. VEP measurements could still be recorded after photoreceptor degeneration.

#### **4.4. Behavioral assessment in photoreceptor degenerated-Thy-I TG rat**

In our virtual optomotor system, a stimulus of blue stripes over a black background was produced according to a sine wave function with variable amplitude and frequency (Fig. 5A). All the photoreceptor-degenerated Thy-I TG and wild-type (normal) rats tracked the virtual rotating blue/black gratings (Fig. 5B). However, tracking stopped when the contrast was reduced below a specific threshold. We observed that contrast sensitivity was small at the minimal spatial frequency of 0.06 cycles per degree (CPD), increased with an increase in spatial frequency, and was negligible at spatial frequencies over 0.52 CPD. Therefore, the relationship followed an inverted U-shaped curve, as noted in previous reports [57]. In photoreceptordegenerated Thy-I TG rats, no reduction of contrast sensitivity was observed at any spatial frequency. Unexpectedly, the contrast sensitivity was instead somewhat enhanced at low spatial frequencies such as 0.09 or 0.18 CPD (Fig. 5C).

#### **5. Discussion**

Following continuous light exposure, photoreceptor cells disappeared (Fig. 4A). ERGs showed no response, indicating that the photoreceptor cells degenerated in the whole retina (Fig. 4B).

**Figure 3.** Transduction efficiencies of ChR2 in retinas of RCS rats. (A) Retinal whole-mount specimens obtained from rats injected with AAV-ChR2 at 6 and 10 months of age. (B) Venus-positive cells expressing the ChR2 gene were ob‐

served in whole-mount specimens. (C) The number of RGCs decreased with age.

**Figure 2.** VEP recordings before and after the injection of AAV-ChR2V. (A) VEP recordings from both 6-month- and 10-month-old RCS rats showed no responses. However, VEPs responses were clearly elicited 8 weeks after injection. (B) The amplitudes and latencies from rats injected with AAV-ChR2 at 6 months of age (n = 8) were significantly larger

and shorter than those injected at 10 months of age (n = 4).

498 Gene Therapy - Tools and Potential Applications

The photo-acquisition system of mammalian photoreceptor cells, which mediates various photoreceptor-specific proteins, is very complicated. In contrast, the corresponding system in green algae such as *Chlamydomonas* and *Volvox* is simpler. ChR2 contains a 13-*cis* retinal that absorbs a photon, inducing a conformational change. The ChR2 functions as a cation-selective ion channel. For this reason, the transfer of a single gene, ChR2, to RGCs allows the generation

**Figure 5.** Behavioral assessment using a digital optomotor. (A) The digital optomotor consisted of 4 displays sur‐ rounding a platform. The number of stripes and the contrast were controlled by software. (B) Superimposed images from movies showed that the rats were able to discriminate the moving stripes. The arrow indicates the direction of the moving stripes. The asterisk indicates the point of the rat's nose. (C) The contrast sensitivities of photoreceptordegenerated Thy-I TG rats were higher at low spatial frequencies compared to those of wild-type (normal) rats (n = 8).

To investigate visual acuity resulting from ChR2-expressing RGCs, we established a TG rat model expressing the ChR2 gene in RGCs. Photoreceptor-degenerated TG rats clearly tracked the rotation of blue-black stripes in a virtual optomotor. However, RCS rats that received the ChR2 gene in the AAV vector did not track the rotation of the virtual optomotor at any spatial frequency. Recently, we tested the behavior of RCS rats using a mechanical optomotor system and showed that the intensity of luminosity the rat received was the most important factor influencing their tracking of the rotation of the column [53]. A luminosity of over 500 lux was needed to induce head tracking in ChR2-expressing RCS rats. However, the maximum luminosity of the virtual optomotor was about 100 lux. It was therefore too low to induce head tracking in RCS rats. The question then arises: what is the difference between the TG and RCS rats? We do not have a reasonable explanation for this. One possibility is that the number of ChR2-expressing RGCs in the TG rat is greater than that in the RCS rat. About 45% of the RGCs expressed ChR2 in the TG rat. Compared to the TG rat, the transduction efficiency in the RCS rat is about 28% independent of the age of the animal. This may affect the light sensitivity. As the another explanation, in the case of TG rats, ChR2 is expressed after birth; therefore, there is a possibility that retinal organization and function might be

**Figure 6.** Summary of the visual pathway in ChR2-expressing RGCs. In the normal visual pathway, light (visual signals) is received by photoreceptor cells located at the end of the retinal layer. Photoreceptor cells produce signals that are transmitted to the inner nuclear layer. Finally, RGCs generate action potentials and thereby transmit signals to the LGN. RGCs play a role in transferring the visual signal to the brain. On the other hand, ChR2-expressing retinal gan‐ glion cells directly receive light, produce action potentials, and transmit them to the LGN. Therefore, there is no need

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 501

RGCs are merely one of the candidate cell types that could receive the ChR2 gene. Lagali et al. [60] succeeded in transferring the ChR2 gene into ON-bipolar cells in the retina and confirmed the restoration of visual and behavioral responses. ON- and OFF-bipolar cells receive synaptic input from photoreceptors. Considering that ChR2 can elicit light-on responses, ON-bipolar cells seem to be the most appropriate cells for the transfer of the ChR2 gene. However, 2 questions arise in this regard. First, how can we deliver the ChR2 gene into ON-bipolar cells for human gene therapy? Lagali et al. [60] transferred the ChR2 gene into neonatal mice by electroporation of the plasmid vector. It is generally difficult to transfer a gene into the depths

altered, that cannot be ruled out.

for mediation by other retinal cells.

of photosensitive RGCs. In the normal visual pathway, the light incident upon the eyes is first received by photoreceptor cells located at the end of the retinal layers. The photoreceptor cells control neurotransmitter release, and second-order neurons located in the inner nuclear layer respond to the neurotransmitter. Finally, RGCs produce action potentials and transmit to the lateral geniculate nucleus (LGN) via the optic nerve (Fig. 6). In RP, the photo-acquisition system is damaged due to the degeneration of photoreceptor cells, even if the other retinal layers remain intact. RGCs that are rendered photosensitive by the transfer of the ChR2 gene can directly respond to light and transmit signals to the brain. In this newly organized photoacquisition system, the other retinal neurons besides the RGCs are not required for the perception of light.

Although VEP responses recovered after ChR2 gene transfer, the amplitudes and waveforms were different between rats injected with AAV-ChR2V at 6 and 10 months of age. One possibility is that RGC activity decayed after photoreceptor degeneration. However, our data show that the number of RGCs decreased after photoreceptor degeneration (Fig. 3C). The calculated RGC transduction efficiencies in 6-month-old rats were the same as those in 10 month-old rats. The differences in the recorded amplitudes and latencies shown in Fig. 2 appear to be due to differences in the number of photosensitive RGCs. We previously reported that the RGC transduction efficiency in 10-month-old rats was about 28% [38]. Subsequently, Isago et al. showed that the RGC transduction efficiencies in 6- and 10-month-old rats were 28.3 and 27.7%, respectively [56]. The data clearly indicates that the transduction efficiency is the same, although the number of ChR2-expressing cells was lower, corresponding to the decrease in the number of RGCs.

**Figure 6.** Summary of the visual pathway in ChR2-expressing RGCs. In the normal visual pathway, light (visual signals) is received by photoreceptor cells located at the end of the retinal layer. Photoreceptor cells produce signals that are transmitted to the inner nuclear layer. Finally, RGCs generate action potentials and thereby transmit signals to the LGN. RGCs play a role in transferring the visual signal to the brain. On the other hand, ChR2-expressing retinal gan‐ glion cells directly receive light, produce action potentials, and transmit them to the LGN. Therefore, there is no need for mediation by other retinal cells.

To investigate visual acuity resulting from ChR2-expressing RGCs, we established a TG rat model expressing the ChR2 gene in RGCs. Photoreceptor-degenerated TG rats clearly tracked the rotation of blue-black stripes in a virtual optomotor. However, RCS rats that received the ChR2 gene in the AAV vector did not track the rotation of the virtual optomotor at any spatial frequency. Recently, we tested the behavior of RCS rats using a mechanical optomotor system and showed that the intensity of luminosity the rat received was the most important factor influencing their tracking of the rotation of the column [53]. A luminosity of over 500 lux was needed to induce head tracking in ChR2-expressing RCS rats. However, the maximum luminosity of the virtual optomotor was about 100 lux. It was therefore too low to induce head tracking in RCS rats. The question then arises: what is the difference between the TG and RCS rats? We do not have a reasonable explanation for this. One possibility is that the number of ChR2-expressing RGCs in the TG rat is greater than that in the RCS rat. About 45% of the RGCs expressed ChR2 in the TG rat. Compared to the TG rat, the transduction efficiency in the RCS rat is about 28% independent of the age of the animal. This may affect the light sensitivity. As the another explanation, in the case of TG rats, ChR2 is expressed after birth; therefore, there is a possibility that retinal organization and function might be altered, that cannot be ruled out.

of photosensitive RGCs. In the normal visual pathway, the light incident upon the eyes is first received by photoreceptor cells located at the end of the retinal layers. The photoreceptor cells control neurotransmitter release, and second-order neurons located in the inner nuclear layer respond to the neurotransmitter. Finally, RGCs produce action potentials and transmit to the lateral geniculate nucleus (LGN) via the optic nerve (Fig. 6). In RP, the photo-acquisition system is damaged due to the degeneration of photoreceptor cells, even if the other retinal layers remain intact. RGCs that are rendered photosensitive by the transfer of the ChR2 gene can directly respond to light and transmit signals to the brain. In this newly organized photoacquisition system, the other retinal neurons besides the RGCs are not required for the

**Figure 5.** Behavioral assessment using a digital optomotor. (A) The digital optomotor consisted of 4 displays sur‐ rounding a platform. The number of stripes and the contrast were controlled by software. (B) Superimposed images from movies showed that the rats were able to discriminate the moving stripes. The arrow indicates the direction of the moving stripes. The asterisk indicates the point of the rat's nose. (C) The contrast sensitivities of photoreceptordegenerated Thy-I TG rats were higher at low spatial frequencies compared to those of wild-type (normal) rats (n = 8).

Although VEP responses recovered after ChR2 gene transfer, the amplitudes and waveforms were different between rats injected with AAV-ChR2V at 6 and 10 months of age. One possibility is that RGC activity decayed after photoreceptor degeneration. However, our data show that the number of RGCs decreased after photoreceptor degeneration (Fig. 3C). The calculated RGC transduction efficiencies in 6-month-old rats were the same as those in 10 month-old rats. The differences in the recorded amplitudes and latencies shown in Fig. 2 appear to be due to differences in the number of photosensitive RGCs. We previously reported that the RGC transduction efficiency in 10-month-old rats was about 28% [38]. Subsequently, Isago et al. showed that the RGC transduction efficiencies in 6- and 10-month-old rats were 28.3 and 27.7%, respectively [56]. The data clearly indicates that the transduction efficiency is the same, although the number of ChR2-expressing cells was lower, corresponding to the

perception of light.

500 Gene Therapy - Tools and Potential Applications

decrease in the number of RGCs.

RGCs are merely one of the candidate cell types that could receive the ChR2 gene. Lagali et al. [60] succeeded in transferring the ChR2 gene into ON-bipolar cells in the retina and confirmed the restoration of visual and behavioral responses. ON- and OFF-bipolar cells receive synaptic input from photoreceptors. Considering that ChR2 can elicit light-on responses, ON-bipolar cells seem to be the most appropriate cells for the transfer of the ChR2 gene. However, 2 questions arise in this regard. First, how can we deliver the ChR2 gene into ON-bipolar cells for human gene therapy? Lagali et al. [60] transferred the ChR2 gene into neonatal mice by electroporation of the plasmid vector. It is generally difficult to transfer a gene into the depths of the retina via intravitreous injection of AAV vectors, in spite of the development of various serotypes of AAV vectors for retinal gene therapy [61-65]. Second, does synaptic transmission remain intact after photoreceptor degeneration? Some studies have reported that retinal remodeling is triggered in bipolar cells and horizontal cells following photoreceptor degen‐ eration [66-70]. Recently, Doroudchi et al. [71] succeeded in transferring the ChR2 gene into ON-bipolar cells by the subretinal injection of a modified AAV vector (AAV8-Y733F) [72] that included a specific promoter for ON-bipolar cells (mGRM6-SV40), and demonstrated the behavioral recovery of the light response. These 2 questions could be resolved by these attractive methods used the specific promoter and the modified AAV vectors if the recovered visual acuity is investigated using a behavioral approach.

**Author details**

**References**

Hiroshi Tomita1,2\*, Eriko Sugano1

368(9549), 1795-1809.

(2007). , 144(6), 791-811.

\*Address all correspondence to: htomita@iwate-u.ac.jp

2 Tohoku University Hospital, Sendai, Miyagi, Japan

analysis. Arch Ophthalmol (1997). , 115(4), 511-515.

mentosa. Arch Ophthalmol (1992). , 110(11), 1634-1639.

retinitis pigmentosa. Nature (1990). , 343(6256), 364-366.

thalmic research. Surv Ophthalmol (1992). , 37(1), 63-68.

Arch Ophthalmol (2004). , 122(7), 1029-1037.

, Hitomi Isago1

1 Department of Chemistry and Bioengineering, Iwate University, Morioka, Iwate, Japan

[1] Humayun, M. S, Prince, M, & De Juan, E. Jr., Barron Y, Moskowitz M, Klock IB, Mi‐ lam AH. Morphometric analysis of the extramacular retina from postmortem eyes

[2] Santos, A, Humayun, M. S, & De Juan, E. Jr., Greenburg RJ, Marsh MJ, Klock IB, Mi‐ lam AH. Preservation of the inner retina in retinitis pigmentosa. A morphometric

[3] Stone, J. L, Barlow, W. E, Humayun, M. S, & De Juan, E. Jr., Milam AH. Morphomet‐ ric analysis of macular photoreceptors and ganglion cells in retinas with retinitis pig‐

[4] Hartong, D. T, Berson, E. L, & Dryja, T. P. Retinitis pigmentosa. Lancet (2006). ,

[5] Dryja, T. P, Mcgee, T. L, Reichel, E, Hahn, L. B, Cowley, G. S, Yandell, D. W, Sand‐ berg, M. A, & Berson, E. L. A point mutation of the rhodopsin gene in one form of

[6] Stone, E. M. Leber congenital amaurosis- a model for efficient genetic testing of het‐ erogeneous disorders: LXIV Edward Jackson Memorial Lecture. Am J Ophthalmol

[7] Blum, M, Hykin, P. G, Sanders, M, & Volcker, H. E. Theodor Leber: a founder of oph‐

[8] Dharmaraj, S, Leroy, B. P, Sohocki, M. M, Koenekoop, R. K, Perrault, I, Anwar, K, Khaliq, S, Devi, R. S, Birch, D. G, De Pool, E, Izquierdo, N, Van Maldergem, L, Ismail, M, Payne, A. M, Holder, G. E, Bhattacharya, S. S, Bird, A. C, Kaplan, J, & Maumenee, I. H. The phenotype of Leber congenital amaurosis in patients with AIPL1 mutations.

with retinitis pigmentosa. Invest Ophthalmol Vis Sci (1999). , 40(1), 143-148.

, Namie Murayama1

and Makoto Tamai2

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 503

Since the discovery of ChR2, bacteriorhodopsins that have similar functions as that of ChR2 derived from *Chlamydomonas* have been identified. Channelrhodopsin-1 from the green alga *Volvox* [73] is a light-activated cation channel that has a different wavelength sensitivity from that of *Chlamydomonas*-derived ChR2. Halorhodopsin, which functions as a light-activated chloride channel, has been identified in *Halobacterium salinarum* [74,75]. Researchers have attempted to discover new light-activated ion channel genes, or to artificially design more functional ones [76-78]. In the future, more effective gene therapy strategies for restoring vision in RP might be developed using newly developed genes and vectors.

#### **6. Conclusion**

Target diseases for gene therapy were previously restricted to lethal and severe diseases that lead to death. In our country (Japan), the gene therapy guidelines were updated in 2002, whereby diseases in which bodily functions are severely impaired, such as loss of arms or legs, blindness, and deafness, were added to the list of target diseases for gene therapy. Based on these guidelines, people suffering from impaired vision caused by RP are eligible for gene therapy. However, gene therapy using genes derived from living organisms other than humans has not previously been tested in clinical trials. Safety studies, especially immuno‐ logical reactions, using appropriate animal models in ChR2-based gene therapy is important before proceeding to clinical trials.

#### **Acknowledgements**

This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 24390393 and 23659804) and the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). We express our heartfelt appreciation to Dr. Ichiro Hagimori in Narita Animal Science Laboratory Co. Ltd., whose enormous support and insightful comments were invaluable during the course of this study.

#### **Author details**

of the retina via intravitreous injection of AAV vectors, in spite of the development of various serotypes of AAV vectors for retinal gene therapy [61-65]. Second, does synaptic transmission remain intact after photoreceptor degeneration? Some studies have reported that retinal remodeling is triggered in bipolar cells and horizontal cells following photoreceptor degen‐ eration [66-70]. Recently, Doroudchi et al. [71] succeeded in transferring the ChR2 gene into ON-bipolar cells by the subretinal injection of a modified AAV vector (AAV8-Y733F) [72] that included a specific promoter for ON-bipolar cells (mGRM6-SV40), and demonstrated the behavioral recovery of the light response. These 2 questions could be resolved by these attractive methods used the specific promoter and the modified AAV vectors if the recovered

Since the discovery of ChR2, bacteriorhodopsins that have similar functions as that of ChR2 derived from *Chlamydomonas* have been identified. Channelrhodopsin-1 from the green alga *Volvox* [73] is a light-activated cation channel that has a different wavelength sensitivity from that of *Chlamydomonas*-derived ChR2. Halorhodopsin, which functions as a light-activated chloride channel, has been identified in *Halobacterium salinarum* [74,75]. Researchers have attempted to discover new light-activated ion channel genes, or to artificially design more functional ones [76-78]. In the future, more effective gene therapy strategies for restoring vision

Target diseases for gene therapy were previously restricted to lethal and severe diseases that lead to death. In our country (Japan), the gene therapy guidelines were updated in 2002, whereby diseases in which bodily functions are severely impaired, such as loss of arms or legs, blindness, and deafness, were added to the list of target diseases for gene therapy. Based on these guidelines, people suffering from impaired vision caused by RP are eligible for gene therapy. However, gene therapy using genes derived from living organisms other than humans has not previously been tested in clinical trials. Safety studies, especially immuno‐ logical reactions, using appropriate animal models in ChR2-based gene therapy is important

This work was partly supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 24390393 and 23659804) and the Program for the Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (NIBIO). We express our heartfelt appreciation to Dr. Ichiro Hagimori in Narita Animal Science Laboratory Co. Ltd., whose enormous support and

insightful comments were invaluable during the course of this study.

visual acuity is investigated using a behavioral approach.

502 Gene Therapy - Tools and Potential Applications

**6. Conclusion**

before proceeding to clinical trials.

**Acknowledgements**

in RP might be developed using newly developed genes and vectors.

Hiroshi Tomita1,2\*, Eriko Sugano1 , Hitomi Isago1 , Namie Murayama1 and Makoto Tamai2

\*Address all correspondence to: htomita@iwate-u.ac.jp


#### **References**


[9] Dryja, T. P, Adams, S. M, Grimsby, J. L, Mcgee, T. L, Hong, D. H, Li, T, Andreasson, S, & Berson, E. L. Null RPGRIP1 alleles in patients with Leber congenital amaurosis. Am J Hum Genet (2001). , 68(5), 1295-1298.

[19] Maguire, A. M, Simonelli, F, Pierce, E. A, & Pugh, E. N. Jr., Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auric‐ chio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 505

[20] Bainbridge, J. W, Smith, A. J, Barker, S. S, Robbie, S, Henderson, R, Balaggan, K, Vis‐ wanathan, A, Holder, G. E, Stockman, A, Tyler, N, Petersen-jones, S, Bhattacharya, S. S, Thrasher, A. J, Fitzke, F. W, Carter, B. J, Rubin, G. S, Moore, A. T, & Ali, R. R. Effect of gene therapy on visual function in Leber's congenital amaurosis. N Engl J Med

[21] Maguire, A. M, High, K. A, Auricchio, A, Wright, J. F, Pierce, E. A, Testa, F, Mingoz‐ zi, F, Bennicelli, J. L, Ying, G. S, Rossi, S, Fulton, A, Marshall, K. A, Banfi, S, Chung, D. C, Morgan, J. I, Hauck, B, Zelenaia, O, Zhu, X, Raffini, L, Coppieters, F, De Baere, E, Shindler, K. S, Volpe, N. J, Surace, E. M, Acerra, C, Lyubarsky, A, Redmond, T. M, Stone, E, Sun, J, Mcdonnell, J. W, Leroy, B. P, Simonelli, F, & Bennett, J. Age-depend‐ ent effects of RPE65 gene therapy for Leber's congenital amaurosis: a phase 1 dose-

[22] Simonelli, F, Maguire, A. M, Testa, F, Pierce, E. A, Mingozzi, F, Bennicelli, J. L, Rossi, S, Marshall, K, Banfi, S, Surace, E. M, Sun, J, Redmond, T. M, Zhu, X, Shindler, K. S, Ying, G. S, Ziviello, C, Acerra, C, Wright, J. F, Mcdonnell, J. W, High, K. A, Bennett, J, & Auricchio, A. Gene therapy for Leber's congenital amaurosis is safe and effective

through 1.5 years after vector administration. Mol Ther (2010). , 18(3), 643-650. [23] Colella, P, & Auricchio, A. Gene Therapy of Inherited Retinopathies: A Long and Successful Road from Viral Vectors to Patients. Hum Gene Ther (2012). , 23(8),

[24] Berson, E. L, Sandberg, M. A, & Dryja, T. P. Autosomal dominant retinitis pigmento‐ sa with rhodopsin, valine-345-methionine. Trans Am Ophthalmol Soc (1991). discus‐

[25] Olsson, J. E, Gordon, J. W, Pawlyk, B. S, Roof, D, Hayes, A, Molday, R. S, Mukai, S, Cowley, G. S, Berson, E. L, & Dryja, T. P. Transgenic mice with a rhodopsin mutation (Pro23His): a mouse model of autosomal dominant retinitis pigmentosa. Neuron

[26] Chang, G. Q, Hao, Y, & Wong, F. Apoptosis: final common pathway of photorecep‐ tor death in rd, rds, and rhodopsin mutant mice. Neuron (1993). , 11(4), 595-605. [27] Ranchon, I. LaVail MM, Kotake Y, Anderson RE. Free radical trap phenyl-N-tert-bu‐ tylnitrone protects against light damage but does not rescue and S334ter rhodopsin

transgenic rats from inherited retinal degeneration. J Neurosci (2003). , 23H.

amaurosis. N Engl J Med (2008). , 358(21), 2240-2248.

escalation trial. Lancet (2009). , 374(9701), 1597-1605.

(2008). , 358(21), 2231-2239.

796-807.

sion 128-130., 89, 117-128.

(1992). , 9(5), 815-830.


[19] Maguire, A. M, Simonelli, F, Pierce, E. A, & Pugh, E. N. Jr., Mingozzi F, Bennicelli J, Banfi S, Marshall KA, Testa F, Surace EM, Rossi S, Lyubarsky A, Arruda VR, Konkle B, Stone E, Sun J, Jacobs J, Dell'Osso L, Hertle R, Ma JX, Redmond TM, Zhu X, Hauck B, Zelenaia O, Shindler KS, Maguire MG, Wright JF, Volpe NJ, McDonnell JW, Auric‐ chio A, High KA, Bennett J. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N Engl J Med (2008). , 358(21), 2240-2248.

[9] Dryja, T. P, Adams, S. M, Grimsby, J. L, Mcgee, T. L, Hong, D. H, Li, T, Andreasson, S, & Berson, E. L. Null RPGRIP1 alleles in patients with Leber congenital amaurosis.

[10] Lotery, A. J, Jacobson, S. G, Fishman, G. A, Weleber, R. G, Fulton, A. B, Namperu‐ malsamy, P, Heon, E, Levin, A. V, Grover, S, Rosenow, J. R, Kopp, K. K, Sheffield, V. C, & Stone, E. M. Mutations in the CRB1 gene cause Leber congenital amaurosis.

[11] Marlhens, F, Bareil, C, Griffoin, J. M, Zrenner, E, Amalric, P, Eliaou, C, Liu, S. Y, Har‐ ris, E, Redmond, T. M, Arnaud, B, Claustres, M, & Hamel, C. P. Mutations in RPE65

[12] Perrault, I, Rozet, J. M, Calvas, P, Gerber, S, Camuzat, A, Dollfus, H, Chatelin, S, Sou‐ ied, E, Ghazi, I, Leowski, C, & Bonnemaison, M. Le Paslier D, Frezal J, Dufier JL, Pit‐ tler S, Munnich A, Kaplan J. Retinal-specific guanylate cyclase gene mutations in

[13] Perrault, I, Hanein, S, Zanlonghi, X, Serre, V, Nicouleau, M, Defoort-delhemmes, S, Delphin, N, Fares-taie, L, Gerber, S, Xerri, O, Edelson, C, Goldenberg, A, & Dun‐ combe, A. Le Meur G, Hamel C, Silva E, Nitschke P, Calvas P, Munnich A, Roche O, Dollfus H, Kaplan J, Rozet JM. Mutations in NMNAT1 cause Leber congenital amau‐ rosis with early-onset severe macular and optic atrophy. Nat Genet (2012). in press.

[14] Cremers, F. P. van den Hurk JA, den Hollander AI. Molecular genetics of Leber con‐

[15] Acland, G. M, Aguirre, G. D, Ray, J, Zhang, Q, Aleman, T. S, Cideciyan, A. V, Pearcekelling, S. E, Anand, V, Zeng, Y, Maguire, A. M, Jacobson, S. G, Hauswirth, W. W, & Bennett, J. Gene therapy restores vision in a canine model of childhood blindness.

[16] Acland, G. M, Aguirre, G. D, Bennett, J, Aleman, T. S, Cideciyan, A. V, Bennicelli, J, Dejneka, N. S, Pearce-kelling, S. E, Maguire, A. M, Palczewski, K, Hauswirth, W. W, & Jacobson, S. G. Long-term restoration of rod and cone vision by single dose rAAVmediated gene transfer to the retina in a canine model of childhood blindness. Mol

[17] Simonelli, F, Ziviello, C, Testa, F, Rossi, S, Fazzi, E, Bianchi, P. E, Fossarello, M, Si‐ gnorini, S, Bertone, C, Galantuomo, S, Brancati, F, Valente, E. M, Ciccodicola, A, Ri‐ naldi, E, Auricchio, A, & Banfi, S. Clinical and molecular genetics of Leber's congenital amaurosis: a multicenter study of Italian patients. Invest Ophthalmol Vis

[18] Cideciyan, A. V, Hauswirth, W. W, Aleman, T. S, Kaushal, S, Schwartz, S. B, Boye, S. L, Windsor, E. A, Conlon, T. J, Sumaroka, A, Roman, A. J, Byrne, B. J, & Jacobson, S. G. Vision 1 year after gene therapy for Leber's congenital amaurosis. N Engl J Med

cause Leber's congenital amaurosis. Nat Genet (1997). , 17(2), 139-141.

Leber's congenital amaurosis. Nat Genet (1996). , 14(4), 461-464.

genital amaurosis. Hum Mol Genet (2002). , 11(10), 1169-1176.

Nat Genet (2001). , 28(1), 92-95.

Ther (2005). , 12(6), 1072-1082.

Sci (2007). , 48(9), 4284-4290.

(2009). , 361(7), 725-727.

Am J Hum Genet (2001). , 68(5), 1295-1298.

504 Gene Therapy - Tools and Potential Applications

Arch Ophthalmol (2001). , 119(3), 415-420.


[28] Tomita, H, Kotake, Y, & Anderson, R. E. Mechanism of protection from light-induced retinal degeneration by the synthetic antioxidant phenyl-N-tert-butylnitrone. Invest Ophthalmol Vis Sci (2005). , 46(2), 427-434.

[41] Ishizuka, T, Kakuda, M, Araki, R, & Yawo, H. Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neu‐

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 507

[42] Li, X, Gutierrez, D. V, Hanson, M. G, Han, J, Mark, M. D, Chiel, H, Hegemann, P, Landmesser, L. T, & Herlitze, S. Fast noninvasive activation and inhibition of neural and network activity by vertebrate rhodopsin and green algae channelrhodopsin.

[43] LaVail MM, Sidman RL, O'Neil D.Photoreceptor-pigment epithelial cell relationships in rats with inherited retinal degeneration. Radioautographic and electron micro‐ scope evidence for a dual source of extra lamellar material. J Cell Biol (1972). , 53(1),

[44] Mullen, R. J. LaVail MM. Inherited retinal dystrophy: primary defect in pigment epi‐ thelium determined with experimental rat chimeras. Science (1976). , 192(4241),

[45] Cruz, D, Yasumura, PM, Weir, D, Matthes, J, Abderrahim, MT, & La, H. , Vollrath D. Mutation of the receptor tyrosine kinase gene Mertk in the retinal dystrophic RCS

[46] Gal, A, Li, Y, Thompson, D. A, Weir, J, Orth, U, Jacobson, S. G, Apfelstedt-sylla, E, & Vollrath, D. Mutations in MERTK, the human orthologue of the RCS rat retinal dys‐

[47] Tomita, H, Sugano, E, Fukazawa, Y, Isago, H, Sugiyama, Y, Hiroi, T, Ishizuka, T, Mushiake, H, Kato, M, Hirabayashi, M, Shigemoto, R, Yawo, H, & Tamai, M. Visual properties of transgenic rats harboring the channelrhodopsin-2 gene regulated by the

[48] Barclay, A. N, & Hyden, H. Localizatin of the Thy-1 antigen in rat brain and spinal

[49] Mason, D. W, & Williams, A. F. The kinetics of antibody binding to membrane anti‐

[50] Barnstable, C. J, & Drager, U. C. Thy-1 antigen: a ganglion cell specific marker in ro‐

[51] Perry, V. H, Morris, R. J, & Raisman, G. Is Thy-1 expressed only by ganglion cells and their axons in the retina and optic nerve? J Neurocytol (1984). , 13(5), 809-824.

[52] Sugano, E, Tomita, H, Ishiguro, S, Abe, T, & Tamai, M. Establishment of effective methods for transducing genes into iris pigment epithelial cells by using adeno-asso‐

cord by immunofluorescence. J Neurochem (1978). , 31(6), 1375-1391.

gens in solution and at the cell surface. Biochem J (1980). , 187(1), 1-20.

ciated virus type 2. Invest Ophthalmol Vis Sci (2005). , 46(9), 3341-3348.

trophy gene, cause retinitis pigmentosa. Nat Genet (2000). , 26(3), 270-271.

Proc Natl Acad Sci U S A (2005). , 102(49), 17816-17821.

rosci Res (2006). , 54(2), 85-94.

rat. Hum Mol Genet 2000;9(4):645-651.

thy-1.2 promoter. PLoS One (2009). e7679.

dent retina. Neuroscience (1984). , 11(4), 847-855.

185-209.

799-801.


[41] Ishizuka, T, Kakuda, M, Araki, R, & Yawo, H. Kinetic evaluation of photosensitivity in genetically engineered neurons expressing green algae light-gated channels. Neu‐ rosci Res (2006). , 54(2), 85-94.

[28] Tomita, H, Kotake, Y, & Anderson, R. E. Mechanism of protection from light-induced retinal degeneration by the synthetic antioxidant phenyl-N-tert-butylnitrone. Invest

[29] Cayouette, M, & Gravel, C. Adenovirus-mediated gene transfer of ciliary neurotro‐ phic factor can prevent photoreceptor degeneration in the retinal degeneration (rd)

[30] Jomary, C, Vincent, K. A, Grist, J, Neal, M. J, & Jones, S. E. Rescue of photoreceptor function by AAV-mediated gene transfer in a mouse model of inherited retinal de‐

[31] Bennett, J, Zeng, Y, Bajwa, R, Klatt, L, Li, Y, & Maguire, A. M. Adenovirus-mediated delivery of rhodopsin-promoted bcl-2 results in a delay in photoreceptor cell death in

[32] Chow, A. Y, & Peachey, N. The subretinal microphotodiode array retinal prosthesis

[33] Dobelle, W. H. Artificial vision for the blind by connecting a television camera to the

[34] Rizzo, J. F. rd, Wyatt J, Loewenstein J, Kelly S, Shire D. Perceptual efficacy of electri‐ cal stimulation of human retina with a microelectrode array during short-term surgi‐

[35] Gekeler, F, Kobuch, K, Schwahn, H. N, Stett, A, Shinoda, K, & Zrenner, E. Subretinal electrical stimulation of the rabbit retina with acutely implanted electrode arrays.

[36] Weiland, J. D, Cho, A. K, & Humayun, M. S. Retinal prostheses: current clinical re‐

[37] Bi, A, Cui, J, Ma, Y. P, Olshevskaya, E, Pu, M, Dizhoor, A. M, & Pan, Z. H. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with pho‐

[38] Tomita, H, Sugano, E, Yawo, H, Ishizuka, T, Isago, H, Narikawa, S, Kugler, S, & Tamai, M. Restoration of visual response in aged dystrophic RCS rats using AAVmediated channelopsin-2 gene transfer. Invest Ophthalmol Vis Sci (2007). , 48(8),

[39] Nagel, G, Szellas, T, Huhn, W, Kateriya, S, Adeishvili, N, Berthold, P, Ollig, D, He‐ gemann, P, & Bamberg, E. Channelrhodopsin-2, a directly light-gated cation-selective

[40] Boyden, E. S, Zhang, F, Bamberg, E, Nagel, G, & Deisseroth, K. Millisecond-time‐ scale, genetically targeted optical control of neural activity. Nat Neurosci (2005). ,

membrane channel. Proc Natl Acad Sci U S A (2003). , 100(24), 13940-13945.

cal trials. Invest Ophthalmol Vis Sci (2003). , 44(12), 5362-5369.

Graefes Arch Clin Exp Ophthalmol (2004). , 242(7), 587-596.

toreceptor degeneration. Neuron (2006). , 50(1), 23-33.

sults and future needs. Ophthalmology (2011). , 118(11), 2227-2237.

Ophthalmol Vis Sci (2005). , 46(2), 427-434.

mouse. Hum Gene Ther (1997). , 8(4), 423-430.

generation. Gene Ther (1997). , 4(7), 683-690.

visual cortex. ASAIO J (2000). , 46(1), 3-9.

II. Ophthalmic Res (1999).

506 Gene Therapy - Tools and Potential Applications

3821-3826.

8(9), 1263-1268.

the rd/rd mouse. Gene Ther (1998). , 5(9), 1156-1164.


[53] Tomita, H, Sugano, E, Isago, H, Hiroi, T, Wang, Z, Ohta, E, & Tamai, M. Channelrho‐ dopsin-2 gene transduced into retinal ganglion cells restores functional vision in ge‐ netically blind rats. Exp Eye Res (2010). , 90(3), 429-436.

recombinant feline immunodeficiency virus vectors. Hum Gene Ther (2002). , 13(6),

Gene Therapy for Retinitis Pigmentosa http://dx.doi.org/10.5772/52987 509

[65] Allocca, M, Mussolino, C, Garcia-hoyos, M, Sanges, D, Iodice, C, Petrillo, M, Vanden‐ berghe, L. H, Wilson, J. M, Marigo, V, Surace, E. M, & Auricchio, A. Novel adenoassociated virus serotypes efficiently transduce murine photoreceptors. J Virol

[66] Marc, R. E, Jones, B. W, Anderson, J. R, Kinard, K, Marshak, D. W, Wilson, J. H, Wen‐ sel, T, & Lucas, R. J. Neural reprogramming in retinal degeneration. Invest Ophthal‐

[67] Marc, R. E, Jones, B. W, Watt, C. B, & Strettoi, E. Neural remodeling in retinal degen‐

[68] Strettoi, E, & Pignatelli, V. Modifications of retinal neurons in a mouse model of reti‐

[69] Strettoi, E, Pignatelli, V, Rossi, C, Porciatti, V, & Falsini, B. Remodeling of second-or‐ der neurons in the retina of rd/rd mutant mice. Vision Res (2003). , 43(8), 867-877. [70] Strettoi, E, Porciatti, V, Falsini, B, Pignatelli, V, & Rossi, C. Morphological and func‐ tional abnormalities in the inner retina of the rd/rd mouse. J Neurosci (2002). , 22(13),

[71] Doroudchi, M. M, Greenberg, K. P, Liu, J, Silka, K. A, Boyden, E. S, Lockridge, J. A, Arman, A. C, Janani, R, Boye, S. E, Boye, S. L, Gordon, G. M, Matteo, B. C, Sampath, A. P, Hauswirth, W. W, & Horsager, A. Virally delivered channelrhodopsin-2 safely and effectively restores visual function in multiple mouse models of blindness. Mol

[72] Pang, J. J, Dai, X, Boye, S. E, Barone, I, Boye, S. L, Mao, S, Everhart, D, Dinculescu, A, Liu, L, Umino, Y, Lei, B, Chang, B, Barlow, R, Strettoi, E, & Hauswirth, W. W. Longterm retinal function and structure rescue using capsid mutant AAV8 vector in the rd10 mouse, a model of recessive retinitis pigmentosa. Mol Ther (2011). , 19(2),

[73] Zhang, F, Prigge, M, Beyriere, F, Tsunoda, S. P, Mattis, J, Yizhar, O, Hegemann, P, & Deisseroth, K. Red-shifted optogenetic excitation: a tool for fast neural control de‐

[74] Zhang, Y, Ivanova, E, Bi, A, & Pan, Z. H. Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor de‐

[75] Kolbe, M, Besir, H, Essen, L. O, & Oesterhelt, D. Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution. Science (2000). , 288(5470), 1390-1396.

rived from Volvox carteri. Nat Neurosci (2008). , 11(6), 631-633.

generation. J Neurosci (2009). , 29(29), 9186-9196.

nitis pigmentosa. Proc Natl Acad Sci U S A (2000). , 97(20), 11020-11025.

689-696.

5492-5504.

234-242.

Ther (2011). , 19(7), 1220-1229.

(2007). , 81(20), 11372-11380.

mol Vis Sci (2007). , 48(7), 3364-3371.

eration. Prog Retin Eye Res (2003). , 22(5), 607-655.


recombinant feline immunodeficiency virus vectors. Hum Gene Ther (2002). , 13(6), 689-696.

[65] Allocca, M, Mussolino, C, Garcia-hoyos, M, Sanges, D, Iodice, C, Petrillo, M, Vanden‐ berghe, L. H, Wilson, J. M, Marigo, V, Surace, E. M, & Auricchio, A. Novel adenoassociated virus serotypes efficiently transduce murine photoreceptors. J Virol (2007). , 81(20), 11372-11380.

[53] Tomita, H, Sugano, E, Isago, H, Hiroi, T, Wang, Z, Ohta, E, & Tamai, M. Channelrho‐ dopsin-2 gene transduced into retinal ganglion cells restores functional vision in ge‐

[54] Iwamura, Y, Fujii, Y, & Kamei, C. The effects of certain H(1)-antagonists on visual

[55] Papathanasiou, E. S, Peachey, N. S, Goto, Y, Neafsey, E. J, Castro, A. J, & Kartje, G. L. Visual cortical plasticity following unilateral sensorimotor cortical lesions in the neo‐

[56] Isago, H, Sugano, E, Wang, Z, Murayama, N, Koyanagi, E, Tamai, M, & Tomita, H. Age-dependent differences in recovered visual responses in Royal College of Sur‐ geons rats transduced with the Channelrhodopsin-2 gene. J Mol Neurosci (2012). ,

[57] Prusky, G. T, Alam, N. M, Beekman, S, & Douglas, R. M. Rapid quantification of adult and developing mouse spatial vision using a virtual optomotor system. Invest

[58] Sugano, E, Isago, H, Wang, Z, Murayama, N, Tamai, M, & Tomita, H. Immune re‐ sponses to adeno-associated virus type 2 encoding channelrhodopsin-2 in a geneti‐

[59] Rapp, L. M, & Smith, S. C. Morphologic comparisons between rhodopsin-mediated and short-wavelength classes of retinal light damage. Invest Ophthalmol Vis Sci

[60] Lagali, P. S, Balya, D, Awatramani, G. B, Munch, T. A, Kim, D. S, Busskamp, V, Cep‐ ko, C. L, & Roska, B. Light-activated channels targeted to ON bipolar cells restore

[61] Auricchio, A, Kobinger, G, Anand, V, Hildinger, M, Connor, O, Maguire, E, Wilson, A. M, & Bennett, J. M. J. Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum Mol Genet

[62] Yang, P, Seiler, M. J, Aramant, R. B, & Whittemore, S. R. Differential lineage restric‐ tion of rat retinal progenitor cells in vitro and in vivo. J Neurosci Res (2002). , 69(4),

[63] Weber, M, Rabinowitz, J, Provost, N, Conrath, H, Folliot, S, Briot, D, Cherel, Y, Che‐ nuaud, P, Samulski, J, Moullier, P, & Rolling, F. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigment‐ ed epithelium in rat, dog, and nonhuman primate after subretinal delivery. Mol Ther

[64] Lotery, A. J, Derksen, T. A, Russell, S. R, Mullins, R. F, Sauter, S, Affatigato, L. M, Stone, E. M, & Davidson, B. L. Gene transfer to the nonhuman primate retina with

visual function in retinal degeneration. Nat Neurosci (2008). , 11(6), 667-675.

cally blind rat model for gene therapy. Gene Ther (2011). , 18(3), 266-274.

netically blind rats. Exp Eye Res (2010). , 90(3), 429-436.

natal rat. Exp Neurol (2006). , 199(1), 122-129.

Ophthalmol Vis Sci (2004). , 45(12), 4611-4616.

46(2), 393-400.

508 Gene Therapy - Tools and Potential Applications

(1992). , 33(12), 3367-3377.

(2001). , 10(26), 3075-3081.

(2003). , 7(6), 774-781.

466-476.

evoked potential in rats. Brain Res Bull (2003). , 61(4), 393-398.


[76] Govorunova, E. G, Spudich, E. N, Lane, C. E, Sineshchekov, O. A, & Spudich, J. L. New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Meso‐ stigma viride. MBio (2011). ee00111., 00115.

**Chapter 21**

**Gene Therapy for Erythroid Metabolic Inherited**

Gene therapy is becoming a powerful tool to treat genetic diseases. Clinical trials performed during last two decades have demonstrated its usefulness in the treatment of several genetic diseases [1] but also the need to improve vector delivery, expression and safety [2]. New vectors should reduce genotoxicity (genomic alteration due to vector integration), immuno‐ genicity (immune response to gene delivery vectors and/or trangenes) and cytotoxicity (in‐

In mature erythrocytes, most metabolic needs are covered by glycolysis, oxidative pentose phosphate pathway and glutathione cycle. Hereditary enzyme deficiencies of all these path‐ ways have been identified, being most of them associated with chronic non-spherocytic he‐ molitic anemia (CNSHA). Hereditary hemolytic anemia exhibits a high molecular heterogeneity with a wide number of different mutations involved in the structural genes of nearly all affected enzymes. Deficiency in metabolic enzymes impairs energy balance in the erythrocytes, with or without changes in oxygen affinity of hemoglobin and delivery to the tissues. Despite of having a better understanding of their molecular basis, definitive curative

Conventional bone marrow transplantation allows the generation of donor-derived functional hematopoietic cells of all lineages in the host, and represents the standard of care or at least a val‐ id therapeutic option for many inherited diseases [3]. However, complications associated to al‐ logeneic transplantation can be as severe as the enzymatic deficiency. The recessive inheriting trait of most of these metabolic diseases and the confined enzymatic defect to the hematopoietic/

> © 2013 Garcia-Gomez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

duced by ectopic expression and/or overexpression of the transgene).

therapy for Red Blood Cells (RBC) enzyme defects still remains undeveloped.

Maria Garcia-Gomez, Oscar Quintana-Bustamante, Maria Garcia-Bravo, S. Navarro, Zita Garate and

Additional information is available at the end of the chapter

**Diseases**

Jose C. Segovia

**1. Introduction**

http://dx.doi.org/10.5772/52531


### **Gene Therapy for Erythroid Metabolic Inherited Diseases**

[76] Govorunova, E. G, Spudich, E. N, Lane, C. E, Sineshchekov, O. A, & Spudich, J. L. New channelrhodopsin with a red-shifted spectrum and rapid kinetics from Meso‐

[77] Prigge, M, Schneider, F, Tsunoda, S. P, Shilyansky, C, Wietek, J, Deisseroth, K, & He‐ gemann, P. Color-tuned Channelrhodopsins for Multiwavelength Optogenetics. J Bi‐

[78] Wang, H, Sugiyama, Y, Hikima, T, Sugano, E, Tomita, H, Takahashi, T, Ishizuka, T, & Yawo, H. Molecular determinants differentiating photocurrent properties of two channelrhodopsins from chlamydomonas. J Biol Chem (2009). , 284(9), 5685-5696.

stigma viride. MBio (2011). ee00111., 00115.

ol Chem (2012). in press.

510 Gene Therapy - Tools and Potential Applications

Maria Garcia-Gomez, Oscar Quintana-Bustamante, Maria Garcia-Bravo, S. Navarro, Zita Garate and Jose C. Segovia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52531

#### **1. Introduction**

Gene therapy is becoming a powerful tool to treat genetic diseases. Clinical trials performed during last two decades have demonstrated its usefulness in the treatment of several genetic diseases [1] but also the need to improve vector delivery, expression and safety [2]. New vectors should reduce genotoxicity (genomic alteration due to vector integration), immuno‐ genicity (immune response to gene delivery vectors and/or trangenes) and cytotoxicity (in‐ duced by ectopic expression and/or overexpression of the transgene).

In mature erythrocytes, most metabolic needs are covered by glycolysis, oxidative pentose phosphate pathway and glutathione cycle. Hereditary enzyme deficiencies of all these path‐ ways have been identified, being most of them associated with chronic non-spherocytic he‐ molitic anemia (CNSHA). Hereditary hemolytic anemia exhibits a high molecular heterogeneity with a wide number of different mutations involved in the structural genes of nearly all affected enzymes. Deficiency in metabolic enzymes impairs energy balance in the erythrocytes, with or without changes in oxygen affinity of hemoglobin and delivery to the tissues. Despite of having a better understanding of their molecular basis, definitive curative therapy for Red Blood Cells (RBC) enzyme defects still remains undeveloped.

Conventional bone marrow transplantation allows the generation of donor-derived functional hematopoietic cells of all lineages in the host, and represents the standard of care or at least a val‐ id therapeutic option for many inherited diseases [3]. However, complications associated to al‐ logeneic transplantation can be as severe as the enzymatic deficiency. The recessive inheriting trait of most of these metabolic diseases and the confined enzymatic defect to the hematopoietic/

© 2013 Garcia-Gomez et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

erythropoietic system, make them suitable diseases to be treated by gene therapy. Correction by gene therapy requires the stable transfer of a functional gene into the autologous self-renewing Hematopoietic stem cells (HSCs) and their mature progeny. Autologous BM transplantation of genetically corrected cells shows several advantages over the allogeneic procedure. First, it overcomes the limitation of human leukocyte antigen (HLA)-compatible donor availability, so it can be applied to every patient. Second, the reduction of morbidity and mortality associated with the transplant procedure, as there is no risk of graft versus host disease (GvHD) and conse‐ quently no need for post-transplant immunosuppression.

mother), whereas in homozygous females the mutations are transmitted from both parents. Thereby, female heterozygotes represent a red blood cell mosaic population, causing a wide

> jaundice, spleno- and hepatomegaly, hemoglobinuria, leukocyte disfunction, and susceptibility to infections

Reticulocytosis, splenomegaly, *hidrops foetalis*, and death in neonatal period

disturbances

neuromuscular disorders, mental retardation, frecuent infections and death *in utero*

disease type VII

5-oxoprolinuria, metabolic acidosis, central nervous system impairment

**Table 1.** Most Common Erythroid Metabolic Inherited Diseases. BM transplantation and gene therapy approaches

*GPI* 19q13.1 A.R neuromuscular

*BPGM* 7q31-q34 A.R erythrocytosis

A.R, autosomic recessive; D, donor; R, receptor; C, conditioning; P, protocol

*TPI1* 12p13 A.R

Deficiency (HK) *HK1* 10q22 A.R defects in platelets

se Deficiency (PFK) *PFKL* 21q22.3 A.R myopathy, storage

*GSS* 20q11.2 A.R

**Bone Marrow**

D: normal CBA/N+/+ mice + 5FU R: CBA Pk-1slc/ PK-1slc mice C: minimal (100 or 400 cGy) [6]

D: normal CBA/N+/+ mice R: CBA Pk-1slc/ PK-1slc mice C: no conditioning [8]

D: normal Basenji dogs R: PKD Basenji dogs C: sublethal dose (200 cGy) + mycophenolate memofetil + cyclosporine [10]

D: HLA-identical sister R: PKD severe patient C: busulfan + cyclophosphamide [12]

**Transplantation Gene Therapy**

Gene Therapy for Erythroid Metabolic Inherited Diseases

D: C57BL/6 mice P: Transduction of 5-FU treated BM cells with MMLV-hG6PD or MPSV-hG6PD vectors and subsequent transplantation. R: lethally irradiated C57BL/6 mice [5]

http://dx.doi.org/10.5772/52531

513

D: WT mice P: Transduction of 5-FU treated BM cells with pMNSM-hLPK retroviral vector and subsequent transplantation R: lethally irradiated mice [7]

D: CBA PK-1slc/PK-1slc mice P: Transgenic rescue using the μLCR-PKLR-hRPK construct [9]

D: WT mice P: Transduction of Lin-

cells with a MSFV-hRPK retroviral vector and subsequent transplantation R: lethally irradiated WT mice [11]

D: AcB55 mice P: Transduction of Lin-

cells with a MSFV-hRPK retroviral vector and subsequent transplantation R: lethally irradiated AcB55 mice [13]

Sca1+ BM

Sca1+ BM

range clinical picture.

Glucose-6 Phosphate Dehydrogenase Deficiency (G6PD)

Pyruvate Kinase

Glucose Phosphate Isomerase Deficiency (GPI)

Triose Phosphate Isomerase Deficiency (TPI)

Hexokinase

Phosphofructokina

Bisphosphoglycerat e Mutase Deficiency (BPGM)

Glutathion Synthetase Deficiency (GSD)

Deficiency (PKD) *PKLR* 1q21 A.R

**Disease Gene Chrom. Inheritance Other sympthoms**

*G6PD* Xq28 X-linked

To date, gene therapy approaches for the treatment of inherited metabolic deficiencies are still limited, mainly because of the frequent lack of selective advantage of genetically corrected cells. This implies that high levels of transgene expression are required, as well as an efficient trans‐ duction of HSCs. This requirement have already been described in different RBC diseases as in the erytropoietic protoporphyria (EPP) [4] caused by the deficiency of the last enzyme of the heme biosintesis pathway or in the piruvate kinase deficiency (PKD) [3], where there is an im‐ pairment in the final yield of ATP in RBC. Additionally, some RBC pathologies require switch‐ ing on expression of the transgene at only the proper stage of differentiation, which represents another challenge in the development of new gene therapy protocols.

#### **2. Gene therapy attempts for inherited metabolic diseases of erythrocytes**

Although more than 14 metabolic deficiencies have been identified causing CNSHA, ap‐ proaches of gene therapy have been done only in a few of them (Table 1). Below, we are in‐ cluding a short description of the different diseases and the attempts addressed.

Among glycolytic defects causing CNSHA, Glucose 6-phosphate dehydrogenase (G6PD) de‐ ficiency is the most common genetic disease. More than 400 million people are affected world wide, showing a vast variability of clinical features. G6PD catalyzes the first reaction of the pentose phosphate pathway, in which Glucose 6-phosphate (G6P) is oxidized and Nicotinamide adenine dinucleotide phosphate is reduced (NADPH) resulting in decarboxy‐ lation of CO2 and pentose phosphate. G6PD plays a central role in the cellular physiology as it is the major source of NADPH, required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system and others. Indeed, G6PD is essential for cell survival. *G6PD* is a 20 kb X-linked gene that maps to the Xq28 region, consisting of 13 exons and 12 introns, which encode a 514 amino acids protein with ubiquitous expression. More than 100 missense mutations in the *G6PD* gene have been identified [14], being most of them single-point mutations causing an amino acid substitution. Frequently, these mutations cause mild symptoms or no disease, except when patients are challenged by increased oxidative stress or fava beans. However, some muta‐ tions provoke severe instability of the G6PD and, as a result, lifelong CNSHA with a varia‐ ble severity [15,16]. Through genetic studies it has been observed that severe clinical manifestations appear preferentially in exons 7, 10 and 11. As *G6PD* is X-linked, the defect is fully expressed in affected males (hemizygotes who inherit the mutation only from the mother), whereas in homozygous females the mutations are transmitted from both parents. Thereby, female heterozygotes represent a red blood cell mosaic population, causing a wide range clinical picture.

erythropoietic system, make them suitable diseases to be treated by gene therapy. Correction by gene therapy requires the stable transfer of a functional gene into the autologous self-renewing Hematopoietic stem cells (HSCs) and their mature progeny. Autologous BM transplantation of genetically corrected cells shows several advantages over the allogeneic procedure. First, it overcomes the limitation of human leukocyte antigen (HLA)-compatible donor availability, so it can be applied to every patient. Second, the reduction of morbidity and mortality associated with the transplant procedure, as there is no risk of graft versus host disease (GvHD) and conse‐

To date, gene therapy approaches for the treatment of inherited metabolic deficiencies are still limited, mainly because of the frequent lack of selective advantage of genetically corrected cells. This implies that high levels of transgene expression are required, as well as an efficient trans‐ duction of HSCs. This requirement have already been described in different RBC diseases as in the erytropoietic protoporphyria (EPP) [4] caused by the deficiency of the last enzyme of the heme biosintesis pathway or in the piruvate kinase deficiency (PKD) [3], where there is an im‐ pairment in the final yield of ATP in RBC. Additionally, some RBC pathologies require switch‐ ing on expression of the transgene at only the proper stage of differentiation, which represents

**2. Gene therapy attempts for inherited metabolic diseases of erythrocytes**

Although more than 14 metabolic deficiencies have been identified causing CNSHA, ap‐ proaches of gene therapy have been done only in a few of them (Table 1). Below, we are in‐

Among glycolytic defects causing CNSHA, Glucose 6-phosphate dehydrogenase (G6PD) de‐ ficiency is the most common genetic disease. More than 400 million people are affected world wide, showing a vast variability of clinical features. G6PD catalyzes the first reaction of the pentose phosphate pathway, in which Glucose 6-phosphate (G6P) is oxidized and Nicotinamide adenine dinucleotide phosphate is reduced (NADPH) resulting in decarboxy‐ lation of CO2 and pentose phosphate. G6PD plays a central role in the cellular physiology as it is the major source of NADPH, required by many essential cellular systems including the antioxidant pathways, nitric oxide synthase, NADPH oxidase, cytochrome p450 system and others. Indeed, G6PD is essential for cell survival. *G6PD* is a 20 kb X-linked gene that maps to the Xq28 region, consisting of 13 exons and 12 introns, which encode a 514 amino acids protein with ubiquitous expression. More than 100 missense mutations in the *G6PD* gene have been identified [14], being most of them single-point mutations causing an amino acid substitution. Frequently, these mutations cause mild symptoms or no disease, except when patients are challenged by increased oxidative stress or fava beans. However, some muta‐ tions provoke severe instability of the G6PD and, as a result, lifelong CNSHA with a varia‐ ble severity [15,16]. Through genetic studies it has been observed that severe clinical manifestations appear preferentially in exons 7, 10 and 11. As *G6PD* is X-linked, the defect is fully expressed in affected males (hemizygotes who inherit the mutation only from the

cluding a short description of the different diseases and the attempts addressed.

quently no need for post-transplant immunosuppression.

512 Gene Therapy - Tools and Potential Applications

another challenge in the development of new gene therapy protocols.


**Table 1.** Most Common Erythroid Metabolic Inherited Diseases. BM transplantation and gene therapy approaches

Patients with CNSHA suffer anemia and jaundice, but often tolerate their condition well. However, G6PD variants with low activity are related with alterations in the erythrocyte membrane facilitating its breakdown and causing intravasal hemolysis. These symptoms are often accompanied by spleno- and hepatomegaly and hemoglobinuria. Besides, leukocyte dysfunctions caused by lower concentration of NADPH appear when G6PD activity is be‐ low 5% of the normal activity, leading to an immune depression [17]. Vives *et al.* and other groups have also observed an increased susceptibility to infections [18,19].

intestine) and RPK (restricted to erythrocytes) through the use of alternative promoters [24]. PK-M1 is expressed in adult nomal tissue, like brain or muscle. The PK-M2 isoform is typically expressed in proliferating tissues like fetal, tumoral and several other adult tissues [25] and dur‐ ing the maturation of the erythroblasts, gradually decreases, giving rise to the RPK isoform.

Gene Therapy for Erythroid Metabolic Inherited Diseases

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515

The codifying region of *PK-LR* gene is split into twelve exons, ten of which are shared by the two isoforms, while exons 1 and 2 are specific for the erythrocyte and the hepatic isoenzyme respec‐ tively [26]. However, clinical symptoms caused by *PK-LR* mutations are confined to RBC be‐ cause the hepatic deficiency is usually compensated by the persistent enzyme synthesis in hepatocytes [27]. To date, more than 150 different mutations in the *PK-LR* have been associated with CNSHA, being most of them missense mutations, splicing and codon stop. Only two var‐ iants, -72 G and -83 C, have been identified in the promoter regions so far [26,27]. Molecular studies indicate that severe syndrome is commonly associated with disruptive mutations and

PK deficiency is transmitted as an autosomal recessive trait and although its global inci‐ dence is still unknown, it has been estimated in 1:20000 in the general caucasian population [29]. Clinical symptoms appear in homozygous and compound heterozygous patients, which lead to a very variable clinical picture, ranging from mild or fully compensated forms to life-treating neonatal anemia necessitating exchange transfusions and subsequent contin‐ uous support [28]. Pathological manifestations are usually observed when enzyme activity falls below 25% of normal PK activity [30], and severe disease has been associated with a high degree of reticulocytosis [31]. *Hydrops foetalis* and death in the neonatal period have al‐ so been reported in rare cases [32,33]. PK deficiency treatment is based on supportive meas‐ ures since no specific therapy for severe cases is available to date. Periodic cell transfusions may be required in severe anemic cases, often impairing their quality of life. Splenectomy can be clinically useful in some patients increasing the hemoglobin levels, as well as iron chelation to decrease the common iron overload observed in PKD patients [34]. However, in some severe cases, allogeneic bone marrow transplantation is required and it has been suc‐

The feasibility of gene therapy in PKD was first reported by the group of Asano, who intro‐ duced the human LPK cDNA into C57BL/6 mouse bone marrow cells using a retroviral vec‐ tor [7]. They demonstrated the expression of the LPK transgene mRNA in both peripheral blood and hematopoietic organs after bone marrow transplantation. However, viral-derived expression in peripheral blood was detectable no longer than 30 days post-transplantation, indicating an insufficient transduction efficacy of the retroviral vector used or transduction of non-pluripotent BM cells. In a hemolytic anemia dog model, bone marrow transplanta‐ tion of minimal conditioned receptors failed to correct the hematological symptoms [10]. Other approaches to rescue RPK phenotype through a gene addition strategy have been also addressed using a PKD transgenic mouse model (*CBA/N PK-1SLC/PK-1SLC*) [9]. In this assay, the hemolytic anemia and reticulocytosis was fully corrected when the human gene was highly expressed by means of pronuclear injection, although splenomegaly was still present. Interestingly, the authors observed a negative correlation between RBC PK activity and the number of apoptotic erythroid progenitors in the spleen, providing evidence that the meta‐

missense mutations involving the active site or protein stability [28].

cessfully performed in one severe affected child [12].

Preclinical work from Rovira et al demonstrates that *hG6PD* gene transfer into HSCs may be a viable strategy for the treatment of severe G6PD deficiency [5]. Through the transplantation of pluripotent hematopetic stem cells transduced with γ-retroviral vectors carrying the wild type human G6PD cDNA, they achieved a stable and lifelong expression of hG6PD in all the hemato‐ poietic tissues of primary and secondary receptor mice. In this study, transgene expression was driven by the 3' LTR from either the Moloney murine leukemia virus (MMLV) or the myelopro‐ liferative sarcoma virus (MPSV), obtaining an efficient transduction in murine hematopoietic progenitors. The corrected cells were then injected into lethally irradiated syngeneic mice, in‐ creasing 2-fold the enzyme activity in peripheral blood cells in comparison with non-trans‐ planted control mice. Long-term hG6PD expression derived from the vector was also observed, which was similar to that of the endogenous enzyme activity. Similar expression was detected in RBC and in White Blood Cells (WBC) in different hematopoietic organs, as expected due to the use of a viral ubiquitous promoter. These results support gene therapy as a suitable strategy for the treatment of severe CNSHA due to G6PD deficiency. Additionally, they also demon‐ strated the efficacy of this gene therapy vector in human embryonic stem cells (hESC) in which the *G6PD* gene had been inactivated by targeted homologous recombination, which implies the potential application of gene therapy to G6PD hESCs. Moreover, although a selective advant‐ age in favor of G6PD corrected cells has not been reported because the mice used showed nor‐ mal G6PD activity, Rovira et al observed a strong selection after transduction of G6PD-deficient ES cells with their vectors. In this regard, the development of G6PD deficient mouse models would be a valuable tool to test new protocols. Furthermore, the mouse strain recently devel‐ oped by Hay Ko et al may be useful, although it does not reproduce all the features of the human G6PD-deficiency [20].

Pyruvate kinase deficiency (PKD), the second most frequent abnormality of glycolysis causing CNSHA, has also been proposed as a potential disease to be treated by gene therapy. Pyruvate kinase (PK) catalyzes the second ATP generation reaction of the glycolysis pathway by convert‐ ing phosphoenolpyruvate (PEP) into pyruvate, yielding nearly 50% of the total ATP production in red blood cells. PK plays a crucial role in erythrocyte metabolism, since mature RBC are abso‐ lutely dependent on the ATP generated by glycolysis, giving the loss of mitochondria, nucleus and endoplasmic reticulum in their mature state. RPK is therefore necessary for maintaining cell integrity and function. Reduced levels of erythrocyte Pyruvate kinase (RPK) lead to an accu‐ mulation of glycolytic intermediates that ultimately shortens the life span of mature RBC by metabolic block [21]. Four tissue-specific isoenzymes of PK (M1, M2, R and L) encoded by two different genes (*PK-M* and *PK-LR*) have been identified in humans [22]. The *PK-LR* gene, locat‐ ed on chromosome 1 (1q21) [23] encodes for both LPK (expressed in liver, renal cortex and small intestine) and RPK (restricted to erythrocytes) through the use of alternative promoters [24]. PK-M1 is expressed in adult nomal tissue, like brain or muscle. The PK-M2 isoform is typically expressed in proliferating tissues like fetal, tumoral and several other adult tissues [25] and dur‐ ing the maturation of the erythroblasts, gradually decreases, giving rise to the RPK isoform.

Patients with CNSHA suffer anemia and jaundice, but often tolerate their condition well. However, G6PD variants with low activity are related with alterations in the erythrocyte membrane facilitating its breakdown and causing intravasal hemolysis. These symptoms are often accompanied by spleno- and hepatomegaly and hemoglobinuria. Besides, leukocyte dysfunctions caused by lower concentration of NADPH appear when G6PD activity is be‐ low 5% of the normal activity, leading to an immune depression [17]. Vives *et al.* and other

Preclinical work from Rovira et al demonstrates that *hG6PD* gene transfer into HSCs may be a viable strategy for the treatment of severe G6PD deficiency [5]. Through the transplantation of pluripotent hematopetic stem cells transduced with γ-retroviral vectors carrying the wild type human G6PD cDNA, they achieved a stable and lifelong expression of hG6PD in all the hemato‐ poietic tissues of primary and secondary receptor mice. In this study, transgene expression was driven by the 3' LTR from either the Moloney murine leukemia virus (MMLV) or the myelopro‐ liferative sarcoma virus (MPSV), obtaining an efficient transduction in murine hematopoietic progenitors. The corrected cells were then injected into lethally irradiated syngeneic mice, in‐ creasing 2-fold the enzyme activity in peripheral blood cells in comparison with non-trans‐ planted control mice. Long-term hG6PD expression derived from the vector was also observed, which was similar to that of the endogenous enzyme activity. Similar expression was detected in RBC and in White Blood Cells (WBC) in different hematopoietic organs, as expected due to the use of a viral ubiquitous promoter. These results support gene therapy as a suitable strategy for the treatment of severe CNSHA due to G6PD deficiency. Additionally, they also demon‐ strated the efficacy of this gene therapy vector in human embryonic stem cells (hESC) in which the *G6PD* gene had been inactivated by targeted homologous recombination, which implies the potential application of gene therapy to G6PD hESCs. Moreover, although a selective advant‐ age in favor of G6PD corrected cells has not been reported because the mice used showed nor‐ mal G6PD activity, Rovira et al observed a strong selection after transduction of G6PD-deficient ES cells with their vectors. In this regard, the development of G6PD deficient mouse models would be a valuable tool to test new protocols. Furthermore, the mouse strain recently devel‐ oped by Hay Ko et al may be useful, although it does not reproduce all the features of the human

Pyruvate kinase deficiency (PKD), the second most frequent abnormality of glycolysis causing CNSHA, has also been proposed as a potential disease to be treated by gene therapy. Pyruvate kinase (PK) catalyzes the second ATP generation reaction of the glycolysis pathway by convert‐ ing phosphoenolpyruvate (PEP) into pyruvate, yielding nearly 50% of the total ATP production in red blood cells. PK plays a crucial role in erythrocyte metabolism, since mature RBC are abso‐ lutely dependent on the ATP generated by glycolysis, giving the loss of mitochondria, nucleus and endoplasmic reticulum in their mature state. RPK is therefore necessary for maintaining cell integrity and function. Reduced levels of erythrocyte Pyruvate kinase (RPK) lead to an accu‐ mulation of glycolytic intermediates that ultimately shortens the life span of mature RBC by metabolic block [21]. Four tissue-specific isoenzymes of PK (M1, M2, R and L) encoded by two different genes (*PK-M* and *PK-LR*) have been identified in humans [22]. The *PK-LR* gene, locat‐ ed on chromosome 1 (1q21) [23] encodes for both LPK (expressed in liver, renal cortex and small

groups have also observed an increased susceptibility to infections [18,19].

G6PD-deficiency [20].

514 Gene Therapy - Tools and Potential Applications

The codifying region of *PK-LR* gene is split into twelve exons, ten of which are shared by the two isoforms, while exons 1 and 2 are specific for the erythrocyte and the hepatic isoenzyme respec‐ tively [26]. However, clinical symptoms caused by *PK-LR* mutations are confined to RBC be‐ cause the hepatic deficiency is usually compensated by the persistent enzyme synthesis in hepatocytes [27]. To date, more than 150 different mutations in the *PK-LR* have been associated with CNSHA, being most of them missense mutations, splicing and codon stop. Only two var‐ iants, -72 G and -83 C, have been identified in the promoter regions so far [26,27]. Molecular studies indicate that severe syndrome is commonly associated with disruptive mutations and missense mutations involving the active site or protein stability [28].

PK deficiency is transmitted as an autosomal recessive trait and although its global inci‐ dence is still unknown, it has been estimated in 1:20000 in the general caucasian population [29]. Clinical symptoms appear in homozygous and compound heterozygous patients, which lead to a very variable clinical picture, ranging from mild or fully compensated forms to life-treating neonatal anemia necessitating exchange transfusions and subsequent contin‐ uous support [28]. Pathological manifestations are usually observed when enzyme activity falls below 25% of normal PK activity [30], and severe disease has been associated with a high degree of reticulocytosis [31]. *Hydrops foetalis* and death in the neonatal period have al‐ so been reported in rare cases [32,33]. PK deficiency treatment is based on supportive meas‐ ures since no specific therapy for severe cases is available to date. Periodic cell transfusions may be required in severe anemic cases, often impairing their quality of life. Splenectomy can be clinically useful in some patients increasing the hemoglobin levels, as well as iron chelation to decrease the common iron overload observed in PKD patients [34]. However, in some severe cases, allogeneic bone marrow transplantation is required and it has been suc‐ cessfully performed in one severe affected child [12].

The feasibility of gene therapy in PKD was first reported by the group of Asano, who intro‐ duced the human LPK cDNA into C57BL/6 mouse bone marrow cells using a retroviral vec‐ tor [7]. They demonstrated the expression of the LPK transgene mRNA in both peripheral blood and hematopoietic organs after bone marrow transplantation. However, viral-derived expression in peripheral blood was detectable no longer than 30 days post-transplantation, indicating an insufficient transduction efficacy of the retroviral vector used or transduction of non-pluripotent BM cells. In a hemolytic anemia dog model, bone marrow transplanta‐ tion of minimal conditioned receptors failed to correct the hematological symptoms [10]. Other approaches to rescue RPK phenotype through a gene addition strategy have been also addressed using a PKD transgenic mouse model (*CBA/N PK-1SLC/PK-1SLC*) [9]. In this assay, the hemolytic anemia and reticulocytosis was fully corrected when the human gene was highly expressed by means of pronuclear injection, although splenomegaly was still present. Interestingly, the authors observed a negative correlation between RBC PK activity and the number of apoptotic erythroid progenitors in the spleen, providing evidence that the meta‐ bolic alteration in PK deficiency affects not only the survival of RBC, but also the maturation of erythroid progenitors, resulting in ineffective erythropoiesis [35]. Further studies from this group indicate that RPK plays an important role as an antioxidant during erythrocyte differentiation, since glycolytic inhibition by mutations in *Pklr* gene increased the oxidative stress in SLC3 cells (established from *Pk-1slc* mouse) and led to the activation of hypoxia-in‐ ducible factor-1 (HIF1), as well as the expression of downstream proapoptotic genes [36].

ization from glucose 6-phosphate to fructose 6-phosphate, an equilibrium reaction of the glycolysis pathway. Glucose turnover is affected only in deficiencies below a very low criti‐ cal residual GPI activity, but with a drastic decline of lactate formation. As no isoenzyme does exist, patients suffer not only from CNSHA and tissue hypoxia, but also from neuro‐ muscular disturbances. In some cases, GPI deficiency has been found in PKD patients, in‐ creasing the severity of the clinical scenario and reflecting the degree of the perturbation of glycolysis. The lack of ATP leads to a destabilization of the erythrocyte membrane causing earlier lysis of the RBC and hemolytic anemia of variable degrees [38]. Animal models of GPI deficiency have been described, showing similar symptoms to the human disease [39].

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517

Other enzyme deficiencies causing CNSHA are Triose phosphate isomerase (TPI) deficiency, associated with neuromuscular disorders, mental retardation and frequent infections, Hexo‐ kinase deficiency (HK), affecting also platelet metabolism, phosphofructokinase (PFK) defi‐ ciency, 2,3-bisphosphoglycerate mutase (BPGM) deficiency and Glutathione synthetase (GS) deficiency (reviewed in [17,40,41]). Although the incidence of these diseases can be high (ie. TPI is considered as a frequent enzymopathy affecting 0.1% for caucasian populations and even 4.6% for black populations), they are considered rare or very rare diseases, because on‐ ly few cases (~25 patients in the case of TPI) are diagnosed due to the severity of the clinical manifestations. No gene therapy approaches have been addressed up to now to treat these enzymopaties. However, due to their common characteristics, strategies developed in the

other enzyme deficiencies could be applied directly to the treatment of all of them.

**diseases: Erythroid specific expression vectors**

**3. Optimization of vectors for the gene therapy of metabolic erythroid**

The introduction of a cDNA, encoding for the correct version of the target mutated gene into patient cells using retroviral vectors has been successful for several inherited diseases. The initial integrative vectors for gene therapy design and used in clinical trials were based on Gamma(γ)-retroviral vectors in which the transgene expression was driven by the viral LTR promoter. γ-retroviruses preferably integrate in regions adjacent to the transcription initia‐ tion site [42]. The expression of the transgene is promoted by the viral LTR, which drives a high expression that can affect gene regulation of the surrounding genes. Although a high efficiency of transduction and therapeutic effects have been described with these vectors in various monogenic disorders such as immunodeficiencies, adverse effects associated with insertional mutagenesis have also been observed. This has led to the development of the next generation of integrative vectors using self-inactivating-LTR lentiviral backbones. SIN-Lentiviral vectors tend to integrate in intergenic transcribing areas, which represent a safer integrative pattern than γ-retroviral vectors. Aditionally, the expression of the transgene is driven by internal promoters, offering a more physiological expression and a less genotoxic profile when using weak promoters [43]. Current efforts to reduce the mutagenic potential of gene therapy vectors are focussed on not only the use of new viral backbones [44] but also on tissue-specific promoters to restrict the transgene expression to target cells [45] and insu‐

Until now, no gene therapy attempt has been applied to this deficiency.

In addition, our work carried out in mouse models supported the therapeutic potential of viral vectors for the gene therapy of PK deficiency. Throughout the transduction of bone marrow cells using γ-retroviral vectors that carry the human RPK cDNA and subse‐ quent transplantation, we reported a long-term expression of the human protein in RBC obtained from primary and secondary receptor mice, without detectable adverse effects [11]. Recently, we have also reported a successful gene therapy approach using the same retroviral vectors in the congenital mouse strain AcB55, identified by Min-Oo in studies of alleles involved in malaria susceptibility [37]. These mice carry a loss-of-function mu‐ tation (269T-> A) resulting in the amino acid substitution I90N in the *Pklr* gene, which yields a similar RBC phenotype to that observed in PKD patients, including splenomega‐ ly and constitutive reticulocytosis. Retroviral-derived expression was capable of fully re‐ solving the pathological phenotype in terms of hematological parameters, anemia, reticulocytosis and splenomegaly, together with normalization of bone marrow and spleen erythroid progenitors, erythropoietin (EPO), PK activity and ATP levels. Interest‐ ingly, despite a strong viral promoter was used to drive the expression of the transgene, metabolic energy balance was no modified in white blood cells. Moreover, we observed that values above 25% of genetically corrected cells were needed to fully rescue the defi‐ ciency [3], suggesting that RPK transfer protocols will always require a significant extent of gene-complemented HSC. Nevertheless, other experiments performed in the *CBA/N PK-1SLC/PK-1SLC* mouse model of PKD have reveled that 10% of normal BM renders RBC expressing nearly normal RPK protein levels [5]. Differences in the genetic defect of the mouse models used could account for these discrepancies, reinforcing the need for high transduction efficiencies to address the disease in the heterogeneous human population. Additionally, we have proposed the *in utero* transplantation of gene corrected cells as an alternative option for the treatment of PKD. The transplantation of RPK deficient lineage negative fetal liver cells transduced with lentiviruses (LVs) expressing the human wild type version of the RPK in 14.5 day-old fetuses partially restored the anemic phenotype, mainly due to a low engraftment of corrected cells [13]. Improved *in utero* cell transfer would allow therapeutic levels, thus offering an alternative therapeutic option for prena‐ tally diagnosed severe PKD. Following our results in the AcB55 mouse model of PKD, phenotype correction could be reached if the percentage of engraftment of corrected cells is significant. We are currently developing improved lentiviral vectors that could be ap‐ plied in future clinical settings.

Glucose phosphate isomerase (GPI) deficiency is the third most common hereditary cause of CNSHA, due to mutations in *GPI* gene located on the long arm of chromosome 19. The prevalence of this disease is still unknown, with no more than 50 cases reported so far, and with a higher incidence in the black population. The enzyme catalyzes the reversible isomer‐ ization from glucose 6-phosphate to fructose 6-phosphate, an equilibrium reaction of the glycolysis pathway. Glucose turnover is affected only in deficiencies below a very low criti‐ cal residual GPI activity, but with a drastic decline of lactate formation. As no isoenzyme does exist, patients suffer not only from CNSHA and tissue hypoxia, but also from neuro‐ muscular disturbances. In some cases, GPI deficiency has been found in PKD patients, in‐ creasing the severity of the clinical scenario and reflecting the degree of the perturbation of glycolysis. The lack of ATP leads to a destabilization of the erythrocyte membrane causing earlier lysis of the RBC and hemolytic anemia of variable degrees [38]. Animal models of GPI deficiency have been described, showing similar symptoms to the human disease [39]. Until now, no gene therapy attempt has been applied to this deficiency.

bolic alteration in PK deficiency affects not only the survival of RBC, but also the maturation of erythroid progenitors, resulting in ineffective erythropoiesis [35]. Further studies from this group indicate that RPK plays an important role as an antioxidant during erythrocyte differentiation, since glycolytic inhibition by mutations in *Pklr* gene increased the oxidative stress in SLC3 cells (established from *Pk-1slc* mouse) and led to the activation of hypoxia-in‐ ducible factor-1 (HIF1), as well as the expression of downstream proapoptotic genes [36].

In addition, our work carried out in mouse models supported the therapeutic potential of viral vectors for the gene therapy of PK deficiency. Throughout the transduction of bone marrow cells using γ-retroviral vectors that carry the human RPK cDNA and subse‐ quent transplantation, we reported a long-term expression of the human protein in RBC obtained from primary and secondary receptor mice, without detectable adverse effects [11]. Recently, we have also reported a successful gene therapy approach using the same retroviral vectors in the congenital mouse strain AcB55, identified by Min-Oo in studies of alleles involved in malaria susceptibility [37]. These mice carry a loss-of-function mu‐ tation (269T-> A) resulting in the amino acid substitution I90N in the *Pklr* gene, which yields a similar RBC phenotype to that observed in PKD patients, including splenomega‐ ly and constitutive reticulocytosis. Retroviral-derived expression was capable of fully re‐ solving the pathological phenotype in terms of hematological parameters, anemia, reticulocytosis and splenomegaly, together with normalization of bone marrow and spleen erythroid progenitors, erythropoietin (EPO), PK activity and ATP levels. Interest‐ ingly, despite a strong viral promoter was used to drive the expression of the transgene, metabolic energy balance was no modified in white blood cells. Moreover, we observed that values above 25% of genetically corrected cells were needed to fully rescue the defi‐ ciency [3], suggesting that RPK transfer protocols will always require a significant extent of gene-complemented HSC. Nevertheless, other experiments performed in the *CBA/N PK-1SLC/PK-1SLC* mouse model of PKD have reveled that 10% of normal BM renders RBC expressing nearly normal RPK protein levels [5]. Differences in the genetic defect of the mouse models used could account for these discrepancies, reinforcing the need for high transduction efficiencies to address the disease in the heterogeneous human population. Additionally, we have proposed the *in utero* transplantation of gene corrected cells as an alternative option for the treatment of PKD. The transplantation of RPK deficient lineage negative fetal liver cells transduced with lentiviruses (LVs) expressing the human wild type version of the RPK in 14.5 day-old fetuses partially restored the anemic phenotype, mainly due to a low engraftment of corrected cells [13]. Improved *in utero* cell transfer would allow therapeutic levels, thus offering an alternative therapeutic option for prena‐ tally diagnosed severe PKD. Following our results in the AcB55 mouse model of PKD, phenotype correction could be reached if the percentage of engraftment of corrected cells is significant. We are currently developing improved lentiviral vectors that could be ap‐

Glucose phosphate isomerase (GPI) deficiency is the third most common hereditary cause of CNSHA, due to mutations in *GPI* gene located on the long arm of chromosome 19. The prevalence of this disease is still unknown, with no more than 50 cases reported so far, and with a higher incidence in the black population. The enzyme catalyzes the reversible isomer‐

plied in future clinical settings.

516 Gene Therapy - Tools and Potential Applications

Other enzyme deficiencies causing CNSHA are Triose phosphate isomerase (TPI) deficiency, associated with neuromuscular disorders, mental retardation and frequent infections, Hexo‐ kinase deficiency (HK), affecting also platelet metabolism, phosphofructokinase (PFK) defi‐ ciency, 2,3-bisphosphoglycerate mutase (BPGM) deficiency and Glutathione synthetase (GS) deficiency (reviewed in [17,40,41]). Although the incidence of these diseases can be high (ie. TPI is considered as a frequent enzymopathy affecting 0.1% for caucasian populations and even 4.6% for black populations), they are considered rare or very rare diseases, because on‐ ly few cases (~25 patients in the case of TPI) are diagnosed due to the severity of the clinical manifestations. No gene therapy approaches have been addressed up to now to treat these enzymopaties. However, due to their common characteristics, strategies developed in the other enzyme deficiencies could be applied directly to the treatment of all of them.

#### **3. Optimization of vectors for the gene therapy of metabolic erythroid diseases: Erythroid specific expression vectors**

The introduction of a cDNA, encoding for the correct version of the target mutated gene into patient cells using retroviral vectors has been successful for several inherited diseases. The initial integrative vectors for gene therapy design and used in clinical trials were based on Gamma(γ)-retroviral vectors in which the transgene expression was driven by the viral LTR promoter. γ-retroviruses preferably integrate in regions adjacent to the transcription initia‐ tion site [42]. The expression of the transgene is promoted by the viral LTR, which drives a high expression that can affect gene regulation of the surrounding genes. Although a high efficiency of transduction and therapeutic effects have been described with these vectors in various monogenic disorders such as immunodeficiencies, adverse effects associated with insertional mutagenesis have also been observed. This has led to the development of the next generation of integrative vectors using self-inactivating-LTR lentiviral backbones. SIN-Lentiviral vectors tend to integrate in intergenic transcribing areas, which represent a safer integrative pattern than γ-retroviral vectors. Aditionally, the expression of the transgene is driven by internal promoters, offering a more physiological expression and a less genotoxic profile when using weak promoters [43]. Current efforts to reduce the mutagenic potential of gene therapy vectors are focussed on not only the use of new viral backbones [44] but also on tissue-specific promoters to restrict the transgene expression to target cells [45] and insu‐ lators to confer position-independent expression [46]. Additional regulatory DNA elements such as locus control regions (LCR), enhancers, or silencers have also been used to increase lineage specificity.

(cHS4), an insulator sequence of the chicken -like globin cluster. Studies performed by Chung et al with the γ-globin promoter and the neo reporter gene on selected cells lines, demonstrated the ability of cHS4 to insulate the expression cassette from the effects of a strong -globin LCR element [63] and therefore reducing its genotoxicity. Experiments from Arumugam et al showed a two-fold reduction in transforming activity with insulated LCR-

**Erythroid tissue-specific vectors**

ΔLNGFR and NeoR / EGFP

HS1 to HS2 GATA-1 enhancer within the LTR EGFP and hΔLNGFR SFCM retroviral vector [48]

**Promoter / enhancer transgene Vector type Reference**

promoters hβ-globin cDNA Sleeping beauty transposon [54]

**Promoter / enhancer transgene Vector type Reference**

**Physiologically regulated vectors**

HSFE and β-globin promoter hβ-globin cDNA MSCV retroviral vector [55] LCR and β-globin promoter hβ-globin cDNA or EGFP HIV-1 based vectors [56,57]

LCR, cHS4 and β-globin promoter hβ-globin cDNA HIV-1 based vectors [46] β-globin promoter, LCR HS2, HS3, HS4 hβ-globin cDNA AAV2 [59]

LTR, long terminal repeats; HS: hypersensitive site; IHK, human *ALAS*2 intron 8 enhancer, HS40 from αLCR and an‐ kyrin-1 promoter; Ihβp, human *ALAS*2 intron 8 enhancer, HS40 from αLCR and β-globin promoter; HS3βp, HS3 core element form human βLCR and β-globin promoter; LCR, locus control region. Modified from Toscano *et al.*,

SFCM retroviral vector [47]

Gene Therapy for Erythroid Metabolic Inherited Diseases

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519

EGFP HIV-1 based vectors [53]

EGFP HIV-1 based vectors [53]

EGFP HIV-1 based vectors [58]

GFP / *FECH*cDNA HIV-1 based vectors [4]

containing lentiviral vectors comparing with vectors lacking the cHS4 element [68].

HS2 GATA-1 enhancer within the LTR

Ankyrin-1 and α-spectrin promoters combined or not with HS40, GATA-1, ARE and intron 8 enhancers

α-globin HS40 enhancer and Ankyrin-1 promoter

IHK, IHβp and HS3βp chimeric enhancers/

β-globin and θ-globin promoters combined or not with HS40, GATA-1, ARE and intron 8 enhancers

LCR HS4, HS3, HS2, β-globin promoter and truncated β-globin intron 2

**Table 2.** Specific vectors for gene therapy of erythroid inherited diseases.

2011

Gene therapy for RBC disorders requires, ideally, high erythroid-specific transgene expres‐ sion in order to avoid side effects in progenitors or hematopoietic lineages other than the erythroid one. In inherited enzymophaties, the overexpression of metabolic enzymes in nonerythroid cells could provide these cells with a potential energetic advantage, with the con‐ sequent risk of disturbing the physiological generation of ATP in WBC. Also, the restriction of transcriptional activity to target cells with the use of either tissue-specific or physiologi‐ cally regulated vectors decreasees the effect of the integrative vectors in the host genome. This goal is particularly important for erythrocyte metabolic deficiencies, as all the affected enzymes are highly regulated and connected with central metabolic pathways. Indeed, an expression limited to the erythroid progeny would reduce the genotoxic risk, as RBC be‐ come transcriptionally inactive during differentiation, and finally extrude their nucleus. To study tissue-specific gene therapy strategies for RBC diseases, hemoglobinophaties have been the most widely used.

Erythroid regulatory elements have been extensively used to manage targeted expression to RBC using reporter genes (Table 2). The Locus Control Regions (LCR), defined by their ability to enhance the expression of linked genes to physiological levels in a tissuespecific and copy number-dependent manner at ectopic chromatin sites are commonly used. The components of the LCR normally colocalize to sites of DNase I hypersensitivi‐ ty (HS) in the chromatin of expressing cells. Individual HS are composed of arrays of multiple ubiquitous and lineage-specific transcription factor-binding sites. In early experi‐ ments performed with retroviral backbones, the group of Ferrari developed an erythroidspecific vector by the replacement of the constitutive retroviral enhancer in the U3 region of the 3' LTR with the HS2 autoregulatory enhancer of the erythroid GATA-1 transcrip‐ tion factor gene. The expression of this vector was restricted to the erythroblastic proge‐ ny of both human progenitors and mouse-repopulating stem cells [47,48]. Later, they showed that the addition of the HS1 enhancer to HS2, both from the GATA-1 gene, with‐ in the LTR of the retroviral vector significantly improved the expression of the reporter gene. Another enhancer element that has been used to achieve erythroid-specific expres‐ sion is HS40, located upstream of the ζ-globin gene, since it is able to enhance the activi‐ ty of heterologous promoters in a tissue-specific manner [49]. It has been shown to be genetically stable in MMLV vectors and enhances expression comparable to that of a sin‐ gle -globin gene [50], although HS40 lacks some of the properties of the LCR, like posi‐ tion independence [51] or copy number dependence [52].

An additional improvement to provide safer vectors for RBC gene therapy was provided by the use of insulators elements, which have been shown to reduce position effects in trans‐ genic animals [60]. Insulators are genomic elements that can shelter genes from their sur‐ rounding chromosomal environment, by either blocking the action of a distal enhancer on a promoter [60,61], or by acting as barriers that protect the gene from the silencing effect of heterochromatin [61]. The most well studied element is the chicken hypersensitive site 4 (cHS4), an insulator sequence of the chicken -like globin cluster. Studies performed by Chung et al with the γ-globin promoter and the neo reporter gene on selected cells lines, demonstrated the ability of cHS4 to insulate the expression cassette from the effects of a strong -globin LCR element [63] and therefore reducing its genotoxicity. Experiments from Arumugam et al showed a two-fold reduction in transforming activity with insulated LCRcontaining lentiviral vectors comparing with vectors lacking the cHS4 element [68].

lators to confer position-independent expression [46]. Additional regulatory DNA elements such as locus control regions (LCR), enhancers, or silencers have also been used to increase

Gene therapy for RBC disorders requires, ideally, high erythroid-specific transgene expres‐ sion in order to avoid side effects in progenitors or hematopoietic lineages other than the erythroid one. In inherited enzymophaties, the overexpression of metabolic enzymes in nonerythroid cells could provide these cells with a potential energetic advantage, with the con‐ sequent risk of disturbing the physiological generation of ATP in WBC. Also, the restriction of transcriptional activity to target cells with the use of either tissue-specific or physiologi‐ cally regulated vectors decreasees the effect of the integrative vectors in the host genome. This goal is particularly important for erythrocyte metabolic deficiencies, as all the affected enzymes are highly regulated and connected with central metabolic pathways. Indeed, an expression limited to the erythroid progeny would reduce the genotoxic risk, as RBC be‐ come transcriptionally inactive during differentiation, and finally extrude their nucleus. To study tissue-specific gene therapy strategies for RBC diseases, hemoglobinophaties have

Erythroid regulatory elements have been extensively used to manage targeted expression to RBC using reporter genes (Table 2). The Locus Control Regions (LCR), defined by their ability to enhance the expression of linked genes to physiological levels in a tissuespecific and copy number-dependent manner at ectopic chromatin sites are commonly used. The components of the LCR normally colocalize to sites of DNase I hypersensitivi‐ ty (HS) in the chromatin of expressing cells. Individual HS are composed of arrays of multiple ubiquitous and lineage-specific transcription factor-binding sites. In early experi‐ ments performed with retroviral backbones, the group of Ferrari developed an erythroidspecific vector by the replacement of the constitutive retroviral enhancer in the U3 region of the 3' LTR with the HS2 autoregulatory enhancer of the erythroid GATA-1 transcrip‐ tion factor gene. The expression of this vector was restricted to the erythroblastic proge‐ ny of both human progenitors and mouse-repopulating stem cells [47,48]. Later, they showed that the addition of the HS1 enhancer to HS2, both from the GATA-1 gene, with‐ in the LTR of the retroviral vector significantly improved the expression of the reporter gene. Another enhancer element that has been used to achieve erythroid-specific expres‐ sion is HS40, located upstream of the ζ-globin gene, since it is able to enhance the activi‐ ty of heterologous promoters in a tissue-specific manner [49]. It has been shown to be genetically stable in MMLV vectors and enhances expression comparable to that of a sin‐ gle -globin gene [50], although HS40 lacks some of the properties of the LCR, like posi‐

An additional improvement to provide safer vectors for RBC gene therapy was provided by the use of insulators elements, which have been shown to reduce position effects in trans‐ genic animals [60]. Insulators are genomic elements that can shelter genes from their sur‐ rounding chromosomal environment, by either blocking the action of a distal enhancer on a promoter [60,61], or by acting as barriers that protect the gene from the silencing effect of heterochromatin [61]. The most well studied element is the chicken hypersensitive site 4

lineage specificity.

518 Gene Therapy - Tools and Potential Applications

been the most widely used.

tion independence [51] or copy number dependence [52].



LTR, long terminal repeats; HS: hypersensitive site; IHK, human *ALAS*2 intron 8 enhancer, HS40 from αLCR and an‐ kyrin-1 promoter; Ihβp, human *ALAS*2 intron 8 enhancer, HS40 from αLCR and β-globin promoter; HS3βp, HS3 core element form human βLCR and β-globin promoter; LCR, locus control region. Modified from Toscano *et al.*, 2011

**Table 2.** Specific vectors for gene therapy of erythroid inherited diseases.

Tissue-specific expression using alternative human promoters can be convenient or more effi‐ cient for some diseases, but driving the expression of the therapeutic genes using own promot‐ ers is still the most physiological approach to reduce the genotoxic risk of integrating gene vectors [62]. The use of physiologically regulated vectors has been limited mainly because the promoter and the enhancer elements have to be obtained from the affected genes and they are often too large to be included in a lentiviral backbone, and also because the gene expression pat‐ tern depends partially on chromatin positioning [63]. -globin LCR has been widely used when attempting to solve this limitation. The -globin LCR consists of 5 HS regions located upstream of the entire cluster of human -like globin genes, each containing a high density of erythroid-spe‐ cific and ubiquitous transcription binding elements [64]. Much of the transcriptional activity of the -globin LCR resides in HS2 and HS3 sites, but site 4 is important in adult globin expression [65]. Previous studies *in vitro* and *in vivo* have shown that -globin LCR can enhance erythroidspecific expression from heterologous non-erythroid promoters [66,67]. First approaches using -globin LCR and 3' enhancers were based on murine γretroviral vectors [74,75], but the limited packaging capacity of these vectors (up to 8 kb) did not allow the presence of such as large regu‐ latory sequences. Several vector designs including different combinations of regulatory se‐ quences and a deletion of a cryptic polyadenylation site within intron 2 of -globin gene [68], flanked by an extended promoter sequence and the -globin 3' proximal enhancer were devel‐ oped. The combination of the LCR elements (3'2 kb) spanning HS2, 3 and 4, were the best amongst several possibilities [69] to achieve a high titer retroviral vector capable of expressing high levels of the transgene.

endogenous microRNA cellular machinery. Following this strategy, engineered microRNA tar‐ get sequences in the vector (miRTs-vector) are recognized by a cell specific microRNA (miR‐ NA), avoiding the expression of the therapeutic gene in undesired cell populations [63]. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regu‐ lates key functional proteins involved in hematopoietic lineage differentiation. Particularly, miR-223 has been proposed as a myeloid-specific regulator that negatively regulates progenitor proliferation and granulocyte differentiation and activation [73]. Moreover, Felli et al observed that hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that down-modulation of this miRNA is re‐ quired for erythroid progenitor recruitment and commitment [79]. Further studies may deter‐ mine if the use of miRNA-223 target in lentiviral vectors could be useful to achieve a desirable

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

521

In addition, the erythroid-specificity of short segments of the -globin LCR element has been documented in adeno-associated virus 2 (AAV2) system. Their efficacy to mediate an eryth‐ roid-restricted expression has been proved by Tan *et al.*, who reported a successful AAV2-medi‐ ated high and stable transduction of the human -globin gene in HSCs from -thalassemia mouse model, which were then transplanted into recipient and rescued them of the disease [59]. These vectors have gained attention as potential useful vectors for human gene therapy, mainly be‐ cause of their non-pathogenic nature in humans and their relativly easy production. Besides, AAV2 vectors are easily purified to high titers and are able to transduce dividing and non-di‐ viding cells. However, most of proviral AAV2 genomes remain episomal and the insert size is restricted to just over 4kb. Further studies are still needed to know whether they would be a bet‐ ter option than current lentiviral vectors. Also, long-term genotoxic risk of recombinant AAV2

In addition, the efficacy of some of these erythroid-specific elements and promoters has also been tested in non-viral vectors, such as transposons. Zhu et al, for instance, studied several hybrid promoters driving the expression of the human -globin gene using the sleeping beau‐ ty transposon (SB-Tn). They combined several erythroid elements to develop different chi‐ meric promoters. Their results indicated that the ankyrin-1 minimum promoter was stronger than -globin's, and the hALAS I8 enhancer (IH) was significantly more powerful that HS3 core element from -LCR and -globin promoter [54]. SB-Tn system is a promising non-viral vector for efficient genomic insertion, even with erythroid-specificity. However, its efficiency for delivering transgenes into HSCs is still much lower than other engineering vi‐

**4. Overcoming conventional gene therapy pitfalls: gene editing in**

Since Yamanaka et al first reported the generation of mouse induced Pluripotent Stem Cells (iPSC) in 2006 by the ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and

**4.1. Human induced pluripotent stem cells and reprogramming platforms**

erythroid-specific expression for gene therapy of red blood cell diseases.

therapy in human is not known up to the date.

**induced plutipotent stem cells**

ral vectors.

Other approaches to achieve consistent long-term expression of a transgene have been based on the use of HSFE element, an erythroid-specific chromatin remodelling element derived from the human β-globin LCR which contains binding sites for the erythroid-specific factors NF-E2, GATA-1, EKLF and the ubiquitous factor Sp-1, all of which are necessary to establish a hyper‐ sensitive chromatin domain. Work by Nemeth *et al.*, demonstrated that the HSFE can mediate functional tissue-specific "opening" of a minimal human β-globin promoter and increases ex‐ pression of a human β-globin gene in both MEL cell clones and in transgenic mice. Their results indicated that the most effective vector included tandem copies of the HSFE and produced a 5 fold increase in expression compared to the promoter alone [55] in the context of an integrated retroviral vector.

Gene therapy for RBC metabolic diseases can also benefit from the new technologies based on the modification in mRNA stability or translation efficacy of the transgenes. The use of the posttranscriptional regulatory element (Wpre) from the woodchuck hepatitis virus (WHV) has sig‐ nificantly increased transgene expression in target cells [64,65], even in HSC [70] by stabilization of mRNA at post-transcriptional level. However, it may raise safety concerns, since it contains a truncated form of the WHV *X* gene, which has been implicated in animal liver cancer [71]. Therefore, Wpre has subsequently been improved by a mutation of the open read‐ ing frame of the *X* gene [72]. Combination of erythroid promoters like ankyrin-1 or -spectrin with Wpre sequence increased 2-fold the expression in unilineage erythroid cultures [53], and when combined also with erythroid enhancers inserted in tandem: HS40 and GATA-1 or HS40 and I8 enhancers [53]. RNA targeting strategies have mainly been used to down regulate ex‐ pression of cellular genes using vectors expressing interference RNAs (iRNAs). They can be al‐ so used to control the expression of integrating vectors knocking down the transgene by the endogenous microRNA cellular machinery. Following this strategy, engineered microRNA tar‐ get sequences in the vector (miRTs-vector) are recognized by a cell specific microRNA (miR‐ NA), avoiding the expression of the therapeutic gene in undesired cell populations [63]. Several miRNAs are differentially expressed during hematopoiesis and their specific expression regu‐ lates key functional proteins involved in hematopoietic lineage differentiation. Particularly, miR-223 has been proposed as a myeloid-specific regulator that negatively regulates progenitor proliferation and granulocyte differentiation and activation [73]. Moreover, Felli et al observed that hematopoietic progenitor cells transduced with miR-223 showed a significant reduction of their erythroid clonogenic capacity, suggesting that down-modulation of this miRNA is re‐ quired for erythroid progenitor recruitment and commitment [79]. Further studies may deter‐ mine if the use of miRNA-223 target in lentiviral vectors could be useful to achieve a desirable erythroid-specific expression for gene therapy of red blood cell diseases.

Tissue-specific expression using alternative human promoters can be convenient or more effi‐ cient for some diseases, but driving the expression of the therapeutic genes using own promot‐ ers is still the most physiological approach to reduce the genotoxic risk of integrating gene vectors [62]. The use of physiologically regulated vectors has been limited mainly because the promoter and the enhancer elements have to be obtained from the affected genes and they are often too large to be included in a lentiviral backbone, and also because the gene expression pat‐ tern depends partially on chromatin positioning [63]. -globin LCR has been widely used when attempting to solve this limitation. The -globin LCR consists of 5 HS regions located upstream of the entire cluster of human -like globin genes, each containing a high density of erythroid-spe‐ cific and ubiquitous transcription binding elements [64]. Much of the transcriptional activity of the -globin LCR resides in HS2 and HS3 sites, but site 4 is important in adult globin expression [65]. Previous studies *in vitro* and *in vivo* have shown that -globin LCR can enhance erythroidspecific expression from heterologous non-erythroid promoters [66,67]. First approaches using -globin LCR and 3' enhancers were based on murine γretroviral vectors [74,75], but the limited packaging capacity of these vectors (up to 8 kb) did not allow the presence of such as large regu‐ latory sequences. Several vector designs including different combinations of regulatory se‐ quences and a deletion of a cryptic polyadenylation site within intron 2 of -globin gene [68], flanked by an extended promoter sequence and the -globin 3' proximal enhancer were devel‐ oped. The combination of the LCR elements (3'2 kb) spanning HS2, 3 and 4, were the best amongst several possibilities [69] to achieve a high titer retroviral vector capable of expressing

Other approaches to achieve consistent long-term expression of a transgene have been based on the use of HSFE element, an erythroid-specific chromatin remodelling element derived from the human β-globin LCR which contains binding sites for the erythroid-specific factors NF-E2, GATA-1, EKLF and the ubiquitous factor Sp-1, all of which are necessary to establish a hyper‐ sensitive chromatin domain. Work by Nemeth *et al.*, demonstrated that the HSFE can mediate functional tissue-specific "opening" of a minimal human β-globin promoter and increases ex‐ pression of a human β-globin gene in both MEL cell clones and in transgenic mice. Their results indicated that the most effective vector included tandem copies of the HSFE and produced a 5 fold increase in expression compared to the promoter alone [55] in the context of an integrated

Gene therapy for RBC metabolic diseases can also benefit from the new technologies based on the modification in mRNA stability or translation efficacy of the transgenes. The use of the posttranscriptional regulatory element (Wpre) from the woodchuck hepatitis virus (WHV) has sig‐ nificantly increased transgene expression in target cells [64,65], even in HSC [70] by stabilization of mRNA at post-transcriptional level. However, it may raise safety concerns, since it contains a truncated form of the WHV *X* gene, which has been implicated in animal liver cancer [71]. Therefore, Wpre has subsequently been improved by a mutation of the open read‐ ing frame of the *X* gene [72]. Combination of erythroid promoters like ankyrin-1 or -spectrin with Wpre sequence increased 2-fold the expression in unilineage erythroid cultures [53], and when combined also with erythroid enhancers inserted in tandem: HS40 and GATA-1 or HS40 and I8 enhancers [53]. RNA targeting strategies have mainly been used to down regulate ex‐ pression of cellular genes using vectors expressing interference RNAs (iRNAs). They can be al‐ so used to control the expression of integrating vectors knocking down the transgene by the

high levels of the transgene.

520 Gene Therapy - Tools and Potential Applications

retroviral vector.

In addition, the erythroid-specificity of short segments of the -globin LCR element has been documented in adeno-associated virus 2 (AAV2) system. Their efficacy to mediate an eryth‐ roid-restricted expression has been proved by Tan *et al.*, who reported a successful AAV2-medi‐ ated high and stable transduction of the human -globin gene in HSCs from -thalassemia mouse model, which were then transplanted into recipient and rescued them of the disease [59]. These vectors have gained attention as potential useful vectors for human gene therapy, mainly be‐ cause of their non-pathogenic nature in humans and their relativly easy production. Besides, AAV2 vectors are easily purified to high titers and are able to transduce dividing and non-di‐ viding cells. However, most of proviral AAV2 genomes remain episomal and the insert size is restricted to just over 4kb. Further studies are still needed to know whether they would be a bet‐ ter option than current lentiviral vectors. Also, long-term genotoxic risk of recombinant AAV2 therapy in human is not known up to the date.

In addition, the efficacy of some of these erythroid-specific elements and promoters has also been tested in non-viral vectors, such as transposons. Zhu et al, for instance, studied several hybrid promoters driving the expression of the human -globin gene using the sleeping beau‐ ty transposon (SB-Tn). They combined several erythroid elements to develop different chi‐ meric promoters. Their results indicated that the ankyrin-1 minimum promoter was stronger than -globin's, and the hALAS I8 enhancer (IH) was significantly more powerful that HS3 core element from -LCR and -globin promoter [54]. SB-Tn system is a promising non-viral vector for efficient genomic insertion, even with erythroid-specificity. However, its efficiency for delivering transgenes into HSCs is still much lower than other engineering vi‐ ral vectors.

#### **4. Overcoming conventional gene therapy pitfalls: gene editing in induced plutipotent stem cells**

#### **4.1. Human induced pluripotent stem cells and reprogramming platforms**

Since Yamanaka et al first reported the generation of mouse induced Pluripotent Stem Cells (iPSC) in 2006 by the ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and cMyc) [74] and one year later in human cells together with Thompson's group [75,76], many laboratories around the world have been able to reprogram a large range of somatic cells in‐ to pluripotent stem cells, from neural stem cells [77] to terminally differentiated B-lympho‐ cytes [78]. The reproducibility and potentiallity (unlimited self-renewal and ability to differentiate into any cell type) of this technology has made the iPSC field to advance very rapidly. The human iPSC (hiPSC) technology brings together all the potential of hESC in terms of pluripotency without any ethical issue and the immunotolerance of the autologous cell treatment. Therefore, hiPSC technology is one of the most promising fields for future therapies for many human diseases. Safer reprogramming approaches have been designed and many patient specific hiPSC have been generated both to model human diseases and to correct by gene therapy approaches. Depending on the cell type to be reprogrammed, the number of factors used could be reduced and, what is more important, oncogenes or tumor related proteins included in the reprogramming cocktail, like *c-MYC* or *KLF4* [79] could be removed from the original reprogramming cocktail [80-82]. Several groups developed excis‐ able polycistronic lentiviral vectors [83,84] or transposon-based reprogramming systems [85,86], which could be removed after getting the hiPSC clones. Similar results have been ob‐ tained using recombinant proteins [82], synthetic mRNAs [87], and non integrating RNA Sendai Virus vectors [88]. Except for Sendai viruses, non integrating methods show a re‐ duced reprogramming efficiency and the range of cells reprogrammed is not as large as with lentiviral or retroviral vectors.

iPSC technology makes feasible the availability of patient specific cells to study the biology of the disease and develop advanced tools to cure the phenotype and could potentially be used as a therapeutic option (Figure 1). Focussing on metabolic diseases, the first reported metabolic disease patient specific hiPSC line was obtained one year after the first generation of hiPSCs. It was from a 42-year old female that suffered from Type I Diabetes mellitus [89] and it showed no differences compared to a wild type hiPSC line in terms of pluripotency. Next report in which liver metabolic disease patient samples were reprogrammed was car‐ ried out by the group of Ludovic Vallier [90], and showed the potential of this kind of ap‐ proaches for disease modelling and new drug discovery. They reprogrammed fibroblast obtained from α-1 Antitrypsin deficiency (*A1ATD*), Familiar Hypercholesterolemia (FH), Glucose-6-Phosphate deficiency (G6PD), Crigler-Najjar Syndrome and hereditary Tyrosine‐ mia Type 1 patients, and generated hepatocytes that showed characteristics of mature hepat‐ ic cells, like albumin secretion or cytocrome p450 metabolism. Three of the five cell lines (*A1ATD*, FH, and GSD1a hiPSCs) were capable of recapitulating the disease phenotype in vitro. Disease modelling in erythroid diseased induced pluripotent cell lines has been per‐ formed for -Thalassemia [91,92] and sickle cell anemia [93,94]. In these reports the pheno‐ type was corrected by LVs integrated in areas of the genome that were considered safe for viral integration [83] or by gene editing using homologous recombination of the affected lo‐ cus [91,93,94].

led to the opportunity to control the integration of viral vectors at a clonal level. As we have mentioned before, the analysis of lentiviral integration sites in β-thalassemia hiPSC allowed the identification of corrected hiPSC clones expressing β-globin transgene from a safe ge‐ nomic site (also called Safe harbour), a site in which integration does not disturb the expres‐ sion of any neighbouring genes during their erythroid differentiation [83]. The therapeutic use of patient-specific hiPSC emerges then from the combination of gene and cell therapy.

**Drug testing**

**Therapy development**

**Conventional therapy**

**Gene and Cell therapy**

> **Gene correction**

**Differentiation into desired cells**

http://dx.doi.org/10.5772/52531

523

Gene Therapy for Erythroid Metabolic Inherited Diseases

Gene editing is a process in which a DNA sequence is introduced into a specific locus or a chromosomal sequence is replaced. This site-specific precise introduction requires an accu‐ rate recognition mechanism of the target site on the genome. Under normal conditions, the maintenance of the integrity of the genome requires that the cells repair DNA damage with high fidelity. One of the most harmful DNA damage is the generation of double-strand breaks (DSB). DSB are often resolved by non-homologous end joining (NHEJ), which joins the two ends of the DSB. However this DNA repair mechanism could introduce mutations. On the contrary, homologous recombination (HR) is a truly accurate DNA repair mecha‐ nism because it is basically a "copy and paste" mechanism. This process uses an undamaged

From this new research field,future gene therapy protocols will emerge.

**4.2. Gene editing based on homologous recombination**

**Reprogramming to pluripotency**

**Figure 1.** Potential utilities of hiPSC and iPS technology

**Patient sample**

The future therapeutic application of hiPSC will not only require non-integrative reprog‐ ramming system, but also a more precise gene correction. During last years, the cooperation between hiPSC technology and gene editing is being explored. Human iPSC technology has

**Figure 1.** Potential utilities of hiPSC and iPS technology

cMyc) [74] and one year later in human cells together with Thompson's group [75,76], many laboratories around the world have been able to reprogram a large range of somatic cells in‐ to pluripotent stem cells, from neural stem cells [77] to terminally differentiated B-lympho‐ cytes [78]. The reproducibility and potentiallity (unlimited self-renewal and ability to differentiate into any cell type) of this technology has made the iPSC field to advance very rapidly. The human iPSC (hiPSC) technology brings together all the potential of hESC in terms of pluripotency without any ethical issue and the immunotolerance of the autologous cell treatment. Therefore, hiPSC technology is one of the most promising fields for future therapies for many human diseases. Safer reprogramming approaches have been designed and many patient specific hiPSC have been generated both to model human diseases and to correct by gene therapy approaches. Depending on the cell type to be reprogrammed, the number of factors used could be reduced and, what is more important, oncogenes or tumor related proteins included in the reprogramming cocktail, like *c-MYC* or *KLF4* [79] could be removed from the original reprogramming cocktail [80-82]. Several groups developed excis‐ able polycistronic lentiviral vectors [83,84] or transposon-based reprogramming systems [85,86], which could be removed after getting the hiPSC clones. Similar results have been ob‐ tained using recombinant proteins [82], synthetic mRNAs [87], and non integrating RNA Sendai Virus vectors [88]. Except for Sendai viruses, non integrating methods show a re‐ duced reprogramming efficiency and the range of cells reprogrammed is not as large as with

iPSC technology makes feasible the availability of patient specific cells to study the biology of the disease and develop advanced tools to cure the phenotype and could potentially be used as a therapeutic option (Figure 1). Focussing on metabolic diseases, the first reported metabolic disease patient specific hiPSC line was obtained one year after the first generation of hiPSCs. It was from a 42-year old female that suffered from Type I Diabetes mellitus [89] and it showed no differences compared to a wild type hiPSC line in terms of pluripotency. Next report in which liver metabolic disease patient samples were reprogrammed was car‐ ried out by the group of Ludovic Vallier [90], and showed the potential of this kind of ap‐ proaches for disease modelling and new drug discovery. They reprogrammed fibroblast obtained from α-1 Antitrypsin deficiency (*A1ATD*), Familiar Hypercholesterolemia (FH), Glucose-6-Phosphate deficiency (G6PD), Crigler-Najjar Syndrome and hereditary Tyrosine‐ mia Type 1 patients, and generated hepatocytes that showed characteristics of mature hepat‐ ic cells, like albumin secretion or cytocrome p450 metabolism. Three of the five cell lines (*A1ATD*, FH, and GSD1a hiPSCs) were capable of recapitulating the disease phenotype in vitro. Disease modelling in erythroid diseased induced pluripotent cell lines has been per‐ formed for -Thalassemia [91,92] and sickle cell anemia [93,94]. In these reports the pheno‐ type was corrected by LVs integrated in areas of the genome that were considered safe for viral integration [83] or by gene editing using homologous recombination of the affected lo‐

The future therapeutic application of hiPSC will not only require non-integrative reprog‐ ramming system, but also a more precise gene correction. During last years, the cooperation between hiPSC technology and gene editing is being explored. Human iPSC technology has

lentiviral or retroviral vectors.

522 Gene Therapy - Tools and Potential Applications

cus [91,93,94].

led to the opportunity to control the integration of viral vectors at a clonal level. As we have mentioned before, the analysis of lentiviral integration sites in β-thalassemia hiPSC allowed the identification of corrected hiPSC clones expressing β-globin transgene from a safe ge‐ nomic site (also called Safe harbour), a site in which integration does not disturb the expres‐ sion of any neighbouring genes during their erythroid differentiation [83]. The therapeutic use of patient-specific hiPSC emerges then from the combination of gene and cell therapy. From this new research field,future gene therapy protocols will emerge.

#### **4.2. Gene editing based on homologous recombination**

Gene editing is a process in which a DNA sequence is introduced into a specific locus or a chromosomal sequence is replaced. This site-specific precise introduction requires an accu‐ rate recognition mechanism of the target site on the genome. Under normal conditions, the maintenance of the integrity of the genome requires that the cells repair DNA damage with high fidelity. One of the most harmful DNA damage is the generation of double-strand breaks (DSB). DSB are often resolved by non-homologous end joining (NHEJ), which joins the two ends of the DSB. However this DNA repair mechanism could introduce mutations. On the contrary, homologous recombination (HR) is a truly accurate DNA repair mecha‐ nism because it is basically a "copy and paste" mechanism. This process uses an undamaged homologous segment of DNA that can be exogenously provided as a template to copy the information across the DSB. The fidelity of HR gives us the specificity and accuracy that gene editing requires.

subset of homing endonucleases which recognize a DNA sequence from 14 to 40 nucleotides. Current MNs have been engineered from natural homing endonucleases to increase the num‐

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

525

ZNFs have been widely used for gene editing in hESC and hiPSC. In 2007, Dr. Naldini's labora‐ tory showed the insertion of GFP into the CCR5 safe harbour in human stem cells (HSC and hESC) after inducing HR by ZFN expression. The CCR5-ZFN and donor DNAs were delivered into hESC by intergrative deficient lentiviruses. More interestingly, targeted hESC were able to differentiate into neurons keeping GFP expression [99]. Soon, the proof of principle for the clini‐ cal application of ZFN-mediated gene editing was tested in hiPSC from patients affected by dif‐ ferent genetic diseases. The first pre-clinical use of ZFN for gene therapy of a metabolic disease was performed by Yusa *et al.* In this report, gene correction was performed at the α1-antitrypsin (*A1AT*) locus to revert A1AT deficiency in hiPSC derived from a patient with a point mutation. This group included a Puromycin resistence cassette flanked by piggyBac sites, so that the Puro‐ mycin selection facilitated the isolation of corrected A1ATD-iPSC clones. Afterwards, the selec‐ tion cassette was removed by piggyBac transposon, obtaining corrected hiPS clones without any additional sequence. These corrected hiPS clones were then differentiated into hepatocytelike cells to confirm the complete correction of the A1ATD [101]. Other hiPSC gene editing ap‐ proaches and functional correction of erithroid diseases include gene correction of Sickle Cell

One of the major limitations of ZFN is the generation of "off-target" DSB, due to unspecific sequence recognition. Different studies have highlighted this as a possible limitation in the clinical use of ZFN-mediated HR [100,101]. Recent works have explored the potential of oth‐ er types of DNA-nucleases in order to prevent the "off-target" cleave limitations of the ZFN, being TALEN and MN the most promising ones. The feasibility of TALEN to mediate HR in hESC and hiPSC was assess by Jaenisch's group when they designed TALEN targeting the *PPP1R12C* (at *AAVS1* locus), *POU5F1* and *PITX3* genes at precisely the same positions as the one targeted by ZFN in their previous work [102]. The authors described a gene editing effi‐ ciency similar to the one achieved by ZFN with a low level of "off-targets" [103]. A strategy to minimize the potential number of "off-targets" is to design TALEN to work as obligatory heterodimers, which has beeing already done in the engineered MNs. The application of the TALEN and MN as tools to improve HR is still on going. We are exploring the pre-clinical

use of TALEN and MN to correct erythroid metabolic genetic diseases, such as PKD.

**5. Complementary developments for the application of gene therapy to**

Another challenge for the clinical application of gene therapy relates to vector targeting. To achieve successful gene therapy, the appropriate gene must be delivered to target cells and specifically expressed in them, without harming non-targeted cells. The most common and easiest way to target specific cells is by *ex vivo* infection of the desired cell population. There‐

ber of target DNA sequences.

Anemia [94] and -Thalassemia [91].

**erythroid metabolic diseases**

**5.1.** *In vivo* **transduction using engineered envelopes**

The natural HR process has been adapted by researchers to get the desirable addition of an exogenous cassette into the targeted locus. This techniques have been widely use for the generation of knock-out and knock-in transgenic animals [95]. To correct or insert and ex‐ press a transgene by HR we can consider three different strategies: i) Gene correction, a base or some bases can be substituted from the original strand using an homologous sequence where this base or bases are modified; it is the way to introduce/repair point or small muta‐ tions; ii) Safe harbour integration, a complete expression cassette (promoter, transgene and regulatory signals) is inserted in a safe place of the genome, without altering the expression of the surrounding genes and without being silenced by epigenetic mechanisms; this is the case for *AASV1* and *CCR5* loci. Additionally to these well known safe harbours, there is a wide research focused on finding potential new safe harbour places. iii) Knock-in insertion, the cDNA of a gene is introduced in the same site of the endogenous gene, linked by splic‐ ing mechanisms to the endogenous gene assuring the expression of the inserted sequence by the endogenous regulatory elements of the locus where it is integrated.

Gene editing process can be separated in two different steps, generation of DSB and HR. The efficacy of gene editing in human cells depends on the generation of DSB at the specific tar‐ get site and on the DNA repair mechanism that the cell uses to resolve the DSB. Unfortu‐ nately, NHEJ is the dominant pathway to solve these DNA lesions in human cells. Additionally, HR varies in different cell types and requires transit through S-G2 phase of the cell cycle [96]. These limitations make gene editing in human cells difficult to achieve. How‐ ever, different approaches are being used to improve gene editing by HR, like increasing the length of the DNA sequences homologous to target site (homology arms) [97], the use of ad‐ eno-associated vectors [98], the improvement of selection methods for edited cells or the stimulation of HR by inducing DSB using DNA nucleases.

Recently, engineered DNA nucleases have been developed to specifically induce DSB at a unique and defined sequence in the cell genome. These proteins are formed by a nuclease do‐ main and a DNA binding domain whose sequence specificity can be engineered. The most widely used DNA nucleases are Zinc finger nucleases (ZFN), homing meganucleases (MN) and transcription activator-like effector nucleases (TALEN). They identify a potentially unique se‐ quence in the genome and generate DSBs in the desired genomic site, aiming to promote the re‐ pair of the DSB by the cell machinery and, ideally by HR. The DNA binding domain of a ZFNs is derived from zinc-finger proteins and is linked to the nuclease domain of the restriction enzyme Fok-I. DNA-biding domain is a tandem repeat of Cys2His2 zinc fingers, each of which recogniz‐ es three nucleotides. ZFNs work as pairs of two monomers of ZFN, one in reverse orientation. This ZFN dimer can be designed to bind to genomic sequences of 18-36 nucleotides long. TAL‐ ENs have a similar structure to ZFNs, but the DNA-binding domain comes from transcription activator-like effector proteins. The DNA-binding domain in TALENs is a tandem array of ami‐ no acid repeats. Each of these units is able to bind to one of the four possible nucleotides and this makes that the DNA binding domain can be designed to recognize any desired genomic se‐ quence. TALENs also cleave as dimers. Contrary to these synthetic DNA-nucleases, MNs are a subset of homing endonucleases which recognize a DNA sequence from 14 to 40 nucleotides. Current MNs have been engineered from natural homing endonucleases to increase the num‐ ber of target DNA sequences.

homologous segment of DNA that can be exogenously provided as a template to copy the information across the DSB. The fidelity of HR gives us the specificity and accuracy that

The natural HR process has been adapted by researchers to get the desirable addition of an exogenous cassette into the targeted locus. This techniques have been widely use for the generation of knock-out and knock-in transgenic animals [95]. To correct or insert and ex‐ press a transgene by HR we can consider three different strategies: i) Gene correction, a base or some bases can be substituted from the original strand using an homologous sequence where this base or bases are modified; it is the way to introduce/repair point or small muta‐ tions; ii) Safe harbour integration, a complete expression cassette (promoter, transgene and regulatory signals) is inserted in a safe place of the genome, without altering the expression of the surrounding genes and without being silenced by epigenetic mechanisms; this is the case for *AASV1* and *CCR5* loci. Additionally to these well known safe harbours, there is a wide research focused on finding potential new safe harbour places. iii) Knock-in insertion, the cDNA of a gene is introduced in the same site of the endogenous gene, linked by splic‐ ing mechanisms to the endogenous gene assuring the expression of the inserted sequence by

Gene editing process can be separated in two different steps, generation of DSB and HR. The efficacy of gene editing in human cells depends on the generation of DSB at the specific tar‐ get site and on the DNA repair mechanism that the cell uses to resolve the DSB. Unfortu‐ nately, NHEJ is the dominant pathway to solve these DNA lesions in human cells. Additionally, HR varies in different cell types and requires transit through S-G2 phase of the cell cycle [96]. These limitations make gene editing in human cells difficult to achieve. How‐ ever, different approaches are being used to improve gene editing by HR, like increasing the length of the DNA sequences homologous to target site (homology arms) [97], the use of ad‐ eno-associated vectors [98], the improvement of selection methods for edited cells or the

Recently, engineered DNA nucleases have been developed to specifically induce DSB at a unique and defined sequence in the cell genome. These proteins are formed by a nuclease do‐ main and a DNA binding domain whose sequence specificity can be engineered. The most widely used DNA nucleases are Zinc finger nucleases (ZFN), homing meganucleases (MN) and transcription activator-like effector nucleases (TALEN). They identify a potentially unique se‐ quence in the genome and generate DSBs in the desired genomic site, aiming to promote the re‐ pair of the DSB by the cell machinery and, ideally by HR. The DNA binding domain of a ZFNs is derived from zinc-finger proteins and is linked to the nuclease domain of the restriction enzyme Fok-I. DNA-biding domain is a tandem repeat of Cys2His2 zinc fingers, each of which recogniz‐ es three nucleotides. ZFNs work as pairs of two monomers of ZFN, one in reverse orientation. This ZFN dimer can be designed to bind to genomic sequences of 18-36 nucleotides long. TAL‐ ENs have a similar structure to ZFNs, but the DNA-binding domain comes from transcription activator-like effector proteins. The DNA-binding domain in TALENs is a tandem array of ami‐ no acid repeats. Each of these units is able to bind to one of the four possible nucleotides and this makes that the DNA binding domain can be designed to recognize any desired genomic se‐ quence. TALENs also cleave as dimers. Contrary to these synthetic DNA-nucleases, MNs are a

the endogenous regulatory elements of the locus where it is integrated.

stimulation of HR by inducing DSB using DNA nucleases.

gene editing requires.

524 Gene Therapy - Tools and Potential Applications

ZNFs have been widely used for gene editing in hESC and hiPSC. In 2007, Dr. Naldini's labora‐ tory showed the insertion of GFP into the CCR5 safe harbour in human stem cells (HSC and hESC) after inducing HR by ZFN expression. The CCR5-ZFN and donor DNAs were delivered into hESC by intergrative deficient lentiviruses. More interestingly, targeted hESC were able to differentiate into neurons keeping GFP expression [99]. Soon, the proof of principle for the clini‐ cal application of ZFN-mediated gene editing was tested in hiPSC from patients affected by dif‐ ferent genetic diseases. The first pre-clinical use of ZFN for gene therapy of a metabolic disease was performed by Yusa *et al.* In this report, gene correction was performed at the α1-antitrypsin (*A1AT*) locus to revert A1AT deficiency in hiPSC derived from a patient with a point mutation. This group included a Puromycin resistence cassette flanked by piggyBac sites, so that the Puro‐ mycin selection facilitated the isolation of corrected A1ATD-iPSC clones. Afterwards, the selec‐ tion cassette was removed by piggyBac transposon, obtaining corrected hiPS clones without any additional sequence. These corrected hiPS clones were then differentiated into hepatocytelike cells to confirm the complete correction of the A1ATD [101]. Other hiPSC gene editing ap‐ proaches and functional correction of erithroid diseases include gene correction of Sickle Cell Anemia [94] and -Thalassemia [91].

One of the major limitations of ZFN is the generation of "off-target" DSB, due to unspecific sequence recognition. Different studies have highlighted this as a possible limitation in the clinical use of ZFN-mediated HR [100,101]. Recent works have explored the potential of oth‐ er types of DNA-nucleases in order to prevent the "off-target" cleave limitations of the ZFN, being TALEN and MN the most promising ones. The feasibility of TALEN to mediate HR in hESC and hiPSC was assess by Jaenisch's group when they designed TALEN targeting the *PPP1R12C* (at *AAVS1* locus), *POU5F1* and *PITX3* genes at precisely the same positions as the one targeted by ZFN in their previous work [102]. The authors described a gene editing effi‐ ciency similar to the one achieved by ZFN with a low level of "off-targets" [103]. A strategy to minimize the potential number of "off-targets" is to design TALEN to work as obligatory heterodimers, which has beeing already done in the engineered MNs. The application of the TALEN and MN as tools to improve HR is still on going. We are exploring the pre-clinical use of TALEN and MN to correct erythroid metabolic genetic diseases, such as PKD.

#### **5. Complementary developments for the application of gene therapy to erythroid metabolic diseases**

#### **5.1.** *In vivo* **transduction using engineered envelopes**

Another challenge for the clinical application of gene therapy relates to vector targeting. To achieve successful gene therapy, the appropriate gene must be delivered to target cells and specifically expressed in them, without harming non-targeted cells. The most common and easiest way to target specific cells is by *ex vivo* infection of the desired cell population. There‐

fore, cells can be directly exposed to the viral vectors facilitating viral-cell interaction. These interactions are driven by the envelope protein which can be adapted from other viruses re‐ directing the tropism of the vector. The most widely used vectors are lentiviral vectors pseu‐ dotyped with the attachment glycoprotein of the vesicular stomatitis virus (VSV-G), which allows the production of high-titre vectors and confers a broad host range [104]. In compari‐ son with them, engineered LVs capable of delivering genes of interest to predetermined cells, can reduce the targeting of undesirable cell types and improve the safety profile, which will further enhance the use of this vector system for gene therapy applications [105,106]. As we have mentioned above, the use of promoters and regulatory sequences that are only active in target cells adds lineage specific expression, although integration of the viruses in non desired cells is still possible. *Ex vivo*-targeted gene delivery, as commonly used in HSCs transduction, is associated with a risk of inducing cell differentiation and loss of the engraftment potential of these cells [107]. On the contrary, *in vivo* gene transfer could target HSCs in their stem cell niche, a microenvironment that regulates HSC survival and maintenance [105]. To accomplish this, the vector must display a suitable system to selec‐ tively infect the desired population, for example the introduction of a specific ligand to bind a target-cell receptor [106].

ment and efficiently transduces very immature hCD34+

**5.2.** *In vitro* **production of mature erythrocytes**

for transfusion, avoiding the adverse immune effects.

this possibility a therapeutic option.

**6. Conclusions**

HSC-targeted LVs should improve current gene therapy protocols through the transduction

Periodical blood transfusion is the previous to the last therapeutic option for severe cases of CNSHA patients. However, this clinical practice involves also adverse effects related to the immuneresponse against minor erythrocyte antigens which makes the patients refractory to additional blood transfusions in the long run. The availability of genetically corrected pa‐ tient-specific iPSC would allow the possibility of generating disease free erythrocytes ready

There have been numerous attempts to produce RBC *in vitro* from different sources of stem cells. To date, the most successful protocol has been developed by the group of Luc Douay [113,114]. Using peripheral blood CD34+ cells, these authors were able to expand and gener‐ ate RBC with *in vitro* and *in vivo* features of native RBC, and were also capable of transfusing a patient with *in vitro* generated erythrocytes. Notably, the same authors reported a protocol to generate RBC from hiPSC as an alternative source of HSC [114]. Other groups have de‐ scribed similar protocols to generate erythrocytes from hESC or hiPSC [115-118], although in all these studies the RBC generated from embryonic cells expressed embryonic and foetal hemoglobins but low levels of adult hemoglobin. Additional efforts should be done to make

Erythroid metabolic diseases are well defined and well known diseases which main symp‐ tom is CNSHA. As they are monogenic diseases that can be cured by allogeneic bone mar‐ row transplantation, they are very good candidates to be treated by gene therapy. However, the low number of patients with poor prognosis requiring BM transplantation and the ab‐ sence of an apparent selective advantage of the corrected cells over the diseased ones have made their approach for gene therapy less attractive than other erythropaties. Up to now, no gene therapy clinical trial for erythroid metabolic diseseases has been accomplished. Gene therapy attempts in animal models have been applied to G6PD and PKD with successful re‐ sults, emphasizing the usefulness of a gene therapy approach for these diseases. Although adverse effects due to ectopic expression of the metabolic enzyme have not been observed, an erythroid specific expression is preferred. Many developments have been made for the specific expression of globin genes that could be adapted to vectors developed for the dis‐ cussed erythroid metabolic diseases. Similarly, any attempt directed to the improvement of HSC transduction, including the possibility of *in vivo* targeted gene therapy could be ap‐ plied. On the other hand, the combination of cell reprogramming and gene editing opens a new world of possibilities that could be easily applied to these diseases. hESC and hiPSC are helping in the development of the next generation of gene therapy, which implies a precise

of primitive HSCs directly in the bone marrow of patients with genetic diseases.

HSCs [113]. This new generation of

http://dx.doi.org/10.5772/52531

527

Gene Therapy for Erythroid Metabolic Inherited Diseases

Many attempts have been made to develop targeted transduction systems using retroviral and lentiviral vectors by altering the envelope glycoprotein (Env), which is responsible for the binding of the virus to the cell surface receptors and for mediating viral entry into the cell. The plasticity of the surface domain of Env allows insertion of ligands, peptides or sin‐ gle-chain antibodies that can direct the vectors to specific cell types [108]. However, this type of manipulation negatively affects the fusion domain of Env, resulting in low viral tit‐ ers. To overcome this downside, a method to engineer lentiviral vectors has been developed. These vectors transduce specific cell types by breaking up the binding and fusion functions of the envelope protein into two distinct proteins [108]. Instead of pseudotyping lentiviral vectors with a modified viral envelope protein, these lentiviral vectors co-display a targeting antibody and a fusogenic molecule on the same viral vector surface. Based on molecular rec‐ ognition, the targeting antibody should direct lentiviral vectors to the specific cell type. The binding between the antibody and the corresponding cellular antigen should induce endo‐ cytosis resulting in the transport of lentiviral vectors into the endosomal compartment. Once inside the endosome, the fusogenic molecule should undergo a conformational change in re‐ sponse to the decrease in pH, thereby releasing the viral core into the cytosol [109]. The use of fusion domain of the binding defective Sindbis virus glycoprotein together with an anti-CD20 antibody has been shown to mediate the targeted transduction of lentiviral vectors to CD20-expressing B cells [110].

However, two major challenges for *in vivo* gene delivery are LVthe exposure to the host im‐ mune/complement system and off-target cell transfer after systemic administration. For these reasons, second generation of early-acting-cytokine-displaying LVs has been devel‐ oped, that circumvents these obstacles by specifically targeting hCD34+ cells [111,112]. For example, RDTR/SCFHA-LV, consisting of RD114 glycoprotein and stem cell factor (SCF) fused to the *Influenza hameglutinin* env protein, is resistant to degradation by human comple‐

ment and efficiently transduces very immature hCD34+ HSCs [113]. This new generation of HSC-targeted LVs should improve current gene therapy protocols through the transduction of primitive HSCs directly in the bone marrow of patients with genetic diseases.

#### **5.2.** *In vitro* **production of mature erythrocytes**

Periodical blood transfusion is the previous to the last therapeutic option for severe cases of CNSHA patients. However, this clinical practice involves also adverse effects related to the immuneresponse against minor erythrocyte antigens which makes the patients refractory to additional blood transfusions in the long run. The availability of genetically corrected pa‐ tient-specific iPSC would allow the possibility of generating disease free erythrocytes ready for transfusion, avoiding the adverse immune effects.

There have been numerous attempts to produce RBC *in vitro* from different sources of stem cells. To date, the most successful protocol has been developed by the group of Luc Douay [113,114]. Using peripheral blood CD34+ cells, these authors were able to expand and gener‐ ate RBC with *in vitro* and *in vivo* features of native RBC, and were also capable of transfusing a patient with *in vitro* generated erythrocytes. Notably, the same authors reported a protocol to generate RBC from hiPSC as an alternative source of HSC [114]. Other groups have de‐ scribed similar protocols to generate erythrocytes from hESC or hiPSC [115-118], although in all these studies the RBC generated from embryonic cells expressed embryonic and foetal hemoglobins but low levels of adult hemoglobin. Additional efforts should be done to make this possibility a therapeutic option.

#### **6. Conclusions**

fore, cells can be directly exposed to the viral vectors facilitating viral-cell interaction. These interactions are driven by the envelope protein which can be adapted from other viruses re‐ directing the tropism of the vector. The most widely used vectors are lentiviral vectors pseu‐ dotyped with the attachment glycoprotein of the vesicular stomatitis virus (VSV-G), which allows the production of high-titre vectors and confers a broad host range [104]. In compari‐ son with them, engineered LVs capable of delivering genes of interest to predetermined cells, can reduce the targeting of undesirable cell types and improve the safety profile, which will further enhance the use of this vector system for gene therapy applications [105,106]. As we have mentioned above, the use of promoters and regulatory sequences that are only active in target cells adds lineage specific expression, although integration of the viruses in non desired cells is still possible. *Ex vivo*-targeted gene delivery, as commonly used in HSCs transduction, is associated with a risk of inducing cell differentiation and loss of the engraftment potential of these cells [107]. On the contrary, *in vivo* gene transfer could target HSCs in their stem cell niche, a microenvironment that regulates HSC survival and maintenance [105]. To accomplish this, the vector must display a suitable system to selec‐ tively infect the desired population, for example the introduction of a specific ligand to bind

Many attempts have been made to develop targeted transduction systems using retroviral and lentiviral vectors by altering the envelope glycoprotein (Env), which is responsible for the binding of the virus to the cell surface receptors and for mediating viral entry into the cell. The plasticity of the surface domain of Env allows insertion of ligands, peptides or sin‐ gle-chain antibodies that can direct the vectors to specific cell types [108]. However, this type of manipulation negatively affects the fusion domain of Env, resulting in low viral tit‐ ers. To overcome this downside, a method to engineer lentiviral vectors has been developed. These vectors transduce specific cell types by breaking up the binding and fusion functions of the envelope protein into two distinct proteins [108]. Instead of pseudotyping lentiviral vectors with a modified viral envelope protein, these lentiviral vectors co-display a targeting antibody and a fusogenic molecule on the same viral vector surface. Based on molecular rec‐ ognition, the targeting antibody should direct lentiviral vectors to the specific cell type. The binding between the antibody and the corresponding cellular antigen should induce endo‐ cytosis resulting in the transport of lentiviral vectors into the endosomal compartment. Once inside the endosome, the fusogenic molecule should undergo a conformational change in re‐ sponse to the decrease in pH, thereby releasing the viral core into the cytosol [109]. The use of fusion domain of the binding defective Sindbis virus glycoprotein together with an anti-CD20 antibody has been shown to mediate the targeted transduction of lentiviral vectors to

However, two major challenges for *in vivo* gene delivery are LVthe exposure to the host im‐ mune/complement system and off-target cell transfer after systemic administration. For these reasons, second generation of early-acting-cytokine-displaying LVs has been devel‐

example, RDTR/SCFHA-LV, consisting of RD114 glycoprotein and stem cell factor (SCF) fused to the *Influenza hameglutinin* env protein, is resistant to degradation by human comple‐

cells [111,112]. For

oped, that circumvents these obstacles by specifically targeting hCD34+

a target-cell receptor [106].

526 Gene Therapy - Tools and Potential Applications

CD20-expressing B cells [110].

Erythroid metabolic diseases are well defined and well known diseases which main symp‐ tom is CNSHA. As they are monogenic diseases that can be cured by allogeneic bone mar‐ row transplantation, they are very good candidates to be treated by gene therapy. However, the low number of patients with poor prognosis requiring BM transplantation and the ab‐ sence of an apparent selective advantage of the corrected cells over the diseased ones have made their approach for gene therapy less attractive than other erythropaties. Up to now, no gene therapy clinical trial for erythroid metabolic diseseases has been accomplished. Gene therapy attempts in animal models have been applied to G6PD and PKD with successful re‐ sults, emphasizing the usefulness of a gene therapy approach for these diseases. Although adverse effects due to ectopic expression of the metabolic enzyme have not been observed, an erythroid specific expression is preferred. Many developments have been made for the specific expression of globin genes that could be adapted to vectors developed for the dis‐ cussed erythroid metabolic diseases. Similarly, any attempt directed to the improvement of HSC transduction, including the possibility of *in vivo* targeted gene therapy could be ap‐ plied. On the other hand, the combination of cell reprogramming and gene editing opens a new world of possibilities that could be easily applied to these diseases. hESC and hiPSC are helping in the development of the next generation of gene therapy, which implies a precise gene targeting. Gene editing by HR is the best and safest gene therapy procedure because avoids any perturbation in the targeted genome. Besides the combination of hiPSC and gene editing could be the future therapy for many genetic-based diseases. The hiPSC technology is the springboard for the development of more efficient HR protocols applicable to other types of stem cells such as hematopoietic stem cells. The combination of methods for obtain‐ ing big amounts of RBC from HSC or embryonic cells, along with the improvement of the different gene therapy approaches described in this chapter, opens up the possibility of the therapeutic application involving the infusion of RBC differentiated *in vitro* from genetically corrected patient specific stem cells.

kb kilobases

LV Lentivirus

LCR Locus control region

LTR Long terminal repeats

MN homing meganuclease

PFK phosphofructokinase

RBC Erythrocytes

WT wild-type

NHEJ non-homologous end joining

SIN-LV Self-inactivated lentiviral vector

TPI Triose phosphate isomerase

ZFN zinc finger nuclease

**Aknowledgements**

**Author details**

Zita Garate and Jose C. Segovia

TALEN transcription activator-like effector nuclease

The authors thank L. Cerrato, M.A. Martín and I. Orman for their technical assistance. We would also like to thank Dr. J. Bueren for careful reading and suggestion of the manuscript. M.G.G. was partially supported by a short-term fellowship from the European Molecular Bi‐ ology Organization (EMBO ASTF 188.00-2010). Z.G. is a fellowship of the PhD program of the Departamento de Educación, Universidades e Investigación del Gobierno Vasco. This work was funded by grants from the Ministerio de Economía y Competitividad (SAF2011-30526-C02-01), Fondo de Investigaciones Sanitarias (RD06/0010/0015) and the PERSIST European project. The authors also thank the Fundación Botín for promoting trans‐ lational research at the Hematopoiesis and Gene Therapy Division-CIEMAT/CIBERER.

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

529

Maria Garcia-Gomez, Oscar Quintana-Bustamante, Maria Garcia-Bravo, S. Navarro,

vestigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain

Differentiation and Cytometry Unit, Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de In‐

#### **Nomenclature**

5-FU 5-fluorouracil A1ATD-1 antitripsin deficiency AAV Adeno-associated virus BM Bone marrow BPGM 2,3-bisphosphoglycerate mutase CNSHA Chronic non spherocytic hemaolotyc anemia DSB Double strand breaks Env Viral envelope FH familiar hypercholesterolemia G6P Glucose-6-phosphate G6PD Glucose-6-phopahate dehydrogenase GPI Glucose phosphate isomerase GS Glutathione synthetase hESC human embryonic stem cell hIF1 hypoxia-inducible factor-1 hiPSC Human induced pluripotent stem cell HK Hexokinase HR Homologous recombination HS DNase I hypersensitive sites HSC Hematopoietic stem cell iPSC Induced pluripotent stem cell

kb kilobases LCR Locus control region LTR Long terminal repeats LV Lentivirus MN homing meganuclease NHEJ non-homologous end joining PFK phosphofructokinase RBC Erythrocytes SIN-LV Self-inactivated lentiviral vector TALEN transcription activator-like effector nuclease TPI Triose phosphate isomerase WT wild-type ZFN zinc finger nuclease

#### **Aknowledgements**

gene targeting. Gene editing by HR is the best and safest gene therapy procedure because avoids any perturbation in the targeted genome. Besides the combination of hiPSC and gene editing could be the future therapy for many genetic-based diseases. The hiPSC technology is the springboard for the development of more efficient HR protocols applicable to other types of stem cells such as hematopoietic stem cells. The combination of methods for obtain‐ ing big amounts of RBC from HSC or embryonic cells, along with the improvement of the different gene therapy approaches described in this chapter, opens up the possibility of the therapeutic application involving the infusion of RBC differentiated *in vitro* from genetically

corrected patient specific stem cells.

528 Gene Therapy - Tools and Potential Applications

A1ATD-1 antitripsin deficiency AAV Adeno-associated virus

BPGM 2,3-bisphosphoglycerate mutase

CNSHA Chronic non spherocytic hemaolotyc anemia

**Nomenclature**

5-FU 5-fluorouracil

BM Bone marrow

Env Viral envelope

DSB Double strand breaks

G6P Glucose-6-phosphate

GS Glutathione synthetase

HK Hexokinase

FH familiar hypercholesterolemia

GPI Glucose phosphate isomerase

hESC human embryonic stem cell hIF1 hypoxia-inducible factor-1

HR Homologous recombination HS DNase I hypersensitive sites HSC Hematopoietic stem cell

iPSC Induced pluripotent stem cell

G6PD Glucose-6-phopahate dehydrogenase

hiPSC Human induced pluripotent stem cell

The authors thank L. Cerrato, M.A. Martín and I. Orman for their technical assistance. We would also like to thank Dr. J. Bueren for careful reading and suggestion of the manuscript. M.G.G. was partially supported by a short-term fellowship from the European Molecular Bi‐ ology Organization (EMBO ASTF 188.00-2010). Z.G. is a fellowship of the PhD program of the Departamento de Educación, Universidades e Investigación del Gobierno Vasco. This work was funded by grants from the Ministerio de Economía y Competitividad (SAF2011-30526-C02-01), Fondo de Investigaciones Sanitarias (RD06/0010/0015) and the PERSIST European project. The authors also thank the Fundación Botín for promoting trans‐ lational research at the Hematopoiesis and Gene Therapy Division-CIEMAT/CIBERER.

#### **Author details**

Maria Garcia-Gomez, Oscar Quintana-Bustamante, Maria Garcia-Bravo, S. Navarro, Zita Garate and Jose C. Segovia

Differentiation and Cytometry Unit, Hematopoiesis and Gene Therapy Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de In‐ vestigación Biomédica en Red de Enfermedades Raras (CIBER-ER), Madrid, Spain

#### **References**

[1] Aiuti A, Bachoud-Levi AC, Blesch A, *et al.* Progress and prospects: gene therapy clin‐ ical trials (part 2). Gene Ther. 2007;14:1555-1563.

[14] Bulliamy T, Luzzatto L, Hirono A, Beutler E. Hematologically important mutations: glucose-6-phosphate dehydrogenase. Blood Cells Mol Dis. 1997;23:302-313.

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

531

[15] Beutler E, Kuhl W, Gelbart T, Forman L. DNA sequence abnormalities of human glu‐ cose-6-phosphate dehydrogenase variants. J Biol Chem. 1991;266:4145-4150.

[16] Mason PJ, Sonati MF, MacDonald D, *et al.* New glucose-6-phosphate dehydrogenase

[17] Jacobasch G, Rapoport SM. Hemolytic anemias due to erythrocyte enzyme deficien‐

[18] Vives Corrons JL, Feliu E, Pujades MA, *et al.* Severe-glucose-6-phosphate dehydro‐ genase (G6PD) deficiency associated with chronic hemolytic anemia, granulocyte dysfunction, and increased susceptibility to infections: description of a new molecu‐

[19] Roos D, van Zwieten R, Wijnen JT, *et al.* Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections. Blood.

[20] Ko CH, Li K, Li CL, *et al.* Development of a novel mouse model of severe glucose-6 phosphate dehydrogenase (G6PD)-deficiency for in vitro and in vivo assessment of

[21] Zanella A, Fermo E, Bianchi P, Chiarelli LR, Valentini G. Pyruvate kinase deficiency:

[22] Fothergill-Gilmore LA, Michels PA. Evolution of glycolysis. Prog Biophys Mol Biol.

[23] Satoh H, Tani K, Yoshida MC, Sasaki M, Miwa S, Fujii H. The human liver-type pyr‐ uvate kinase (PKL) gene is on chromosome 1 at band q21. Cytogenet Cell Genet.

[24] Noguchi T, Yamada K, Inoue H, Matsuda T, Tanaka T. The L- and R-type isozymes of rat pyruvate kinase are produced from a single gene by use of different promoters.

[25] Guguen-Guillouzo C, Szajnert MF, Marie J, Delain D, Schapira F. Differentiation in vivo and in vitro of pyruvate kinase isozymes in rat muscle. Biochimie. 1977;59:65-71.

[26] Kanno H, Fujii H, Miwa S. Structural analysis of human pyruvate kinase L-gene and identification of the promoter activity in erythroid cells. Biochem Biophys Res Com‐

[27] Nakashima K, Miwa S, Fujii H, *et al.* Characterization of pyruvate kinase from the liver of a patient with aberrant erythrocyte pyruvate kinase, PK Nagasaki. J Lab Clin

hemolytic toxicity to red blood cells. Blood Cells Mol Dis. 2011;47:176-181.

the genotype-phenotype association. Blood Rev. 2007;21:217-231.

mutations associated with chronic anemia. Blood. 1995;85:1377-1380.

cies. Mol Aspects Med. 1996;17:143-170.

1999;94:2955-2962.

1993;59:105-235.

1988;47:132-133.

mun. 1992;188:516-523.

Med. 1977;90:1012-1020.

J Biol Chem. 1987;262:14366-14371.

lar variant (G6PD Barcelona). Blood. 1982;59:428-434.


[14] Bulliamy T, Luzzatto L, Hirono A, Beutler E. Hematologically important mutations: glucose-6-phosphate dehydrogenase. Blood Cells Mol Dis. 1997;23:302-313.

**References**

[1] Aiuti A, Bachoud-Levi AC, Blesch A, *et al.* Progress and prospects: gene therapy clin‐

[2] Herzog RW, Cao O, Srivastava A. Two decades of clinical gene therapy success is fi‐

[3] Naldini L. Ex vivo gene transfer and correction for cell-based therapies. Nat Rev

[4] Richard E, Mendez M, Mazurier F, *et al.* Gene therapy of a mouse model of protopor‐ phyria with a self-inactivating erythroid-specific lentiviral vector without preselec‐

[5] Rovira A, De Angioletti M, Camacho-Vanegas O, *et al.* Stable in vivo expression of glucose-6-phosphate dehydrogenase (G6PD) and rescue of G6PD deficiency in stem

[6] Richard RE, Weinreich M, Chang KH, Ieremia J, Stevenson MM, Blau CA. Modulat‐ ing erythrocyte chimerism in a mouse model of pyruvate kinase deficiency. Blood.

[7] Tani K, Yoshikubo T, Ikebuchi K, *et al.* Retrovirus-mediated gene transfer of human pyruvate kinase (PK) cDNA into murine hematopoietic cells: implications for gene

[8] Morimoto M, Kanno H, Asai H, *et al.* Pyruvate kinase deficiency of mice associated with nonspherocytic hemolytic anemia and cure of the anemia by marrow transplan‐

[9] Kanno H, Utsugisawa T, Aizawa S, *et al.* Transgenic rescue of hemolytic anemia due to red blood cell pyruvate kinase deficiency. Haematologica. 2007;92:731-737.

[10] Zaucha JA, Yu C, Lothrop CD, Jr., *et al.* Severe canine hereditary hemolytic anemia treated by nonmyeloablative marrow transplantation. Biol Blood Marrow Trans‐

[11] Meza NW, Quintana-Bustamante O, Puyet A, *et al.* In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

[12] Tanphaichitr VS, Suvatte V, Issaragrisil S, *et al.* Successful bone marrow transplanta‐ tion in a child with red blood cell pyruvate kinase deficiency. Bone Marrow Trans‐

[13] Meza NW, Alonso-Ferrero ME, Navarro S, *et al.* Rescue of pyruvate kinase deficiency in mice by gene therapy using the human isoenzyme. Mol Ther. 2009;17:2000-2009.

ical trials (part 2). Gene Ther. 2007;14:1555-1563.

nally mounting. Discov Med. 2010;9:105-111.

cells by gene transfer. Blood. 2000;96:4111-4117.

therapy of human PK deficiency. Blood. 1994;83:2305-2310.

tation without host irradiation. Blood. 1995;86:4323-4330.

Genet. 2011;12:301-315.

530 Gene Therapy - Tools and Potential Applications

2004;103:4432-4439.

plant. 2001;7:14-24.

plant. 2000;26:689-690.

Hum Gene Ther. 2007;18:502-514.

tion. Mol Ther. 2001;4:331-338.


[28] Zanella A, Fermo E, Bianchi P, Valentini G. Red cell pyruvate kinase deficiency: mo‐ lecular and clinical aspects. Br J Haematol. 2005;130:11-25.

[43] Modlich U, Navarro S, Zychlinski D, *et al.* Insertional transformation of hematopoiet‐ ic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther.

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

533

[44] Montini E, Cesana D, Schmidt M, et al. Hematopoietic stem cell gene transfer in a tu‐ mor-prone mouse model uncovers low genotoxicity of lentiviral vector integration.

[45] Montini E, Cesana D, Schmidt M, *et al.* The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model

[46] Puthenveetil G, Scholes J, Carbonell D, et al. Successful correction of the human betathalassemia major phenotype using a lentiviral vector. Blood. 2004;104:3445-3453. [47] Grande A, Piovani B, Aiuti A, Ottolenghi S, Mavilio F, Ferrari G. Transcriptional tar‐ geting of retroviral vectors to the erythroblastic progeny of transduced hematopoiet‐

[48] Testa A, Lotti F, Cairns L, *et al.* Deletion of a negatively acting sequence in a chimeric GATA-1 enhancer-long terminal repeat greatly increases retrovirally mediated eryth‐

[49] Ren S, Wong BY, Li J, Luo XN, Wong PM, Atweh GF. Production of genetically stable high-titer retroviral vectors that carry a human gamma-globin gene under the control

[50] Emery DW, Chen H, Li Q, Stamatoyannopoulos G. Development of a condensed lo‐ cus control region cassette and testing in retrovirus vectors for A gamma-globin.

[51] Robertson G, Garrick D, Wu W, Kearns M, Martin D, Whitelaw E. Position-depend‐ ent variegation of globin transgene expression in mice. Proc Natl Acad Sci U S A.

[52] Sharpe JA, Summerhill RJ, Vyas P, Gourdon G, Higgs DR, Wood WG. Role of up‐ stream DNase I hypersensitive sites in the regulation of human alpha globin gene ex‐

[53] Moreau-Gaudry F, Xia P, Jiang G, *et al*. High-level erythroid-specific gene expression in primary human and murine hematopoietic cells with self-inactivating lentiviral

[54] Zhu J, Kren BT, Park CW, Bilgim R, Wong PY, Steer CJ. Erythroid-specific expression of beta-globin by the sleeping beauty transposon for Sickle cell disease. Biochemistry.

[55] Nemeth MJ, Lowrey CH. An Erythroid-Specific Chromatin Opening Element In‐ creases beta-Globin Gene Expression from Integrated Retroviral Gene Transfer Vec‐

of the alpha-globin locus control region. Blood. 1996;87:2518-2524.

2009;17:1919-1928.

Nat Biotechnol. 2006;24:687-696.

ic stem cells. Blood. 1999;93:3276-3285.

Blood Cells Mol Dis. 1998;24:322-339.

pression. Blood. 1993;82:1666-1671.

vectors. Blood. 2001;98:2664-2672.

tors. Gene Ther Mol Biol. 2004;8:475-486.

1995;92:5371-5375.

2007;46:6844-6858.

of HSC gene therapy. J Clin Invest. 2009;119:964-975.

roid expression. J Biol Chem. 2004;279:10523-10531.


[43] Modlich U, Navarro S, Zychlinski D, *et al.* Insertional transformation of hematopoiet‐ ic cells by self-inactivating lentiviral and gammaretroviral vectors. Mol Ther. 2009;17:1919-1928.

[28] Zanella A, Fermo E, Bianchi P, Valentini G. Red cell pyruvate kinase deficiency: mo‐

[29] Beutler E, Gelbart T. Estimating the prevalence of pyruvate kinase deficiency from the gene frequency in the general white population. Blood. 2000;95:3585-3588.

[30] Diez A, Gilsanz F, Martinez J, Perez-Benavente S, Meza NW, Bautista JM. Life-threat‐ ening nonspherocytic hemolytic anemia in a patient with a null mutation in the PKLR gene and no compensatory PKM gene expression. Blood. 2005;106:1851-1856.

[31] Miwa S, Kanno H, Fujii H. Concise review: pyruvate kinase deficiency: historical per‐ spective and recent progress of molecular genetics. Am J Hematol. 1993;42:31-35.

[32] Gilsanz F, Vega MA, Gomez-Castillo E, Ruiz-Balda JA, Omenaca F. Fetal anaemia

[33] Ferreira P, Morais L, Costa R, *et al*. Hydrops fetalis associated with erythrocyte pyru‐

[34] Zanella A, Bianchi P, Iurlo A, *et al.* Iron status and HFE genotype in erythrocyte pyr‐ uvate kinase deficiency: study of Italian cases. Blood Cells Mol Dis. 2001;27:653-661.

[35] Aizawa S, Kohdera U, Hiramoto M, *et al.* Ineffective erythropoiesis in the spleen of a

[36] Aisaki K, Aizawa S, Fujii H, Kanno J, Kanno H. Glycolytic inhibition by mutation of pyruvate kinase gene increases oxidative stress and causes apoptosis of a pyruvate

[37] Min-Oo G, Fortin A, Tam MF, Nantel A, Stevenson MM, Gros P. Pyruvate kinase de‐

[38] Lakomek M, Winkler H. Erythrocyte pyruvate kinase- and glucose phosphate iso‐ merase deficiency: perturbation of glycolysis by structural defects and functional al‐ terations of defective enzymes and its relation to the clinical severity of chronic

[39] Merkle S, Pretsch W. Glucose-6-phosphate isomerase deficiency associated with non‐ spherocytic hemolytic anemia in the mouse: an animal model for the human disease.

[40] Hoyer JD, Allen SL, Beutler E, Kubik K, West C, Fairbanks VF. Erythrocytosis due to bisphosphoglycerate mutase deficiency with concurrent glucose-6-phosphate dehy‐

[41] Njalsson R. Glutathione synthetase deficiency. Cell Mol Life Sci. 2005;62:1938-1945.

[42] Wu X, Li Y, Crise B, Burgess SM. Transcription start regions in the human genome

drogenase (G-6-PD) deficiency. Am J Hematol. 2004;75:205-208.

are favored targets for MLV integration. Science. 2003;300:1749-1751.

patient with pyruvate kinase deficiency. Am J Hematol. 2003;74:68-72.

ficiency in mice protects against malaria. Nat Genet. 2003;35:357-362.

due to pyruvate kinase deficiency. Arch Dis Child. 1993;69:523-524.

vate kinase deficiency. Eur J Pediatr. 2000;159:481-482.

kinase deficient cell line. Exp Hematol. 2007;35:1190-1200.

hemolytic anemia. Biophys Chem. 1997;66:269-284.

Blood. 1993;81:206-213.

lecular and clinical aspects. Br J Haematol. 2005;130:11-25.

532 Gene Therapy - Tools and Potential Applications


[56] May C, Rivella S, Callegari J, *et al.* Therapeutic haemoglobin synthesis in beta-thalas‐ saemic mice expressing lentivirus-encoded human beta-globin. Nature. 2000;406:82-86.

[69] Sadelain M, Rivella S, Lisowski L, Samakoglu S, Riviere I. Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease. Best Pract Res Clin Haema‐

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

535

[70] Ramezani A, Hawley TS, Hawley RG. Lentiviral vectors for enhanced gene expres‐

[71] Kingsman SM, Mitrophanous K, Olsen JC. Potential oncogene activity of the wood‐ chuck hepatitis post-transcriptional regulatory element (WPRE). Gene Ther.

[72] Zanta-Boussif MA, Charrier S, Brice-Ouzet A, *et al.* Validation of a mutated PRE se‐ quence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther.

[73] Johnnidis JB, Harris MH, Wheeler RT, *et al.* Regulation of progenitor cell prolifera‐ tion and granulocyte function by microRNA-223. Nature. 2008;451:1125-1129.

[74] Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryon‐

[75] Takahashi K, Tanabe K, Ohnuki M, *et al.* Induction of pluripotent stem cells from

[76] Yu J, Vodyanik MA, Smuga-Otto K, *et al.* Induced pluripotent stem cell lines derived

[77] Kim JB, Zaehres H, Arauzo-Bravo MJ, Scholer HR. Generation of induced pluripo‐

[78] Hanna J, Markoulaki S, Schorderet P, *et al.* Direct reprogramming of terminally dif‐ ferentiated mature B lymphocytes to pluripotency. Cell. 2008;133:250-264.

[79] Rowland BD, Bernards R, Peeper DS. The KLF4 tumour suppressor is a transcription‐ al repressor of p53 that acts as a context-dependent oncogene. Nat Cell Biol.

[80] Meng X, Neises A, Su RJ, *et al.* Efficient reprogramming of human cord blood CD34+ cells into induced pluripotent stem cells with OCT4 and SOX2 alone. Mol Ther.

[81] Liu T, Zou G, Gao Y, *et al.* High Efficiency of Reprogramming CD34(+) Cells Derived from Human Amniotic Fluid into Induced Pluripotent Stem Cells with Oct4. Stem

[82] Kim D, Kim CH, Moon JI, *et al.* Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 2009;4:472-476.

ic and adult fibroblast cultures by defined factors. Cell. 2006;126:663-676.

adult human fibroblasts by defined factors. Cell. 2007;131:861-872.

tent stem cells from neural stem cells. Nat Protoc. 2009;4:1464-1470.

from human somatic cells. Science. 2007;318:1917-1920.

sion in human hematopoietic cells. Mol Ther. 2000;2:458-469.

tol. 2004;17:517-534.

2005;12:3-4.

2009;16:605-619.

2005;7:1074-1082.

2012;20:408-416.

Cells Dev. 2012.


[69] Sadelain M, Rivella S, Lisowski L, Samakoglu S, Riviere I. Globin gene transfer for treatment of the beta-thalassemias and sickle cell disease. Best Pract Res Clin Haema‐ tol. 2004;17:517-534.

[56] May C, Rivella S, Callegari J, *et al.* Therapeutic haemoglobin synthesis in beta-thalas‐ saemic mice expressing lentivirus-encoded human beta-globin. Nature.

[57] Pawliuk R, Westerman KA, Fabry ME, et al. Correction of sickle cell disease in trans‐

[58] Hanawa H, Yamamoto M, Zhao H, Shimada T, Persons DA. Optimized lentiviral vector design improves titer and transgene expression of vectors containing the

[59] Tan M, Qing K, Zhou S, Yoder MC, Srivastava A. Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted long-term expression of the human be‐ ta-globin gene in hematopoietic cells from homozygous beta-thalassemic mice. Mol

[60] Lisowski L, Sadelain M. Current status of globin gene therapy for the treatment of

[62] Zychlinski D, Schambach A, Modlich U, *et al.* Physiological promoters reduce the

[63] Toscano MG, Romero Z, Munoz P, Cobo M, Benabdellah K, Martin F. Physiological and tissue-specific vectors for treatment of inherited diseases. Gene Ther.

[64] Levings PP, Bungert J. The human beta-globin locus control region. Eur J Biochem.

[65] Navas PA, Peterson KR, Li Q, McArthur M, Stamatoyannopoulos G. The 5'HS4 core element of the human beta-globin locus control region is required for high-level glo‐ bin gene expression in definitive but not in primitive erythropoiesis. J Mol Biol.

[66] Blom van Assendelft G, Hanscombe O, Grosveld F, Greaves DR. The beta-globin dominant control region activates homologous and heterologous promoters in a tis‐

[67] Montiel-Equihua CA, Zhang L, Knight S, *et al.* The beta-Globin Locus Control Region in Combination With the EF1alpha Short Promoter Allows Enhanced Lentiviral Vec‐ tor-mediated Erythroid Gene Expression With Conserved Multilineage Activity. Mol

[68] Sadelain M, Wang CH, Antoniou M, Grosveld F, Mulligan RC. Generation of a hightiter retroviral vector capable of expressing high levels of the human beta-globin

chicken beta-globin locus HS4 insulator element. Mol Ther. 2009;17:667-674.

genic mouse models by gene therapy. Science. 2001;294:2368-2371.

beta-thalassaemia. Br J Haematol. 2008;141:335-345.

[61] Sun FL, Elgin SC. Putting boundaries on silence. Cell. 1999;99:459-462.

genotoxic risk of integrating gene vectors. Mol Ther. 2008;16:718-725.

2000;406:82-86.

534 Gene Therapy - Tools and Potential Applications

Ther. 2001;3:940-946.

2011;18:117-127.

2001;312:17-26.

Ther. 2012;20:1400-1409.

sue-specific manner. Cell. 1989;56:969-977.

gene. Proc Natl Acad Sci U S A. 1995;92:6728-6732.

2002;269:1589-1599.


[83] Papapetrou EP, Lee G, Malani N, *et al.* Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol. 2010;29:73-78.

[96] Delacote F, Lopez BS. Importance of the cell cycle phase for the choice of the appro‐ priate DSB repair pathway, for genome stability maintenance: the trans-S double-

Gene Therapy for Erythroid Metabolic Inherited Diseases

http://dx.doi.org/10.5772/52531

537

[97] Song H, Chung SK, Xu Y. Modeling disease in human ESCs using an efficient BAC-

[98] Khan IF, Hirata RK, Wang PR, *et al.* Engineering of human pluripotent stem cells by

[99] Lombardo A, Genovese P, Beausejour CM, *et al.* Gene editing in human stem cells us‐ ing zinc finger nucleases and integrase-defective lentiviral vector delivery. Nat Bio‐

[100] Gabriel R, Lombardo A, Arens A, *et al.* An unbiased genome-wide analysis of zinc-

[101] Pattanayak V, Ramirez CL, Joung JK, Liu DR. Revealing off-target cleavage specifici‐ ties of zinc-finger nucleases by in vitro selection. Nat Methods. 2011;8:765-770.

[102] Hockemeyer D, Soldner F, Beard C, *et al.* Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol.

[103] Hockemeyer D, Wang H, Kiani S, *et al.* Genetic engineering of human pluripotent

[104] Zavada J. VSV pseudotype particles with the coat of avian myeloblastosis virus. Nat

[105] Cronin J, Zhang XY, Reiser J. Altering the tropism of lentiviral vectors through pseu‐

[106] Waehler R, Russell SJ, Curiel DT. Engineering targeted viral vectors for gene therapy.

[107] Peled A, Petit I, Kollet O, *et al.* Dependence of human stem cell engraftment and re‐

[108] Yang L, Bailey L, Baltimore D, Wang P. Targeting lentiviral vectors to specific cell

[109] Joo KI, Wang P. Visualization of targeted transduction by engineered lentiviral vec‐

[110] Lei Y, Joo KI, Wang P. Engineering fusogenic molecules to achieve targeted transduc‐

[111] Relander T, Johansson M, Olsson K, *et al.* Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV-, or RD114-pseudotyped HIV-1-based vec‐

population of NOD/SCID mice on CXCR4. Science. 1999;283:845-848.

types in vivo. Proc Natl Acad Sci U S A. 2006;103:11479-11484.

tion of enveloped lentiviral vectors. J Biol Eng. 2009;3:8.

tors from stable producer cells. Mol Ther. 2005;11:452-459.

based homologous recombination system. Cell Stem Cell. 2010;6:80-89.

AAV-mediated gene targeting. Mol Ther. 2010;18:1192-1199.

finger nuclease specificity. Nat Biotechnol. 2011;29:816-823.

cells using TALE nucleases. Nat Biotechnol. 2011;29:731-734.

strand break repair model. Cell Cycle. 2008;7:33-38.

technol. 2007;25:1298-1306.

New Biol. 1972;240:122-124.

Nat Rev Genet. 2007;8:573-587.

tors. Gene Ther. 2008;15:1384-1396.

dotyping. Curr Gene Ther. 2005;5:387-398.

2009;27:851-857.


[96] Delacote F, Lopez BS. Importance of the cell cycle phase for the choice of the appro‐ priate DSB repair pathway, for genome stability maintenance: the trans-S doublestrand break repair model. Cell Cycle. 2008;7:33-38.

[83] Papapetrou EP, Lee G, Malani N, *et al.* Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol.

[84] Sommer CA, Sommer AG, Longmire TA, *et al.* Excision of reprogramming trans‐ genes improves the differentiation potential of iPS cells generated with a single excis‐

[85] Mali P, Chou BK, Yen J, *et al.* Butyrate greatly enhances derivation of human induced pluripotent stem cells by promoting epigenetic remodeling and the expression of

[86] Woltjen K, Hamalainen R, Kibschull M, Mileikovsky M, Nagy A. Transgene-free pro‐ duction of pluripotent stem cells using piggyBac transposons. Methods Mol Biol.

[87] Warren L, Manos PD, Ahfeldt T, *et al.* Highly efficient reprogramming to pluripoten‐ cy and directed differentiation of human cells with synthetic modified mRNA. Cell

[88] Fusaki N, Ban H, Nishiyama A, Saeki K, Hasegawa M. Efficient induction of trans‐ gene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys

[89] Park IH, Arora N, Huo H, *et al.* Disease-specific induced pluripotent stem cells. Cell.

[90] Rashid ST, Corbineau S, Hannan N, *et al.* Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest.

[91] Wang Y, Zheng CG, Jiang Y, *et al.* Genetic correction of beta-thalassemia patient-spe‐ cific iPS cells and its use in improving hemoglobin production in irradiated SCID

[92] Papapetrou EP, Lee G, Malani N, *et al.* Genomic safe harbors permit high beta-globin transgene expression in thalassemia induced pluripotent stem cells. Nat Biotechnol.

[93] Sebastiano V, Maeder ML, Angstman JF, *et al.* In situ genetic correction of the sickle cell anemia mutation in human induced pluripotent stem cells using engineered zinc

[94] Zou J, Mali P, Huang X, Dowey SN, Cheng L. Site-specific gene correction of a point mutation in human iPS cells derived from an adult patient with sickle cell disease.

[95] Robbins J. Gene targeting. The precise manipulation of the mammalian genome. Circ

pluripotency-associated genes. Stem Cells. 2010;28:713-720.

2010;29:73-78.

536 Gene Therapy - Tools and Potential Applications

2011;767:87-103.

Stem Cell. 2010;7:618-630.

Biol Sci. 2009;85:348-362.

2008;134:877-886.

2010;120:3127-3136.

2011;29:73-78.

mice. Cell Res. 2012;22:637-648.

Blood. 2011;118:4599-4608.

Res. 1993;73:3-9.

finger nucleases. Stem Cells. 2011;29:1717-1726.

able vector. Stem Cells. 2010;28:64-74.


[112] Di Nunzio F, Piovani B, Cosset FL, Mavilio F, Stornaiuolo A. Transduction of human hematopoietic stem cells by lentiviral vectors pseudotyped with the RD114-TR chi‐ meric envelope glycoprotein. Hum Gene Ther. 2007;18:811-820.

**Chapter 22**

**Targeting the Lung: Challenges in Gene Therapy for**

Cystic Fibrosis (CF) is the most common fatal autosomal recessive genetic disease in the Caucasians with a frequency of approximately 1 in 2500 newborns (Cystic Fibrosis Founda‐ tion, http://www.cff.org/). It affects several organs including the lungs, the liver, the pan‐ creas, the sweat glands and the gastrointestinal and reproductive tracts [1]. The most severe complications that finally lead to death are those in the airway epithelium [2]. Continuous secretion of mucus causes blockage of the lungs by thick sputum and also makes the lungs susceptible to secondary bacterial infections. Subsequent inflammatory responses by the im‐ mune system damage the lungs and the combination of all these factors leads to cardiac fail‐

The primary defect at the biochemical level that is responsible for the symptoms in the lung

cells are the cause of cystic fibrosis. This gene was identified, cloned and named the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) [4, 5]. Though the exact mechanism of pathogenesis is not fully confirmed, the prevailing theory supports that absence or dra‐ matic decrease in the amount of functional CFTR protein at the airways epithelium results in reduced chloride secretion, increased sodium reabsorption and therefore in insufficient airway luminal fluid due to osmosis [6]. These alterations in the respiratory epithelium sub‐ sequently result in deficient mucus clearance which determines chronic cycles of bacterial infections and inflammation [6]. In addition, the formation of thick stationary mucus traps

> © 2013 Kotzamanis et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

) conductance. Specifically, mutations

channel in the apical membrane of epithelial

**Cystic Fibrosis**

http://dx.doi.org/10.5772/52532

**1. Introduction**

ure and to death [3].

George Kotzamanis, Athanassios Kotsinas, Apostolos Papalois and Vassilis G. Gorgoulis

Additional information is available at the end of the chapter

was found to involve cAMP-mediated chloride ion (Cl-

neutrophils that might otherwise clear the infection [7].

in the gene that encodes a cAMP-regulated Cl-


### **Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis**

George Kotzamanis, Athanassios Kotsinas, Apostolos Papalois and Vassilis G. Gorgoulis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52532

#### **1. Introduction**

[112] Di Nunzio F, Piovani B, Cosset FL, Mavilio F, Stornaiuolo A. Transduction of human hematopoietic stem cells by lentiviral vectors pseudotyped with the RD114-TR chi‐

[113] Frecha C, Costa C, Negre D, *et al.* A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure

[114] Lapillonne H, Kobari L, Mazurier C, *et al.* Red blood cell generation from human in‐ duced pluripotent stem cells: perspectives for transfusion medicine. Haematologica.

[115] Lu SJ, Feng Q, Park JS, *et al.* Biologic properties and enucleation of red blood cells

[116] Ma F, Ebihara Y, Umeda K, *et al.* Generation of functional erythrocytes from human embryonic stem cell-derived definitive hematopoiesis. Proc Natl Acad Sci U S A.

[117] Hatzistavrou T, Micallef SJ, Ng ES, Vadolas J, Stanley EG, Elefanty AG. ErythRED, a hESC line enabling identification of erythroid cells. Nat Methods. 2009;6:659-662.

[118] Dias J, Gumenyuk M, Kang H, *et al.* Generation of red blood cells from human in‐

meric envelope glycoprotein. Hum Gene Ther. 2007;18:811-820.

from human embryonic stem cells. Blood. 2008;112:4475-4484.

duced pluripotent stem cells. Stem Cells Dev. 2011;20:1639-1647.

2010;95:1651-1659.

538 Gene Therapy - Tools and Potential Applications

2008;105:13087-13092.

syndrome and in vivo in humanized mice. Blood. 2011;119:1139-1150.

Cystic Fibrosis (CF) is the most common fatal autosomal recessive genetic disease in the Caucasians with a frequency of approximately 1 in 2500 newborns (Cystic Fibrosis Founda‐ tion, http://www.cff.org/). It affects several organs including the lungs, the liver, the pan‐ creas, the sweat glands and the gastrointestinal and reproductive tracts [1]. The most severe complications that finally lead to death are those in the airway epithelium [2]. Continuous secretion of mucus causes blockage of the lungs by thick sputum and also makes the lungs susceptible to secondary bacterial infections. Subsequent inflammatory responses by the im‐ mune system damage the lungs and the combination of all these factors leads to cardiac fail‐ ure and to death [3].

The primary defect at the biochemical level that is responsible for the symptoms in the lung was found to involve cAMP-mediated chloride ion (Cl- ) conductance. Specifically, mutations in the gene that encodes a cAMP-regulated Cl channel in the apical membrane of epithelial cells are the cause of cystic fibrosis. This gene was identified, cloned and named the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) [4, 5]. Though the exact mechanism of pathogenesis is not fully confirmed, the prevailing theory supports that absence or dra‐ matic decrease in the amount of functional CFTR protein at the airways epithelium results in reduced chloride secretion, increased sodium reabsorption and therefore in insufficient airway luminal fluid due to osmosis [6]. These alterations in the respiratory epithelium sub‐ sequently result in deficient mucus clearance which determines chronic cycles of bacterial infections and inflammation [6]. In addition, the formation of thick stationary mucus traps neutrophils that might otherwise clear the infection [7].

© 2013 Kotzamanis et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. For several reasons including the easy access to the respiratory tract without any interven‐ tion procedures, the cloning and the characterization of the *CFTR* gene and the expectation that even relatively low levels of expression of the gene may have a therapeutic outcome [8], Cystic Fibrosis became an ideal target for gene therapy and an example for gene therapy of other lung diseases. Indeed, the first gene therapy clinical trials for CF started in 1993 and 29 clinical trials have been conducted since then (http://www.wiley.com/legacy/wileychi/ genmed/clinical/). Several trials have demonstrated gene transfer and transgene expression. In some cases, low levels of transient correction of Cl ion transport deficiency has been ob‐ served but overall, no clinical improvement has been achieved. The histological, immuno‐ logical and intracellular barriers that exist in the lung have proven to be more difficult to overcome than what was initially thought. The purpose of this chapter is to analyze these barriers and to present the challenges the gene therapist is faced with when targeting the lung for the treatment of CF.

tiveness of this action depends on the viscosity of the mucus which is determined by the lev‐ el of its hydration [10]. Normal airway mucus consists of 97% water [11] but when luminal fluid is reduced, as in CF, the clearance of mucus by cilia and cough is also reduced. Under the basal lamina lies the lamina propria, which consists of elastic fibers and hosts the sub‐ mucosal glands that together with the Goblet cells produce components of the mucus [9]

Several stem cells populations responsible for the maintenance of the respiratory epithelium have been identified in the lung. Specifically, a population of basal cells, residing at close proximity to the underlying basal lamina in the larger airways, has been shown to have a high multipotency potential allowing regulation of the epithelium homeostasis under nor‐ mal circumstances or after injury [12]. Another type of cells with stem-cell-like properties is the Clara cells. These nonciliated cells are located at the terminal bronchioles and produce a solution similar to the surfactant in the alveoli. Interestingly, Clara cells can multiply and

The respiratory portion of each lung consists of approximately 300 million alveoli. Each al‐ veolus has a thin wall consisting mainly of type I and type II alveolar cells (Figure 2D). Type II alveolar cells are responsible for the secretion of a thin layer of fluid that normally coats the alveolar surface in order to decrease the surface tension at the air-fluid interface, the sur‐ factant. Surfactant turnover is mediated by the phagocytic function of alveolar macrophag‐ es, which are also located in the alveolar wall and are frequently seen in the alveolar lumen.

> **Respiratory epithelium**

**Cilia** 

**Pseudostratified columnar epithelium** 

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

541

**cell** 

**Type II cell Type I cell** 

**Macrophage Capillary** 

**Alveolus** 

**Ciliated epithelial** 

**Goblet cell** 

**x400** 

**x400** 

**Lumen** 

**Lamina propria: Basal lamina** 

**Basallamina** 

**Basalcell** 

**Elastic fibers Blood vessel** 

**Submucosal glands** 

**Basal lamina** 

**Figure 2.** A) Upper respiratory tract section, B) Epithelium of upper respiratory tract, C) Mucus produced in Goblet cells

**Mucus** 

**Lumen** 

differentiate into ciliated cells to regenerate the bronchiolar epithelium [13].

A B

C D

and secreted in the lumen, D) Alveolar section.

**x100** 

**x400** 

(Figure 2A,B,C).

First, the basic histology of the lung will be described so that the reader can identify the po‐ tential target cells for CF gene therapy and realize the complexity of the lung structures that the gene transfer agent needs to penetrate in order to reach these target cells.

#### **2. Basic histology of the lung**

The lung is a complex organ that is divided into the air-conducting portion consisting of the trachea, the bronchi and the bronchioles and the respiratory portion consisting of the alveoli, which is the place of gas exchange (Figure 1).

**Figure 1.** Schematic illustration of the lung.

The trachea and most of the bronchi are covered by pseudostratified columnar ciliated epi‐ thelium (known as the respiratory epithelium). Columnar ciliated cells are the predominant population extending from the basal lamina to the airway lumen. Other cells facing the lu‐ men are the nonciliated Goblet cells which produce mucin polymers [9], forming a thin layer of mucus that covers the airway epithelium. The role of the airway mucus is to trap inhaled particles which are then transferred out of the lung by cilia beating and/or cough. The effec‐ tiveness of this action depends on the viscosity of the mucus which is determined by the lev‐ el of its hydration [10]. Normal airway mucus consists of 97% water [11] but when luminal fluid is reduced, as in CF, the clearance of mucus by cilia and cough is also reduced. Under the basal lamina lies the lamina propria, which consists of elastic fibers and hosts the sub‐ mucosal glands that together with the Goblet cells produce components of the mucus [9] (Figure 2A,B,C).

For several reasons including the easy access to the respiratory tract without any interven‐ tion procedures, the cloning and the characterization of the *CFTR* gene and the expectation that even relatively low levels of expression of the gene may have a therapeutic outcome [8], Cystic Fibrosis became an ideal target for gene therapy and an example for gene therapy of other lung diseases. Indeed, the first gene therapy clinical trials for CF started in 1993 and 29 clinical trials have been conducted since then (http://www.wiley.com/legacy/wileychi/ genmed/clinical/). Several trials have demonstrated gene transfer and transgene expression.

served but overall, no clinical improvement has been achieved. The histological, immuno‐ logical and intracellular barriers that exist in the lung have proven to be more difficult to overcome than what was initially thought. The purpose of this chapter is to analyze these barriers and to present the challenges the gene therapist is faced with when targeting the

First, the basic histology of the lung will be described so that the reader can identify the po‐ tential target cells for CF gene therapy and realize the complexity of the lung structures that

The lung is a complex organ that is divided into the air-conducting portion consisting of the trachea, the bronchi and the bronchioles and the respiratory portion consisting of the alveoli,

The trachea and most of the bronchi are covered by pseudostratified columnar ciliated epi‐ thelium (known as the respiratory epithelium). Columnar ciliated cells are the predominant population extending from the basal lamina to the airway lumen. Other cells facing the lu‐ men are the nonciliated Goblet cells which produce mucin polymers [9], forming a thin layer of mucus that covers the airway epithelium. The role of the airway mucus is to trap inhaled particles which are then transferred out of the lung by cilia beating and/or cough. The effec‐

the gene transfer agent needs to penetrate in order to reach these target cells.

ion transport deficiency has been ob‐

In some cases, low levels of transient correction of Cl-

lung for the treatment of CF.

540 Gene Therapy - Tools and Potential Applications

**2. Basic histology of the lung**

**Figure 1.** Schematic illustration of the lung.

which is the place of gas exchange (Figure 1).

Several stem cells populations responsible for the maintenance of the respiratory epithelium have been identified in the lung. Specifically, a population of basal cells, residing at close proximity to the underlying basal lamina in the larger airways, has been shown to have a high multipotency potential allowing regulation of the epithelium homeostasis under nor‐ mal circumstances or after injury [12]. Another type of cells with stem-cell-like properties is the Clara cells. These nonciliated cells are located at the terminal bronchioles and produce a solution similar to the surfactant in the alveoli. Interestingly, Clara cells can multiply and differentiate into ciliated cells to regenerate the bronchiolar epithelium [13].

The respiratory portion of each lung consists of approximately 300 million alveoli. Each al‐ veolus has a thin wall consisting mainly of type I and type II alveolar cells (Figure 2D). Type II alveolar cells are responsible for the secretion of a thin layer of fluid that normally coats the alveolar surface in order to decrease the surface tension at the air-fluid interface, the sur‐ factant. Surfactant turnover is mediated by the phagocytic function of alveolar macrophag‐ es, which are also located in the alveolar wall and are frequently seen in the alveolar lumen.

**Figure 2.** A) Upper respiratory tract section, B) Epithelium of upper respiratory tract, C) Mucus produced in Goblet cells and secreted in the lumen, D) Alveolar section.

It is not yet clear which of these cells are the target for gene therapy of Cystic Fibrosis. In healthy individuals there is little CFTR expression in the lung, except the submucosal glands and epithelial cells at the small airways where higher expression is observed [14]. Early studies suggested that the submucosal glands are the site of maximal CFTR expres‐ sion [15], but more recent data suggested that this may be the ciliated surface epithelium of the bronchioles [16]. This uncertainty is one of the main obstacles for successful CF gene therapy. Depending on which are the cells that need to be corrected, different ana‐ tomical or immunological barriers apply and therefore different administration methods have to be used.

These mechanisms have evolved to secure the host cell's genetic integrity but in gene thera‐ py they constitute one more hurdle to overcome. Indeed, once the therapeutic DNA enters the target cells in the lung of CF patients, it faces a series of intra-cellular barriers that apply to gene therapy in general and not just to gene therapy for CF. These barriers include degra‐ dation by cytosolic nucleases [29, 30] and degradation inside digestive lysozomes formed by transformation of endosomes following endocytosis [31]. Several methods have been imple‐ mented so that the transgene can escape the endosomes after internalization. Cationic lipids and polycationic polymers like polyethylenimine (PEI) [32] utilised as chemical vectors in complexes with the transfected DNA, protect it from nucleases and enable it to escape the endosomes. Such complexes carrying the *CFTR* gene have been used to correct the ion trans‐ port defect in CF transgenic mice [33] and are currently being tested in clinical practice [34]. Other strategies to protect the therapeutic DNA from the endosomes and therefore to in‐ crease the transfection efficiency include the use of pharmacological endosomelytic agents such as chloroquine [35], endosome-disrupting peptides [36-38] and glycerol [39]. All these aim at destabilizing the endosomal membrane so that the contents are released intact to the

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

543

cytosol but may be of limited clinical value due to safety concerns in the host cells.

**4. Immunological barriers to gene transfer to the lung**

product of the transgene and therefore limit the overall efficacy [46].

treatment of chronic diseases such as CF [46].

for gene therapy for CF [45].

The final physical barrier before the *CFTR* transgene enters the nucleus of the non-dividing airway epithelial cells and undergoes transcription is the nuclear envelope [40]. Many viral vectors can efficiently deliver their cargo in the nucleus by exploiting the nuclear transport systems of the host [41], but non-viral vectors are in most cases ineffective in front of the nu‐ clear envelope. Strategies such as the use of chemical vectors based on PEI [42] and of Nu‐ clear Localization Signals (NLS) which are integrated into the transfected DNA and bind to transporter proteins in order to facilitate nuclear entry [43, 44] have been implemented and found to promote nuclear delivery *in vitro*. However, these have not been validated with large therapeutic genes like the *CFTR* and in non-dividing cells *in vivo* and may not be of use

Apart from the extracellular and intracellular barriers described above, there is a second line of defence consisting of specific and non-specific immune responses that protect the lung cells against foreign particles which are present in the air. During gene therapy for CF, these immunological mechanisms can be activated by the vector carrying the transgene or the

Various immunological responses are directed against the carrier of the therapeutic gene be‐ fore this enters the target cells. Pulmonary macrophages have been shown to ingest adeno‐ viral vectors, but when they were removed before transfection, an increase in transgene expression was observed [47]. Furthermore, humoral immune responses mediated by helper T lymphocytes result in the production of neutralizing antibodies against the vector, which restricts the possibility of re-administration and so, the use of most viral vectors for the

#### **3. Physical barriers to gene transfer to the lung**

As most formulations administered to treat CF, including gene transfer agents, are deliv‐ ered directly to the airways in the form of aerosols [17], the first physical barrier that needs to be overcome before the transgene reaches the target cells is the airways mucus. Trapping in the mucus and clearance by cilia is the main factor reducing transfection ef‐ ficiency in lung cells of all individuals. In Cystic Fibrosis, the lungs are progressively fil‐ led with large amounts of purulent secretions, the sputum, which consists of mucus, DNA, actin, cell debris and inflammatory cells [18]. The most commonly used in gene therapy for CF viral vectors and non-viral liposomal vectors have been proven unable to penetrate the CF sputum [19, 20], suggesting that effective treatment may be achieved only at early stages of the disease before the lungs are filled with sputum [21]. Several improvements can be made at this stage to increase transfection efficiency, such as use of methylcellulose gel formulations to inhibit mucociliary clearance [22] or of mucolytics [23, 24], but these need to be validated in clinical trials.

The second physical barrier to gene transfer to the airway epithelium is the composition of the apical surface of the cells. First, the presence of specific receptors determines the kind of viral vector to be used. For example, adenoviral vectors have low transfection efficiency due to the low abundance of the required receptors on the apical side of most human airway epi‐ thelial cells [25] and this is one of the reasons why they are not considered in clinical trials any more [21]. Making the basolateral membrane, which is more abundant in adenoviral re‐ ceptors [25], accessible to the adenoviral vector has been proposed as an attractive alterna‐ tive. Indeed, the use of agents such as sodium caprate that can cause transient dissociation of tight junctions, impressively increases transgene delivery and expression in animal mod‐ els [26, 27] but may not have a clinical application due to the risk of systemic bacterial inva‐ sion. Second, the glycocalyx on the apical membrane seems to interfere with the interaction between adenovirus and its few receptors [28]. Removal of sialic acid residues from the gly‐ cocalyx by pretreatment with neuraminidase may be an effective way to overcome this physical barrier [28].

Although non-viral vectors are not affected by the problems described above, they are sub‐ ject to possible destruction by the cell defence mechanisms against foreign DNA invasion. These mechanisms have evolved to secure the host cell's genetic integrity but in gene thera‐ py they constitute one more hurdle to overcome. Indeed, once the therapeutic DNA enters the target cells in the lung of CF patients, it faces a series of intra-cellular barriers that apply to gene therapy in general and not just to gene therapy for CF. These barriers include degra‐ dation by cytosolic nucleases [29, 30] and degradation inside digestive lysozomes formed by transformation of endosomes following endocytosis [31]. Several methods have been imple‐ mented so that the transgene can escape the endosomes after internalization. Cationic lipids and polycationic polymers like polyethylenimine (PEI) [32] utilised as chemical vectors in complexes with the transfected DNA, protect it from nucleases and enable it to escape the endosomes. Such complexes carrying the *CFTR* gene have been used to correct the ion trans‐ port defect in CF transgenic mice [33] and are currently being tested in clinical practice [34]. Other strategies to protect the therapeutic DNA from the endosomes and therefore to in‐ crease the transfection efficiency include the use of pharmacological endosomelytic agents such as chloroquine [35], endosome-disrupting peptides [36-38] and glycerol [39]. All these aim at destabilizing the endosomal membrane so that the contents are released intact to the cytosol but may be of limited clinical value due to safety concerns in the host cells.

It is not yet clear which of these cells are the target for gene therapy of Cystic Fibrosis. In healthy individuals there is little CFTR expression in the lung, except the submucosal glands and epithelial cells at the small airways where higher expression is observed [14]. Early studies suggested that the submucosal glands are the site of maximal CFTR expres‐ sion [15], but more recent data suggested that this may be the ciliated surface epithelium of the bronchioles [16]. This uncertainty is one of the main obstacles for successful CF gene therapy. Depending on which are the cells that need to be corrected, different ana‐ tomical or immunological barriers apply and therefore different administration methods

As most formulations administered to treat CF, including gene transfer agents, are deliv‐ ered directly to the airways in the form of aerosols [17], the first physical barrier that needs to be overcome before the transgene reaches the target cells is the airways mucus. Trapping in the mucus and clearance by cilia is the main factor reducing transfection ef‐ ficiency in lung cells of all individuals. In Cystic Fibrosis, the lungs are progressively fil‐ led with large amounts of purulent secretions, the sputum, which consists of mucus, DNA, actin, cell debris and inflammatory cells [18]. The most commonly used in gene therapy for CF viral vectors and non-viral liposomal vectors have been proven unable to penetrate the CF sputum [19, 20], suggesting that effective treatment may be achieved only at early stages of the disease before the lungs are filled with sputum [21]. Several improvements can be made at this stage to increase transfection efficiency, such as use of methylcellulose gel formulations to inhibit mucociliary clearance [22] or of mucolytics

The second physical barrier to gene transfer to the airway epithelium is the composition of the apical surface of the cells. First, the presence of specific receptors determines the kind of viral vector to be used. For example, adenoviral vectors have low transfection efficiency due to the low abundance of the required receptors on the apical side of most human airway epi‐ thelial cells [25] and this is one of the reasons why they are not considered in clinical trials any more [21]. Making the basolateral membrane, which is more abundant in adenoviral re‐ ceptors [25], accessible to the adenoviral vector has been proposed as an attractive alterna‐ tive. Indeed, the use of agents such as sodium caprate that can cause transient dissociation of tight junctions, impressively increases transgene delivery and expression in animal mod‐ els [26, 27] but may not have a clinical application due to the risk of systemic bacterial inva‐ sion. Second, the glycocalyx on the apical membrane seems to interfere with the interaction between adenovirus and its few receptors [28]. Removal of sialic acid residues from the gly‐ cocalyx by pretreatment with neuraminidase may be an effective way to overcome this

Although non-viral vectors are not affected by the problems described above, they are sub‐ ject to possible destruction by the cell defence mechanisms against foreign DNA invasion.

have to be used.

542 Gene Therapy - Tools and Potential Applications

physical barrier [28].

**3. Physical barriers to gene transfer to the lung**

[23, 24], but these need to be validated in clinical trials.

The final physical barrier before the *CFTR* transgene enters the nucleus of the non-dividing airway epithelial cells and undergoes transcription is the nuclear envelope [40]. Many viral vectors can efficiently deliver their cargo in the nucleus by exploiting the nuclear transport systems of the host [41], but non-viral vectors are in most cases ineffective in front of the nu‐ clear envelope. Strategies such as the use of chemical vectors based on PEI [42] and of Nu‐ clear Localization Signals (NLS) which are integrated into the transfected DNA and bind to transporter proteins in order to facilitate nuclear entry [43, 44] have been implemented and found to promote nuclear delivery *in vitro*. However, these have not been validated with large therapeutic genes like the *CFTR* and in non-dividing cells *in vivo* and may not be of use for gene therapy for CF [45].

#### **4. Immunological barriers to gene transfer to the lung**

Apart from the extracellular and intracellular barriers described above, there is a second line of defence consisting of specific and non-specific immune responses that protect the lung cells against foreign particles which are present in the air. During gene therapy for CF, these immunological mechanisms can be activated by the vector carrying the transgene or the product of the transgene and therefore limit the overall efficacy [46].

Various immunological responses are directed against the carrier of the therapeutic gene be‐ fore this enters the target cells. Pulmonary macrophages have been shown to ingest adeno‐ viral vectors, but when they were removed before transfection, an increase in transgene expression was observed [47]. Furthermore, humoral immune responses mediated by helper T lymphocytes result in the production of neutralizing antibodies against the vector, which restricts the possibility of re-administration and so, the use of most viral vectors for the treatment of chronic diseases such as CF [46].

Other responses are initiated after the transgene is delivered to the lung cells. Particularly when viral vectors are used, cellular immune responses mediated by cytotoxic T lympho‐ cytes eliminate transduced cells expressing viral proteins resulting in parallel loss of trans‐ gene expression [46]. Although in theory non-viral vectors are not associated to such problems as they are less immunogenic than viral vectors, in practice they are usually used in combination with ligands so that they overcome the physical barriers described above and this can provoke immunological reactions similar to those caused by viral vectors [46]. In addition, viral and non-viral vectors can provoke the release of host cytokines which have been shown to inhibit expression of the gene delivered if this is driven by a common viral promoter [48].

serious immune response to the vector that eventually led to his death 98 hours after admin‐ istration [62]. This death was a setback for all gene therapy clinical studies using viral vec‐ tors, as human immune responses cannot be predicted pre-clinically. Apart from the immunological responses caused by the vector, other unfortunate events of different nature have also been found to be associated with reduced safety. Treatment of patients with Xlinked severe combined immune deficiency (SCIDX1) using a retroviral vector carrying the γc gene resulted in the correction of the disease and huge enthusiasm about the future of gene therapy [63]. However, two of the cured patients developed a leukemia-like condition 2-3 years later due to disruption of an endogenous oncogene by integration of the vector [64, 65]. Since vector integration is usually random and uncontrollable, insertional mutagenesis is a general problem that all integrating vectors have. As these problems also apply to most vectors used in gene therapy for Cystic Fibrosis, avoiding unwanted immune response and insertional mutagenesis are two major challenges for the genetic treatment of CF. Strategies to respond to the second challenge of insertional mutagenesis will be discussed in the next

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

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545

section. To address the first problem, the solution is to use less immunogenic vectors.

their good safety profile albeit with low transduction efficiency [70-74].

Although adenoviral vectors administered systemically can cause acute and potentially lifethreatening cytokine response [66] their local administration at mild doses to nose and lung tissues did not result in such unacceptable safety profile [34]. However, extensive use of these vectors during the early times of CF gene therapy has shown that cellular and humoral immune responses against the virus are generated and these limit repeated administration [67, 68]. Another approach to avoid immunologic reactions from the host is to coat the virus capsid with polyethylene glycol (PEG). Such PEGylated viruses, called "stealth viruses", are not recognised by the immune system and can significantly prolong transgene expression [69]. A less immunogenic alternative to adenoviral constructs is the use of adeno-associated virus (AAV) vectors. Indeed, several human CF trials with AAV vectors have confirmed

Despite significant progress made towards the generation of safer viral vectors [75], non-vi‐ ral synthetic vectors containing only human DNA sequences are the vectors of choice when safety is considered as first priority. These vectors generally consist of the therapeutic DNA either naked or mixed with chemical compounds, like cationic lipids or cationic polymers [76]. Naked DNA is in theory the safest gene therapy agent but very difficult to be intro‐ duced into the target cells. Several physical methods have been developed to facilitate DNA entry into the lung of living animals, such as the use of electrical pulses (electroporation) [77], of ultrasound waves (sonoporation) [78] and of magnetic fields (magnetofection) [79] but none of them has reached the clinical use yet. On the other hand, chemical carriers have rapidly been developed and used in 6% (n=110) of gene therapy clinical trials (http:// www.wiley.com/legacy/wileychi/genmed/clinical). These act by forming complexes with the negatively charged DNA. The complexes condense the DNA, protect it from nucleases, al‐ low its entry into the cells and protect it from the endosomes [80]. Indeed, local administra‐ tion of cationic lipid/CFTR-plasmid-DNA complexes in an aerosol formulation to the lungs of cystic fibrosis transgenic mice resulted in correction of the ion transport defect [33]. Simi‐

Several approaches have been developed to overcome the immunological barriers in the lung. The use of immunosuppressant drugs such as cyclophosphamide have been proved very effective in mice allowing both prolonged transgene expression and repeated adminis‐ tration of an adenoviral vector [49]. Similar results were obtained with corticoid steroids such as dexamethasone [50] and budesonide [51] which were found to decrease inflamma‐ tion mediated by viral vectors. Other strategies include the co-administration of IL-12 [52] and blockade of CD4+ T cells [53-56]. However, all these approaches are likely to cause more damage than benefit, considering that the lungs of CF patients are colonized by pathogenic bacteria, and so they are not applicable in the clinical setting.

On the other hand, non-viral chemical vectors based on cationic lipids can be re-adminis‐ tered without the need to be combined with immunosuppressants [57]. From that aspect, these are safer than viral vectors for gene therapy of CF but still not absolutely harmless as they have been associated with lung toxicity due to provocation of inflammation [58]. An‐ other mediator of inflammation in the lung can be the CpG motifs on the bacterial plasmid DNA which is usually used to clone the therapeutic gene in non-viral gene therapy [59, 60]. Unlike eukaryotic DNA, this dinucleotide is relatively unmethylated in bacteria and can be inflammatory through recognition by toll-like receptor 9 on B cells [61]. As methylation of the CpG motifs prior to gene delivery may decrease the expression of the transgene, the ex‐ clusion of any bacteria-derived DNA from the therapeutic construct is a more promising al‐ ternative.

#### **5. Safety concerns**

Immunological responses elicited by a gene therapy vector do not only pose a barrier to effi‐ cient delivery and expression of the therapeutic gene in the target cells but more important‐ ly, they can raise very serious safety issues. This lesson has been learned from a gene therapy human trial where lethal complications were experienced [62]. In that study, an ade‐ noviral vector containing the cDNA of the gene encoding ornithine transcarbamylase (OTC) was administered to 18 patients with partial OTC deficiency, a disease caused by a defect in urea synthesis. The adenoviral vector provoked immunologic and other side effects, such as fever, myalgia and nausea in 17 out of the 18 participants but the 18th patient developed a serious immune response to the vector that eventually led to his death 98 hours after admin‐ istration [62]. This death was a setback for all gene therapy clinical studies using viral vec‐ tors, as human immune responses cannot be predicted pre-clinically. Apart from the immunological responses caused by the vector, other unfortunate events of different nature have also been found to be associated with reduced safety. Treatment of patients with Xlinked severe combined immune deficiency (SCIDX1) using a retroviral vector carrying the γc gene resulted in the correction of the disease and huge enthusiasm about the future of gene therapy [63]. However, two of the cured patients developed a leukemia-like condition 2-3 years later due to disruption of an endogenous oncogene by integration of the vector [64, 65]. Since vector integration is usually random and uncontrollable, insertional mutagenesis is a general problem that all integrating vectors have. As these problems also apply to most vectors used in gene therapy for Cystic Fibrosis, avoiding unwanted immune response and insertional mutagenesis are two major challenges for the genetic treatment of CF. Strategies to respond to the second challenge of insertional mutagenesis will be discussed in the next section. To address the first problem, the solution is to use less immunogenic vectors.

Other responses are initiated after the transgene is delivered to the lung cells. Particularly when viral vectors are used, cellular immune responses mediated by cytotoxic T lympho‐ cytes eliminate transduced cells expressing viral proteins resulting in parallel loss of trans‐ gene expression [46]. Although in theory non-viral vectors are not associated to such problems as they are less immunogenic than viral vectors, in practice they are usually used in combination with ligands so that they overcome the physical barriers described above and this can provoke immunological reactions similar to those caused by viral vectors [46]. In addition, viral and non-viral vectors can provoke the release of host cytokines which have been shown to inhibit expression of the gene delivered if this is driven by a common viral

Several approaches have been developed to overcome the immunological barriers in the lung. The use of immunosuppressant drugs such as cyclophosphamide have been proved very effective in mice allowing both prolonged transgene expression and repeated adminis‐ tration of an adenoviral vector [49]. Similar results were obtained with corticoid steroids such as dexamethasone [50] and budesonide [51] which were found to decrease inflamma‐ tion mediated by viral vectors. Other strategies include the co-administration of IL-12 [52] and blockade of CD4+ T cells [53-56]. However, all these approaches are likely to cause more damage than benefit, considering that the lungs of CF patients are colonized by pathogenic

On the other hand, non-viral chemical vectors based on cationic lipids can be re-adminis‐ tered without the need to be combined with immunosuppressants [57]. From that aspect, these are safer than viral vectors for gene therapy of CF but still not absolutely harmless as they have been associated with lung toxicity due to provocation of inflammation [58]. An‐ other mediator of inflammation in the lung can be the CpG motifs on the bacterial plasmid DNA which is usually used to clone the therapeutic gene in non-viral gene therapy [59, 60]. Unlike eukaryotic DNA, this dinucleotide is relatively unmethylated in bacteria and can be inflammatory through recognition by toll-like receptor 9 on B cells [61]. As methylation of the CpG motifs prior to gene delivery may decrease the expression of the transgene, the ex‐ clusion of any bacteria-derived DNA from the therapeutic construct is a more promising al‐

Immunological responses elicited by a gene therapy vector do not only pose a barrier to effi‐ cient delivery and expression of the therapeutic gene in the target cells but more important‐ ly, they can raise very serious safety issues. This lesson has been learned from a gene therapy human trial where lethal complications were experienced [62]. In that study, an ade‐ noviral vector containing the cDNA of the gene encoding ornithine transcarbamylase (OTC) was administered to 18 patients with partial OTC deficiency, a disease caused by a defect in urea synthesis. The adenoviral vector provoked immunologic and other side effects, such as fever, myalgia and nausea in 17 out of the 18 participants but the 18th patient developed a

bacteria, and so they are not applicable in the clinical setting.

promoter [48].

544 Gene Therapy - Tools and Potential Applications

ternative.

**5. Safety concerns**

Although adenoviral vectors administered systemically can cause acute and potentially lifethreatening cytokine response [66] their local administration at mild doses to nose and lung tissues did not result in such unacceptable safety profile [34]. However, extensive use of these vectors during the early times of CF gene therapy has shown that cellular and humoral immune responses against the virus are generated and these limit repeated administration [67, 68]. Another approach to avoid immunologic reactions from the host is to coat the virus capsid with polyethylene glycol (PEG). Such PEGylated viruses, called "stealth viruses", are not recognised by the immune system and can significantly prolong transgene expression [69]. A less immunogenic alternative to adenoviral constructs is the use of adeno-associated virus (AAV) vectors. Indeed, several human CF trials with AAV vectors have confirmed their good safety profile albeit with low transduction efficiency [70-74].

Despite significant progress made towards the generation of safer viral vectors [75], non-vi‐ ral synthetic vectors containing only human DNA sequences are the vectors of choice when safety is considered as first priority. These vectors generally consist of the therapeutic DNA either naked or mixed with chemical compounds, like cationic lipids or cationic polymers [76]. Naked DNA is in theory the safest gene therapy agent but very difficult to be intro‐ duced into the target cells. Several physical methods have been developed to facilitate DNA entry into the lung of living animals, such as the use of electrical pulses (electroporation) [77], of ultrasound waves (sonoporation) [78] and of magnetic fields (magnetofection) [79] but none of them has reached the clinical use yet. On the other hand, chemical carriers have rapidly been developed and used in 6% (n=110) of gene therapy clinical trials (http:// www.wiley.com/legacy/wileychi/genmed/clinical). These act by forming complexes with the negatively charged DNA. The complexes condense the DNA, protect it from nucleases, al‐ low its entry into the cells and protect it from the endosomes [80]. Indeed, local administra‐ tion of cationic lipid/CFTR-plasmid-DNA complexes in an aerosol formulation to the lungs of cystic fibrosis transgenic mice resulted in correction of the ion transport defect [33]. Simi‐ lar studies in human patients demonstrated some transgene expression, but not at sufficient levels to provide a clinical benefit [57, 81-86].

and also *in vivo* in genetically modified pigs [95] making them very attractive for use in CF

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547

An alternative to the use of episomal vectors described above, that still satisfies both re‐ quirements for permanent transgene expression and elimination of genotoxic effects is the controlled integration of the therapeutic DNA at a specific site in the host genome where no active genes are present. Several vector systems have been developed to achieve this, with each one of them having its own limitations [96]. From these, vectors based on the ΦC31 in‐ tegrase [97] and on transposase enzymes [98] are the most promising for use in CF gene therapy as they have a preference for specific sequences that already exist in the human ge‐

An additional problem to achieving long-term expression of the *CFT*R transgene delivered to the lung is the life span of the target cells. Depending on the rhythm of natural turnover of these cells, transgene expression can last for as long as these cells are alive. A more effec‐ tive approach would be to either target putative stem cells with the capacity to differentiate to airway epithelial cells [34] or to deliver exogenous heterologous or corrected autologous

Airway basal stem cells are a candidate target for CF stem cell therapy. However, the facts that this population is estimated to represent only a minor part of the total airway epitheli‐ um [12] and that it is quite inaccessible as it is not exposed to the airway lumen make such a

*Ex vivo* gene therapy using stem cells may not only provide permanent cure without the need for re-administration, but also solve the hurdle of the low *in vivo* gene delivery efficien‐ cy. In *ex vivo* cell therapy using Embryonic Stem Cells or foetal Mesenchymal Stem Cells from a healthy embryo there is no need for transfection of a therapeutic gene. However, these stem cells are used in an allogeneic fashion which requires the parallel use of immuno‐ suppressive drugs and are therefore not applicable for the treatment of CF. A more attrac‐ tive strategy would be the transfer of the *CFTR* gene to patient-derived autologous stem cells such as Mesenchymal Stem Cells (MSCs), which can easily be isolated from the bone marrow or adipose tissue of adults [100] or Induced Pluripotent Stem Cells that can be gen‐ erated by reprogramming of adult somatic cells [101, 102]. In that case, the transfection pro‐ cedure would be performed *in vitro* before reimplantation of the cells back to the donor, which is far more efficient than *in vivo* delivery. Furthermore, bone marrow-derived MSCs have been shown to be able to express transgenes [103] and to differentiate to several types of cells including airway epithelial cells [104]. Nevertheless, there is little literature on how the *ex vivo* corrected MSCs can be administered and engrafted in the lung of Cystic Fibrosis patients. Both systemic and topical lung administration of bone marrow-derived cells have been applied and shown to result in some engraftment into the airways [105], but there are several challenges to be addressed, before *ex vivo* cell therapy becomes part of the CF clinical research. These challenges include the very low efficiency of engraftment (<1%) and the fact that previous damage to the surface epithelium caused by epithelia-injuring reagents seems

clinical trials.

nome and have been shown to work *in vivo*.

stem cells to the lungs of CF patients *ex vivo* [99].

therapy approach very challenging.

to be required for the engraftment [75].

#### **6. Duration of transgene expression**

Provided that all obstacles to gene delivery to the lung are overcome and the *CFTR* trans‐ gene finally reaches the target cells, a clinical benefit for CF can only be achieved by lifelong expression of the gene. As repeated administration is in most cases restricted by immune re‐ sponses generated by the patient against the vector, other strategies have been employed for efficient retention and long-term expression.

Integration into the host genome has widely been used in gene therapy to fulfil this require‐ ment. However, the dangers of integration due to insertional mutagenesis have become a widely publicised issue as a result of the SCIDX1 clinical trial, where some patients devel‐ oped leukaemia due to deregulation of the growth-promoting LIM domain only 2 (LMO2) proto-oncogene caused by integration of the vector [64, 65]. The safety concerns regarding uncontrolled integration of the therapeutic gene into the host genome have been strength‐ ened by observations that there is a preference of integrating vectors for the regulatory re‐ gions of transcriptionally active genes [87]. Given the need for long-term expression and the problems associated with vector integration, vectors that persist in the nucleus by being maintained episomally without integrating, could be highly advantageous. Among the sys‐ tems developed to achieve extra-chromosomal maintenance of the vectors carrying the ther‐ apeutic gene, two are considered safe enough for clinical application in the future: artificial chromosomes and systems based on scaffold/matrix attachment region (S/MAR).

Human Artificial Chromosomes (HACs) are vectors able to replicate and segregate in paral‐ lel with the endogenous chromosomes in human cells. To achieve this, they must contain the minimal elements required for chromosome function, namely an origin of replication, te‐ lomeres and centromeres [88]. HACs can be generated by a method similar to the one ap‐ plied for YAC construction in yeast and involves assembling the functional chromosomal elements and building up a HAC *de novo* in human cells. Different strategies have been fol‐ lowed to generate *de novo* HACs, the most convenient of which is to transfect a BAC carry‐ ing only a large array of α-satellite (alphoid) DNA and some marker genes into HT1080 cells[89]. HACs generated this way exist as single (or low copy) chromosomes in the nucleus and have a high mitotic stability (close to 100%) in the absence of selection. The potential use of these vectors in gene therapy has been demonstrated by expression of large therapeutic genes from them [90, 91].

S/MARs are diverse sequences found in all eukaryotic genomes where they are involved in many aspects of chromatin function such as organization of chromatin into loops, which seems to be mediated by the interaction between S/MARs and the nuclear matrix [92]. Vec‐ tors containing an S/MAR element have the ability to remain episomally at low copy num‐ ber for more than 100 generations in the absence of selection and with a mitotic stability of 98% [93]. This ability has been demonstrated in several cell lines and in primary cells [94] and also *in vivo* in genetically modified pigs [95] making them very attractive for use in CF clinical trials.

lar studies in human patients demonstrated some transgene expression, but not at sufficient

Provided that all obstacles to gene delivery to the lung are overcome and the *CFTR* trans‐ gene finally reaches the target cells, a clinical benefit for CF can only be achieved by lifelong expression of the gene. As repeated administration is in most cases restricted by immune re‐ sponses generated by the patient against the vector, other strategies have been employed for

Integration into the host genome has widely been used in gene therapy to fulfil this require‐ ment. However, the dangers of integration due to insertional mutagenesis have become a widely publicised issue as a result of the SCIDX1 clinical trial, where some patients devel‐ oped leukaemia due to deregulation of the growth-promoting LIM domain only 2 (LMO2) proto-oncogene caused by integration of the vector [64, 65]. The safety concerns regarding uncontrolled integration of the therapeutic gene into the host genome have been strength‐ ened by observations that there is a preference of integrating vectors for the regulatory re‐ gions of transcriptionally active genes [87]. Given the need for long-term expression and the problems associated with vector integration, vectors that persist in the nucleus by being maintained episomally without integrating, could be highly advantageous. Among the sys‐ tems developed to achieve extra-chromosomal maintenance of the vectors carrying the ther‐ apeutic gene, two are considered safe enough for clinical application in the future: artificial

chromosomes and systems based on scaffold/matrix attachment region (S/MAR).

Human Artificial Chromosomes (HACs) are vectors able to replicate and segregate in paral‐ lel with the endogenous chromosomes in human cells. To achieve this, they must contain the minimal elements required for chromosome function, namely an origin of replication, te‐ lomeres and centromeres [88]. HACs can be generated by a method similar to the one ap‐ plied for YAC construction in yeast and involves assembling the functional chromosomal elements and building up a HAC *de novo* in human cells. Different strategies have been fol‐ lowed to generate *de novo* HACs, the most convenient of which is to transfect a BAC carry‐ ing only a large array of α-satellite (alphoid) DNA and some marker genes into HT1080 cells[89]. HACs generated this way exist as single (or low copy) chromosomes in the nucleus and have a high mitotic stability (close to 100%) in the absence of selection. The potential use of these vectors in gene therapy has been demonstrated by expression of large therapeutic

S/MARs are diverse sequences found in all eukaryotic genomes where they are involved in many aspects of chromatin function such as organization of chromatin into loops, which seems to be mediated by the interaction between S/MARs and the nuclear matrix [92]. Vec‐ tors containing an S/MAR element have the ability to remain episomally at low copy num‐ ber for more than 100 generations in the absence of selection and with a mitotic stability of 98% [93]. This ability has been demonstrated in several cell lines and in primary cells [94]

levels to provide a clinical benefit [57, 81-86].

546 Gene Therapy - Tools and Potential Applications

**6. Duration of transgene expression**

efficient retention and long-term expression.

genes from them [90, 91].

An alternative to the use of episomal vectors described above, that still satisfies both re‐ quirements for permanent transgene expression and elimination of genotoxic effects is the controlled integration of the therapeutic DNA at a specific site in the host genome where no active genes are present. Several vector systems have been developed to achieve this, with each one of them having its own limitations [96]. From these, vectors based on the ΦC31 in‐ tegrase [97] and on transposase enzymes [98] are the most promising for use in CF gene therapy as they have a preference for specific sequences that already exist in the human ge‐ nome and have been shown to work *in vivo*.

An additional problem to achieving long-term expression of the *CFT*R transgene delivered to the lung is the life span of the target cells. Depending on the rhythm of natural turnover of these cells, transgene expression can last for as long as these cells are alive. A more effec‐ tive approach would be to either target putative stem cells with the capacity to differentiate to airway epithelial cells [34] or to deliver exogenous heterologous or corrected autologous stem cells to the lungs of CF patients *ex vivo* [99].

Airway basal stem cells are a candidate target for CF stem cell therapy. However, the facts that this population is estimated to represent only a minor part of the total airway epitheli‐ um [12] and that it is quite inaccessible as it is not exposed to the airway lumen make such a therapy approach very challenging.

*Ex vivo* gene therapy using stem cells may not only provide permanent cure without the need for re-administration, but also solve the hurdle of the low *in vivo* gene delivery efficien‐ cy. In *ex vivo* cell therapy using Embryonic Stem Cells or foetal Mesenchymal Stem Cells from a healthy embryo there is no need for transfection of a therapeutic gene. However, these stem cells are used in an allogeneic fashion which requires the parallel use of immuno‐ suppressive drugs and are therefore not applicable for the treatment of CF. A more attrac‐ tive strategy would be the transfer of the *CFTR* gene to patient-derived autologous stem cells such as Mesenchymal Stem Cells (MSCs), which can easily be isolated from the bone marrow or adipose tissue of adults [100] or Induced Pluripotent Stem Cells that can be gen‐ erated by reprogramming of adult somatic cells [101, 102]. In that case, the transfection pro‐ cedure would be performed *in vitro* before reimplantation of the cells back to the donor, which is far more efficient than *in vivo* delivery. Furthermore, bone marrow-derived MSCs have been shown to be able to express transgenes [103] and to differentiate to several types of cells including airway epithelial cells [104]. Nevertheless, there is little literature on how the *ex vivo* corrected MSCs can be administered and engrafted in the lung of Cystic Fibrosis patients. Both systemic and topical lung administration of bone marrow-derived cells have been applied and shown to result in some engraftment into the airways [105], but there are several challenges to be addressed, before *ex vivo* cell therapy becomes part of the CF clinical research. These challenges include the very low efficiency of engraftment (<1%) and the fact that previous damage to the surface epithelium caused by epithelia-injuring reagents seems to be required for the engraftment [75].

#### **7. Pattern of transgene expression**

For gene therapy of some diseases it is important to achieve expression of the therapeutic gene at specific levels. Expression at lower levels than normal might not be sufficient to cor‐ rect the defect and at higher levels could result in undesirable effects. In other cases, tissuespecific expression may be very important. The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of minigene/cDNA constructs in gene therapy for certain genetic diseases where precise levels of the gene product are required [88]. There is evidence that CF is such a disease.

**8. Clinical research for cystic fibrosis**

Joseph *et. al.*, 2001 [129] Perricone *et. al.*, 2001 [130]

**Table 1.** List of CF gene therapy clinical trials.

gene transfer efficacy at the clinical level [34].

Gene therapy clinical trials for CF started in 1993 and over 26 viral and non-viral trials have been conducted or are in progress to date. Viral trials were based on engineered adenovirus and adeno-associated virus and non-viral on various cationic lipids, with GL67 being the

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

549

The trials have confirmed several of the safety concerns associated to the use of viral vec‐ tors. However, other challenges and questions raised pre-clinically still remain to be an‐ swered. In general, proof-of-principle for gene transfer to the airways has been

some of the trials but clinically meaningful outcomes such as improvement in pulmonary function and decrease of bacterial colonies have not been clearly shown in any. In con‐ trast, poor results with regards to clinical benefit, obtained so far, revealed another chal‐ lenge in CF clinical research. This is the need to develop more accurate tools to assess

Historically, adenoviral vectors were the first to be used. However, due to the absence of ad‐ enoviral receptors on the apical side of most human airway epithelial cells [25], which re‐ sults in low transduction efficiency, and due to induction of immune responses that exclude repeated administration [67, 68], adeno-associated viral vectors became an alternative. These vectors were soon found to have their own problems. First it is their small packaging capaci‐ ty which barely holds the whole human *CFTR* gene and therefore restricts the use of strong promoters. Then, at least serotype 2 AAV vectors that were used in initial studies could not be re-administered due to stimulation of immune reactions [72, 73]. Suggestions made to

transport defect in

demonstrated by transgene expression or partial correction of the Cl-

most predominant. The references for the published studies are listed in Table 1.

**Adenoviral clinical trials AAV clinical trials Non-viral clinical trials using cationic lipids**

Zabner *et. al.*, 1993 [119] Wagner *et. al.*, 1999 [120] Sorcher *et. al.*, 1994 [121] Crystal *et. al.*, 1994 [122] Aitken *et. al.*, 2001 [70] Caplen *et. al.*, 1995 [82] Boucher *et. al.*, 1994 [123] Wagner *et. al.*, 2002 [74] Gill *et. al.*, 1997 [83] Knowles *et. al.*, 1995 [124] Flotte *et. al.*, 2003 [71] Porteous *et. al.*, 1997 [84] Hay *et. al.*, 1995 [125] Moss *et. al.*, 2004 [73] Zabner *et. al.*, 1997 [86] Zabner *et. al.*, 1996 [68] Moss *et. al.*, 2007 [72] Alton *et. al.* 1999 [81] Bellon *et. al.*, 1997 [126] Hyde *et. al.*, 2000 [57] Harvey *et. al.*, 1999 [67] Noone *et. al.*, 2000 [127] Zuckerman *et. al.*, 1999 [128] Ruiz *et. al.*, 2001 [85]

Although some studies have shown that expression of as little as 5-10% of endogenous *CFTR* levels may suffice to observe a clinical benefit [106], other studies have shown that dif‐ ferent functions of CFTR like Cl transport and Na+ absorption, when they are abnormal they can be restored by different levels of *CFTR* expression [107]. Moreover, restoration of mucus transport at normal rates requires transduction of at least 25% of target cells [108]. These da‐ ta indicate that CF gene therapy may require *CFTR* expression at the right levels, at the right time and in the right population of cells, which can be achieved only if it is driven and con‐ trolled by the gene's natural promoter and regulatory elements present on a genomic thera‐ peutic construct.

The *CFTR* gene is located on chromosome 7, is 200-250 kb long [5] and comprises 27 exons. It shows a tightly regulated temporal and spatial pattern of expression [109, 110], which was not found to be regulated by any tissue-specific regulatory elements, suggest‐ ing that other elements outside the proximal promoter are probably involved in tissue specific regulation of transcription. Several DNase I Hypersensitive Sites (DHS), usually associated with regulation of transcription, have been identified across 400 kb of DNA flanking the *CFTR* gene. These lie 5' to the gene at -79.5 and -20.9 kb with respect to the translation start site [111], in introns 1 [112], 2, 3, 10, 16, 17a, 18, 20 and 21 [113] and 3' to the gene at +5.4, +6.8, +7, +7.4 and +15.6 kb [114]. Most of these DHS have been found to be involved in tissue-specific *CFTR* expression [114-117]. Therefore, a large genomic construct spanning ~300 kb from the -79.5 kb to the +15.6 kb DHS would include all the known long-range controlling elements of the *CFTR* gene and should give full levels of tissue specific expression which would be advantageous for gene therapy of cystic fibro‐ sis. This big in size region has recently been cloned on a single Bacterial Artificial Chro‐ mosome (BAC) vector and is currently available [118].

As the majority of recombinant viruses, commonly utilized as carriers for transfer of plas‐ mid DNA, apart from evoking unwanted immune responses, have a maximum packaging capacity and cannot be used to deliver large genomic-DNA-containing constructs, gene ther‐ apy using genomic loci of therapeutic genes should be non-viral. This restriction raises again the issue of efficiency of delivery which is even more challenging to deal with than when using smaller constructs.

#### **8. Clinical research for cystic fibrosis**

**7. Pattern of transgene expression**

548 Gene Therapy - Tools and Potential Applications

ferent functions of CFTR like Cl-

mosome (BAC) vector and is currently available [118].

peutic construct.

using smaller constructs.

For gene therapy of some diseases it is important to achieve expression of the therapeutic gene at specific levels. Expression at lower levels than normal might not be sufficient to cor‐ rect the defect and at higher levels could result in undesirable effects. In other cases, tissuespecific expression may be very important. The elements responsible for controlled and tissue-specific expression of a gene usually lie within the introns and the sequences before and after the gene. Therefore, the use of genomic constructs which contain the introns and flanking DNA of the therapeutic gene is expected to be more effective than that of minigene/cDNA constructs in gene therapy for certain genetic diseases where precise levels of

Although some studies have shown that expression of as little as 5-10% of endogenous *CFTR* levels may suffice to observe a clinical benefit [106], other studies have shown that dif‐

can be restored by different levels of *CFTR* expression [107]. Moreover, restoration of mucus transport at normal rates requires transduction of at least 25% of target cells [108]. These da‐ ta indicate that CF gene therapy may require *CFTR* expression at the right levels, at the right time and in the right population of cells, which can be achieved only if it is driven and con‐ trolled by the gene's natural promoter and regulatory elements present on a genomic thera‐

The *CFTR* gene is located on chromosome 7, is 200-250 kb long [5] and comprises 27 exons. It shows a tightly regulated temporal and spatial pattern of expression [109, 110], which was not found to be regulated by any tissue-specific regulatory elements, suggest‐ ing that other elements outside the proximal promoter are probably involved in tissue specific regulation of transcription. Several DNase I Hypersensitive Sites (DHS), usually associated with regulation of transcription, have been identified across 400 kb of DNA flanking the *CFTR* gene. These lie 5' to the gene at -79.5 and -20.9 kb with respect to the translation start site [111], in introns 1 [112], 2, 3, 10, 16, 17a, 18, 20 and 21 [113] and 3' to the gene at +5.4, +6.8, +7, +7.4 and +15.6 kb [114]. Most of these DHS have been found to be involved in tissue-specific *CFTR* expression [114-117]. Therefore, a large genomic construct spanning ~300 kb from the -79.5 kb to the +15.6 kb DHS would include all the known long-range controlling elements of the *CFTR* gene and should give full levels of tissue specific expression which would be advantageous for gene therapy of cystic fibro‐ sis. This big in size region has recently been cloned on a single Bacterial Artificial Chro‐

As the majority of recombinant viruses, commonly utilized as carriers for transfer of plas‐ mid DNA, apart from evoking unwanted immune responses, have a maximum packaging capacity and cannot be used to deliver large genomic-DNA-containing constructs, gene ther‐ apy using genomic loci of therapeutic genes should be non-viral. This restriction raises again the issue of efficiency of delivery which is even more challenging to deal with than when

absorption, when they are abnormal they

the gene product are required [88]. There is evidence that CF is such a disease.

transport and Na+

Gene therapy clinical trials for CF started in 1993 and over 26 viral and non-viral trials have been conducted or are in progress to date. Viral trials were based on engineered adenovirus and adeno-associated virus and non-viral on various cationic lipids, with GL67 being the most predominant. The references for the published studies are listed in Table 1.


**Table 1.** List of CF gene therapy clinical trials.

The trials have confirmed several of the safety concerns associated to the use of viral vec‐ tors. However, other challenges and questions raised pre-clinically still remain to be an‐ swered. In general, proof-of-principle for gene transfer to the airways has been demonstrated by transgene expression or partial correction of the Cl transport defect in some of the trials but clinically meaningful outcomes such as improvement in pulmonary function and decrease of bacterial colonies have not been clearly shown in any. In con‐ trast, poor results with regards to clinical benefit, obtained so far, revealed another chal‐ lenge in CF clinical research. This is the need to develop more accurate tools to assess gene transfer efficacy at the clinical level [34].

Historically, adenoviral vectors were the first to be used. However, due to the absence of ad‐ enoviral receptors on the apical side of most human airway epithelial cells [25], which re‐ sults in low transduction efficiency, and due to induction of immune responses that exclude repeated administration [67, 68], adeno-associated viral vectors became an alternative. These vectors were soon found to have their own problems. First it is their small packaging capaci‐ ty which barely holds the whole human *CFTR* gene and therefore restricts the use of strong promoters. Then, at least serotype 2 AAV vectors that were used in initial studies could not be re-administered due to stimulation of immune reactions [72, 73]. Suggestions made to overcome these limitations still need to be validated in humans. Non-viral vectors were found compatible with repeated administration [57], but their efficiency were variable and transgene expression was shown only in some studies. In addition, flu-like symptoms were reported [81, 85], which were associated to the presence of unmethylated CpG motifs on the plasmid DNA that was delivered. The use of genomic constructs containing only human DNA may overcome this limitation but this also needs to be shown in future clinical trials.

[3] Boucher RC. An overview of the pathogenesis of cystic fibrosis lung disease. Adv

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

551

[4] Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, et al. Identifi‐ cation of the cystic fibrosis gene: cloning and characterization of complementary

[5] Rommens JM, Iannuzzi MC, Kerem B, Drumm ML, Melmer G, Dean M, et al. Identi‐ fication of the cystic fibrosis gene: chromosome walking and jumping. Science

[6] Clunes MT, Boucher RC. Cystic Fibrosis: The Mechanisms of Pathogenesis of an In‐

[7] Lyczak JB, Cannon CL, Pier GB. Lung infections associated with cystic fibrosis. Clin

[8] Dorin JR, Farley R, Webb S, Smith SN, Farini E, Delaney SJ, et al. A demonstration using mouse models that successful gene therapy for cystic fibrosis requires only

[9] Mrsny RJ. Lessons from nature: "Pathogen-Mimetic" systems for mucosal nano-medi‐

[10] Lai SK, Wang YY, Wirtz D, Hanes J. Micro- and macrorheology of mucus. Adv Drug

[11] Cu Y, Saltzman WM. Mathematical modeling of molecular diffusion through mucus.

[12] Rock JR, Randell SH, Hogan BL. Airway basal stem cells: a perspective on their roles in epithelial homeostasis and remodeling. Dis Model Mech 2010;3(9-10):545-56. [13] Evans MJ, Cabral-Anderson LJ, Freeman G. Role of the Clara cell in renewal of the

[14] Engelhardt JF, Zepeda M, Cohn JA, Yankaskas JR, Wilson JM. Expression of the cyst‐

[15] Engelhardt JF, Yankaskas JR, Ernst SA, Yang Y, Marino CR, Boucher RC, et al. Sub‐ mucosal glands are the predominant site of CFTR expression in the human bronchus.

[16] Kreda SM, Mall M, Mengos A, Rochelle L, Yankaskas J, Riordan JR, et al. Characteri‐ zation of wild-type and deltaF508 cystic fibrosis transmembrane regulator in human

[17] Proesmans M, Vermeulen F, De Boeck K. What's new in cystic fibrosis? From treating symptoms to correction of the basic defect. Eur J Pediatr 2008 Aug;167(8):839-49. [18] Rubin BK. Mucus, phlegm, and sputum in cystic fibrosis. Respir Care 2009;54(6):

ic fibrosis gene in adult human lung. J Clin Invest 1994;93(2):737-49.

herited Lung Disorder. Drug Discov Today Dis Mech 2007;4(2):63-72.

Drug Deliv Rev 2002;54(11):1359-71.

DNA. Science 1989;245(4922):1066-73.

Microbiol Rev 2002;15(2):194-222.

Deliv Rev 2009;61(2):86-100.

Nat Genet 1992;2(3):240-8.

726-32; discussion 32.

partial gene correction. Gene Ther 1996;3(9):797-801.

bronchiolar epithelium. Lab Invest 1978;38(6):648-53.

respiratory epithelia. Mol Biol Cell 2005;16(5):2154-67.

cines. Adv Drug Deliv Rev 2009;61(2):172-92.

Adv Drug Deliv Rev 2009;61(2):101-14.

1989;245(4922):1059-65.

#### **9. Conclusion**

Almost 20 years have passed since the beginning of gene therapy trials for CF. Despite ini‐ tial enthusiasm, only little progress has been made during that time. In contrast, the main conclusion was that the lung is more difficult to target than initially anticipated. Several bar‐ riers were discovered, which led to the development of respective ways to overcome them. The majority of these have not reached the level of validation in clinical trials yet. For exam‐ ple, the use of a non-viral vector with the ability to remain extra-chromosomally containing the whole genomic region of the *CFTR* gene or *ex vivo* stem cell therapy, are two promising approaches that need to be further explored and may be seen in clinical trials in the future.

#### **Acknowledgements**

AK and VG are financially supported from the European Commission FP7 project INsPiRE. The authors would also like to thank Dr. Ioannis Pateras for his kind gift of Figure 2.

#### **Author details**

George Kotzamanis1 , Athanassios Kotsinas1 , Apostolos Papalois2 and Vassilis G. Gorgoulis1


#### **References**


[3] Boucher RC. An overview of the pathogenesis of cystic fibrosis lung disease. Adv Drug Deliv Rev 2002;54(11):1359-71.

overcome these limitations still need to be validated in humans. Non-viral vectors were found compatible with repeated administration [57], but their efficiency were variable and transgene expression was shown only in some studies. In addition, flu-like symptoms were reported [81, 85], which were associated to the presence of unmethylated CpG motifs on the plasmid DNA that was delivered. The use of genomic constructs containing only human DNA may overcome this limitation but this also needs to be shown in future clinical trials.

Almost 20 years have passed since the beginning of gene therapy trials for CF. Despite ini‐ tial enthusiasm, only little progress has been made during that time. In contrast, the main conclusion was that the lung is more difficult to target than initially anticipated. Several bar‐ riers were discovered, which led to the development of respective ways to overcome them. The majority of these have not reached the level of validation in clinical trials yet. For exam‐ ple, the use of a non-viral vector with the ability to remain extra-chromosomally containing the whole genomic region of the *CFTR* gene or *ex vivo* stem cell therapy, are two promising approaches that need to be further explored and may be seen in clinical trials in the future.

AK and VG are financially supported from the European Commission FP7 project INsPiRE.

[1] Grubb BR, Boucher RC. Pathophysiology of gene-targeted mouse models for cystic

[2] Welsh MJ, Smith JJ. cAMP stimulation of HCO3- secretion across airway epithelia.

, Apostolos Papalois2

and Vassilis G. Gorgoulis1

The authors would also like to thank Dr. Ioannis Pateras for his kind gift of Figure 2.

, Athanassios Kotsinas1

fibrosis. Physiol Rev 1999;79(1 Suppl):S193-214.

1 University of Athens, Medical School, Greece

JOP 2001;2(4 Suppl):291-3.

2 Experimental Research Center ELPEN SA, Greece

**9. Conclusion**

550 Gene Therapy - Tools and Potential Applications

**Acknowledgements**

**Author details**

George Kotzamanis1

**References**


[19] Hida K, Lai SK, Suk JS, Won SY, Boyle MP, Hanes J. Common gene therapy viral vec‐ tors do not efficiently penetrate sputum from cystic fibrosis patients. PLoS One 2011;6(5):e19919.

[32] Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo:

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

553

[33] Hyde SC, Gill DR, Higgins CF, Trezise AE, MacVinish LJ, Cuthbert AW, et al. Cor‐ rection of the ion transport defect in cystic fibrosis transgenic mice by gene therapy.

[34] Davies JC, Alton EW. Gene therapy for cystic fibrosis. Proc Am Thorac Soc 2010;7(6):

[35] Kollen WJ, Midoux P, Erbacher P, Yip A, Roche AC, Monsigny M, et al. Gluconoylat‐ ed and glycosylated polylysines as vectors for gene transfer into cystic fibrosis air‐

[36] Parente RA, Nir S, Szoka FC, Jr. Mechanism of leakage of phospholipid vesicle con‐

[37] Wagner E, Plank C, Zatloukal K, Cotten M, Birnstiel ML. Influenza virus hemaggluti‐ nin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin-poly‐ lysine-DNA complexes: toward a synthetic virus-like gene-transfer vehicle. Proc Natl

[38] Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC, Jr. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabiliz‐

[39] Zauner W, Kichler A, Schmidt W, Mechtler K, Wagner E. Glycerol and polylysine synergize in their ability to rupture vesicular membranes: a mechanism for increased transferrin-polylysine-mediated gene transfer. Exp Cell Res 1997;232(1):137-45.

[40] Lam AP, Dean DA. Progress and prospects: nuclear import of nonviral vectors. Gene

[41] Whittaker GR. Virus nuclear import. Adv Drug Deliv Rev 2003 Jun 16;55(6):733-47.

[42] Pollard H, Remy JS, Loussouarn G, Demolombe S, Behr JP, Escande D. Polyethyleni‐ mine but not cationic lipids promotes transgene delivery to the nucleus in mammali‐

[43] Escriou V, Carriere M, Scherman D, Wils P. NLS bioconjugates for targeting thera‐

[44] Hebert E. Improvement of exogenous DNA nuclear importation by nuclear localiza‐ tion signal-bearing vectors: a promising way for non-viral gene therapy? Biol Cell

[45] Klink D, Schindelhauer D, Laner A, Tucker T, Bebok Z, Schwiebert EM, et al. Gene delivery systems--gene therapy vectors for cystic fibrosis. J Cyst Fibros 2004;3 Suppl

peutic genes to the nucleus. Adv Drug Deliv Rev 2003;55(2):295-306.

tents induced by the peptide GALA. Biochemistry 1990;29(37):8720-8.

polyethylenimine. Proc Natl Acad Sci U S A 1995;92(16):7297-301.

way epithelial cells. Hum Gene Ther 1996;7(13):1577-86.

Nature 1993;362(6417):250-5.

Acad Sci U S A 1992;89(17):7934-8.

Ther 2010;17(4):439-47.

2003;95(2):59-68.

2:203-12.

es bilayers. Biochemistry 1997;36(10):3008-17.

an cells. J Biol Chem 1998;273(13):7507-11.

408-14.


[32] Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, et al. A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine. Proc Natl Acad Sci U S A 1995;92(16):7297-301.

[19] Hida K, Lai SK, Suk JS, Won SY, Boyle MP, Hanes J. Common gene therapy viral vec‐ tors do not efficiently penetrate sputum from cystic fibrosis patients. PLoS One

[20] Sanders NN, Van Rompaey E, De Smedt SC, Demeester J. Structural alterations of gene complexes by cystic fibrosis sputum. Am J Respir Crit Care Med 2001;164(3):

[21] Griesenbach U, Geddes DM, Alton EW. Gene therapy for cystic fibrosis: an example

[22] Sinn PL, Shah AJ, Donovan MD, McCray PB, Jr. Viscoelastic gel formulations en‐ hance airway epithelial gene transfer with viral vectors. Am J Respir Cell Mol Biol

[23] Ferrari S, Kitson C, Farley R, Steel R, Marriott C, Parkins DA, et al. Mucus altering agents as adjuncts for nonviral gene transfer to airway epithelium. Gene Ther

[24] Kushwah R, Oliver JR, Cao H, Hu J. Nacystelyn enhances adenoviral vector-mediat‐

[25] Walters RW, Grunst T, Bergelson JM, Finberg RW, Welsh MJ, Zabner J. Basolateral localization of fiber receptors limits adenovirus infection from the apical surface of

[26] Gregory LG, Harbottle RP, Lawrence L, Knapton HJ, Themis M, Coutelle C. En‐ hancement of adenovirus-mediated gene transfer to the airways by DEAE dextran

[27] Johnson LG, Vanhook MK, Coyne CB, Haykal-Coates N, Gavett SH. Safety and effi‐ ciency of modulating paracellular permeability to enhance airway epithelial gene

[28] Pickles RJ, Fahrner JA, Petrella JM, Boucher RC, Bergelson JM. Retargeting the cox‐ sackievirus and adenovirus receptor to the apical surface of polarized epithelial cells reveals the glycocalyx as a barrier to adenovirus-mediated gene transfer. J Virol

[29] Lechardeur D, Sohn KJ, Haardt M, Joshi PB, Monck M, Graham RW, et al. Metabolic instability of plasmid DNA in the cytosol: a potential barrier to gene transfer. Gene

[30] Pollard H, Toumaniantz G, Amos JL, Avet-Loiseau H, Guihard G, Behr JP, et al. Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected

[31] Dean DA, Strong DD, Zimmer WE. Nuclear entry of nonviral vectors. Gene Ther

ed gene delivery to mouse airways. Gene Ther 2007;14(16):1243-8.

airway epithelia. J Biol Chem 1999;274(15):10219-26.

and sodium caprate in vivo. Mol Ther 2003;7(1):19-26.

transfer in vivo. Hum Gene Ther 2003;14(8):729-47.

for lung gene therapy. Gene Ther 2004;11 Suppl 1:S43-50.

2011;6(5):e19919.

552 Gene Therapy - Tools and Potential Applications

2005;32(5):404-10.

2001;8(18):1380-6.

2000;74(13):6050-7.

Ther 1999;6(4):482-97.

2005;12(11):881-90.

plasmids. J Gene Med 2001;3(2):153-64.

486-93.


[46] Ferrari S, Griesenbach U, Geddes DM, Alton E. Immunological hurdles to lung gene therapy. Clin Exp Immunol 2003;132(1):1-8.

[59] Schwartz DA, Quinn TJ, Thorne PS, Sayeed S, Yi AK, Krieg AM. CpG motifs in bacte‐ rial DNA cause inflammation in the lower respiratory tract. J Clin Invest 1997;100(1):

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

555

[60] Yew NS, Wang KX, Przybylska M, Bagley RG, Stedman M, Marshall J, et al. Contri‐ bution of plasmid DNA to inflammation in the lung after administration of cationic

[61] Krieg AM. CpG motifs in bacterial DNA and their immune effects. Annu Rev Immu‐

[62] Raper SE, Chirmule N, Lee FS, Wivel NA, Bagg A, Gao GP, et al. Fatal systemic in‐ flammatory response syndrome in a ornithine transcarbamylase deficient patient fol‐

[63] Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P, et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

[64] Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, et al. A serious adverse event after successful gene therapy for X-linked severe com‐

[65] Hacein-Bey-Abina S, Von Kalle C, Schmidt M, McCormack MP, Wulffraat N, Leb‐ oulch P, et al. LMO2-associated clonal T cell proliferation in two patients after gene

[66] Zhang Y, Chirmule N, Gao GP, Qian R, Croyle M, Joshi B, et al. Acute cytokine re‐ sponse to systemic adenoviral vectors in mice is mediated by dendritic cells and mac‐

[67] Harvey BG, Leopold PL, Hackett NR, Grasso TM, Williams PM, Tucker AL, et al. Airway epithelial CFTR mRNA expression in cystic fibrosis patients after repetitive

administration of a recombinant adenovirus. J Clin Invest 1999;104(9):1245-55. [68] Zabner J, Ramsey BW, Meeker DP, Aitken ML, Balfour RP, Gibson RL, et al. Repeat administration of an adenovirus vector encoding cystic fibrosis transmembrane con‐ ductance regulator to the nasal epithelium of patients with cystic fibrosis. J Clin In‐

[69] Croyle MA, Chirmule N, Zhang Y, Wilson JM. "Stealth" adenoviruses blunt cellmediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung. J Virol 2001;75(10):4792-801. [70] Aitken ML, Moss RB, Waltz DA, Dovey ME, Tonelli MR, McNamara SC, et al. A phase I study of aerosolized administration of tgAAVCF to cystic fibrosis subjects

[71] Flotte TR, Zeitlin PL, Reynolds TC, Heald AE, Pedersen P, Beck S, et al. Phase I trial of intranasal and endobronchial administration of a recombinant adeno-associated

with mild lung disease. Hum Gene Ther 2001;12(15):1907-16.

lowing adenoviral gene transfer. Mol Genet Metab 2003;80(1-2):148-58.

lipid:pDNA complexes. Hum Gene Ther 1999;10(2):223-34.

bined immunodeficiency. N Engl J Med 2003;348(3):255-6.

therapy for SCID-X1. Science 2003;302(5644):415-9.

rophages. Mol Ther 2001;3(5 Pt 1):697-707.

vest 1996;97(6):1504-11.

68-73.

nol 2002;20:709-60.

Science 2000;288(5466):669-72.


[59] Schwartz DA, Quinn TJ, Thorne PS, Sayeed S, Yi AK, Krieg AM. CpG motifs in bacte‐ rial DNA cause inflammation in the lower respiratory tract. J Clin Invest 1997;100(1): 68-73.

[46] Ferrari S, Griesenbach U, Geddes DM, Alton E. Immunological hurdles to lung gene

[47] Worgall S, Leopold PL, Wolff G, Ferris B, Van Roijen N, Crystal RG. Role of alveolar macrophages in rapid elimination of adenovirus vectors administered to the epithe‐

[48] Qin L, Ding Y, Pahud DR, Chang E, Imperiale MJ, Bromberg JS. Promoter attenua‐ tion in gene therapy: interferon-gamma and tumor necrosis factor-alpha inhibit

[49] Jooss K, Yang Y, Wilson JM. Cyclophosphamide diminishes inflammation and pro‐ longs transgene expression following delivery of adenoviral vectors to mouse liver

[50] Hazinski TA, Ladd PA, DeMatteo CA. Localization and induced expression of fusion

[51] Kolb M, Inman M, Margetts PJ, Galt T, Gauldie J. Budesonide enhances repeated gene transfer and expression in the lung with adenoviral vectors. Am J Respir Crit

[52] Yang Y, Trinchieri G, Wilson JM. Recombinant IL-12 prevents formation of blocking IgA antibodies to recombinant adenovirus and allows repeated gene therapy to

[53] Chirmule N, Raper SE, Burkly L, Thomas D, Tazelaar J, Hughes JV, et al. Readminis‐ tration of adenovirus vector in nonhuman primate lungs by blockade of CD40-CD40

[54] Chirmule N, Truneh A, Haecker SE, Tazelaar J, Gao G, Raper SE, et al. Repeated ad‐ ministration of adenoviral vectors in lungs of human CD4 transgenic mice treated

[55] Jooss K, Turka LA, Wilson JM. Blunting of immune responses to adenoviral vectors

[56] Scaria A, St George JA, Gregory RJ, Noelle RJ, Wadsworth SC, Smith AE, et al. Anti‐ body to CD40 ligand inhibits both humoral and cellular immune responses to adeno‐ viral vectors and facilitates repeated administration to mouse airway. Gene Ther

[57] Hyde SC, Southern KW, Gileadi U, Fitzjohn EM, Mofford KA, Waddell BE, et al. Re‐ peat administration of DNA/liposomes to the nasal epithelium of patients with cystic

[58] Scheule RK, St George JA, Bagley RG, Marshall J, Kaplan JM, Akita GY, et al. Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mam‐

with a nondepleting CD4 antibody. J Immunol 1999;163(1):448-55.

in mouse liver and lung with CTLA4Ig. Gene Ther 1998;5(3):309-19.

lial surface of the respiratory tract. Hum Gene Ther 1997;8(14):1675-84.

transgene expression. Hum Gene Ther 1997;8(17):2019-29.

genes in the rat lung. Am J Respir Cell Mol Biol 1991;4(3):206-9.

therapy. Clin Exp Immunol 2003;132(1):1-8.

554 Gene Therapy - Tools and Potential Applications

and lung. Hum Gene Ther 1996;7(13):1555-66.

Care Med 2001;164(5):866-72.

1997;4(6):611-7.

mouse lung. Nat Med 1995;1(9):890-3.

fibrosis. Gene Ther 2000;7(13):1156-65.

malian lung. Hum Gene Ther 1997;8(6):689-707.

ligand interactions. J Virol 2000;74(7):3345-52.


virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part clinical study. Hum Gene Ther 2003;14(11):1079-88.

[84] Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

557

[85] Ruiz FE, Clancy JP, Perricone MA, Bebok Z, Hong JS, Cheng SH, et al. A clinical in‐ flammatory syndrome attributable to aerosolized lipid-DNA administration in cystic

[86] Zabner J, Cheng SH, Meeker D, Launspach J, Balfour R, Perricone MA, et al. Com‐ parison of DNA-lipid complexes and DNA alone for gene transfer to cystic fibrosis

[87] Bushman F, Lewinski M, Ciuffi A, Barr S, Leipzig J, Hannenhalli S, et al. Genomewide analysis of retroviral DNA integration. Nat Rev Microbiol 2005;3(11):848-58. [88] Perez-Luz S, Diaz-Nido J. Prospects for the use of artificial chromosomes and mini‐ chromosome-like episomes in gene therapy. J Biomed Biotechnol 2010;pii:642804.. [89] Ebersole TA, Ross A, Clark E, McGill N, Schindelhauer D, Cooke H, et al. Mammali‐ an artificial chromosome formation from circular alphoid input DNA does not re‐

[90] Grimes BR, Schindelhauer D, McGill NI, Ross A, Ebersole TA, Cooke HJ. Stable gene expression from a mammalian artificial chromosome. EMBO Rep 2001;2(10):910-4. [91] Mejia JE, Larin Z. The assembly of large BACs by in vivo recombination. Genomics

[92] Heng HH, Goetze S, Ye CJ, Liu G, Stevens JB, Bremer SW, et al. Chromatin loops are selectively anchored using scaffold/matrix-attachment regions. J Cell Sci 2004;117(Pt

[93] Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ. A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic

[94] Papapetrou EP, Ziros PG, Micheva ID, Zoumbos NC, Athanassiadou A. Gene trans‐ fer into human hematopoietic progenitor cells with an episomal vector carrying an

[95] Manzini S, Vargiolu A, Stehle IM, Bacci ML, Cerrito MG, Giovannoni R, et al. Geneti‐ cally modified pigs produced with a nonviral episomal vector. Proc Natl Acad Sci U

[96] Voigt K, Izsvak Z, Ivics Z. Targeted gene insertion for molecular medicine. J Mol

[97] Bertoni C, Jarrahian S, Wheeler TM, Li Y, Olivares EC, Calos MP, et al. Enhancement of plasmid-mediated gene therapy for muscular dystrophy by directed plasmid inte‐

1997;4(3):210-8.

2000 Dec 1;70(2):165-70.

Acids Res 1999;27(2):426-8.

S A 2006;103(47):17672-7.

Med (Berl) 2008;86(11):1205-19.

S/MAR element. Gene Ther 2006;13(1):40-51.

gration. Proc Natl Acad Sci U S A 2006;103(2):419-24.

7):999-1008.

fibrosis. Hum Gene Ther 2001;12(7):751-61.

airway epithelia in vivo. J Clin Invest 1997;100(6):1529-37.

quire telomere repeats. Hum Mol Genet 2000;9(11):1623-31.


[84] Porteous DJ, Dorin JR, McLachlan G, Davidson-Smith H, Davidson H, Stevenson BJ, et al. Evidence for safety and efficacy of DOTAP cationic liposome mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis. Gene Ther 1997;4(3):210-8.

virus serotype 2 (rAAV2)-CFTR vector in adult cystic fibrosis patients: a two-part

[72] Moss RB, Milla C, Colombo J, Accurso F, Zeitlin PL, Clancy JP, et al. Repeated aero‐ solized AAV-CFTR for treatment of cystic fibrosis: a randomized placebo-controlled

[73] Moss RB, Rodman D, Spencer LT, Aitken ML, Zeitlin PL, Waltz D, et al. Repeated adeno-associated virus serotype 2 aerosol-mediated cystic fibrosis transmembrane regulator gene transfer to the lungs of patients with cystic fibrosis: a multicenter,

[74] Wagner JA, Nepomuceno IB, Messner AH, Moran ML, Batson EP, Dimiceli S, et al. A phase II, double-blind, randomized, placebo-controlled clinical trial of tgAAVCF us‐ ing maxillary sinus delivery in patients with cystic fibrosis with antrostomies. Hum

[75] Griesenbach U, Alton EW. Gene transfer to the lung: lessons learned from more than 2 decades of CF gene therapy. Adv Drug Deliv Rev 2009 Feb 27;61(2):128-39.

[76] Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent prog‐

[77] Dean DA, Machado-Aranda D, Blair-Parks K, Yeldandi AV, Young JL. Electropora‐ tion as a method for high-level nonviral gene transfer to the lung. Gene Ther

[78] Xenariou S, Liang HD, Griesenbach U, Zhu J, Farley R, Somerton L, et al. Low-fre‐ quency ultrasound increases non-viral gene transfer to the mouse lung. Acta Biochim

[79] Xenariou S, Griesenbach U, Ferrari S, Dean P, Scheule RK, Cheng SH, et al. Using magnetic forces to enhance non-viral gene transfer to airway epithelium in vivo.

[80] Tros de Ilarduya C, Sun Y, Duzgunes N. Gene delivery by lipoplexes and polyplexes.

[81] Alton EW, Stern M, Farley R, Jaffe A, Chadwick SL, Phillips J, et al. Cationic lipidmediated CFTR gene transfer to the lungs and nose of patients with cystic fibrosis: a

[82] Caplen NJ, Alton EW, Middleton PG, Dorin JR, Stevenson BJ, Gao X, et al. Liposomemediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis.

[83] Gill DR, Southern KW, Mofford KA, Seddon T, Huang L, Sorgi F, et al. A placebocontrolled study of liposome-mediated gene transfer to the nasal epithelium of pa‐

double-blind placebo-controlled trial. Lancet 1999;353(9157):947-54.

tients with cystic fibrosis. Gene Ther 1997;4(3):199-209.

clinical study. Hum Gene Ther 2003;14(11):1079-88.

phase 2B trial. Hum Gene Ther 2007;18(8):726-32.

Gene Ther 2002;13(11):1349-59.

ress. AAPS J 2009;11(4):671-81.

Gene Ther 2006;13(21):1545-52.

Eur J Pharm Sci 2010;40(3):159-70.

Nat Med 1995;1(1):39-46.

Biophys Sin (Shanghai) 2010;42(1):45-51.

2003;10(18):1608-15.

556 Gene Therapy - Tools and Potential Applications

double-blind, placebo-controlled trial. Chest 2004;125(2):509-21.


[98] Ivics Z, Izsvak Z. The expanding universe of transposon technologies for gene and cell engineering. Mob DNA 2010;1(1):25.

[111] Smith AN, Wardle CJ, Harris A. Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene. Biochem Biophys Res Commun 1995;211(1):274-81.

Targeting the Lung: Challenges in Gene Therapy for Cystic Fibrosis

http://dx.doi.org/10.5772/52532

559

[112] Smith AN, Barth ML, McDowell TL, Moulin DS, Nuthall HN, Hollingsworth MA, et al. A regulatory element in intron 1 of the cystic fibrosis transmembrane conductance

[113] Smith DJ, Nuthall HN, Majetti ME, Harris A. Multiple potential intragenic regulatory

[114] Nuthall HN, Moulin DS, Huxley C, Harris A. Analysis of DNase-I-hypersensitive sites at the 3' end of the cystic fibrosis transmembrane conductance regulator gene

[115] Nuthall HN, Vassaux G, Huxley C, Harris A. Analysis of a DNase I hypersensitive site located -20.9 kb upstream of the CFTR gene. Eur J Biochem 1999;266(2):431-43.

[116] Phylactides M, Rowntree R, Nuthall H, Ussery D, Wheeler A, Harris A. Evaluation of potential regulatory elements identified as DNase I hypersensitive sites in the CFTR

[117] Rowntree RK, Vassaux G, McDowell TL, Howe S, McGuigan A, Phylactides M, et al. An element in intron 1 of the CFTR gene augments intestinal expression in vivo.

[118] Kotzamanis G, Abdulrazzak H, Gifford-Garner J, Haussecker PL, Cheung W, Grillot-Courvalin C, et al. CFTR expression from a BAC carrying the complete human gene

[119] Zabner J, Couture LA, Gregory RJ, Graham SM, Smith AE, Welsh MJ. Adenovirusmediated gene transfer transiently corrects the chloride transport defect in nasal epi‐

[120] Wagner JA, Messner AH, Moran ML, Daifuku R, Kouyama K, Desch JK, et al. Safety and biological efficacy of an adeno-associated virus vector-cystic fibrosis transmem‐ brane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus. Laryngoscope

[121] Sorscher EJ, Logan JJ, Frizzell RA, Lyrene RK, Bebok Z, Dong JY, et al. Informed con‐ sent to participate in a research study -- gene therapy for cystic fibrosis using cationic liposome mediated gene transfer: a phase I trial of safety and efficacy in the nasal air‐

[122] Crystal RG, McElvaney NG, Rosenfeld MA, Chu CS, Mastrangeli A, Hay JG, et al. Administration of an adenovirus containing the human CFTR cDNA to the respirato‐

[123] Boucher RC, Knowles MR, Johnson LG, Olsen JC, Pickles R, Wilson JM, et al. Gene therapy for cystic fibrosis using E1-deleted adenovirus: a phase I trial in the nasal

ry tract of individuals with cystic fibrosis. Nat Genet 1994;8(1):42-51.

and associated regulatory elements. J Cell Mol Med 2009;13(9A):2938-48.

thelia of patients with cystic fibrosis. Cell 1993;75(2):207-16.

regulator gene. J Biol Chem 1996;271(17):9947-54.

(CFTR). Biochem J 1999;341 ( Pt 3):601-11.

gene. Eur J Biochem 2002;269(2):553-9.

Hum Mol Genet 2001;10(14):1455-64.

1999;109(2 Pt 1):266-74.

way. Hum Gene Ther 1994;5(10):1271-7.

elements in the CFTR gene. Genomics 2000;64(1):90-6.


[111] Smith AN, Wardle CJ, Harris A. Characterization of DNASE I hypersensitive sites in the 120kb 5' to the CFTR gene. Biochem Biophys Res Commun 1995;211(1):274-81.

[98] Ivics Z, Izsvak Z. The expanding universe of transposon technologies for gene and

[99] Leblond AL, Naud P, Forest V, Gourden C, Sagan C, Romefort B, et al. Developing cell therapy techniques for respiratory disease: intratracheal delivery of genetically engineered stem cells in a murine model of airway injury. Hum Gene Ther

[100] Abdallah BM, Kassem M. Human mesenchymal stem cells: from basic biology to

[101] Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell

[102] Yu J, Vodyanik MA, Smuga-Otto K, Antosiewicz-Bourget J, Frane JL, Tian S, et al. In‐ duced pluripotent stem cell lines derived from human somatic cells. Science

[103] Aluigi M, Fogli M, Curti A, Isidori A, Gruppioni E, Chiodoni C, et al. Nucleofection is an efficient nonviral transfection technique for human bone marrow-derived mes‐

[104] Wang G, Bunnell BA, Painter RG, Quiniones BC, Tom S, Lanson NA, Jr., et al. Adult stem cells from bone marrow stroma differentiate into airway epithelial cells: poten‐

[105] Conese M, Ascenzioni F, Boyd AC, Coutelle C, De Fino I, De Smedt S, et al. Gene and cell therapy for cystic fibrosis: from bench to bedside. J Cyst Fibros 2011;10 Suppl

[106] Gan KH, Veeze HJ, van den Ouweland AM, Halley DJ, Scheffer H, van der Hout A, et al. A cystic fibrosis mutation associated with mild lung disease. N Engl J Med

[107] Johnson LG, Boyles SE, Wilson J, Boucher RC. Normalization of raised sodium ab‐ sorption and raised calcium-mediated chloride secretion by adenovirus-mediated ex‐ pression of cystic fibrosis transmembrane conductance regulator in primary human

[108] Zhang L, Button B, Gabriel SE, Burkett S, Yan Y, Skiadopoulos MH, et al. CFTR de‐ livery to 25% of surface epithelial cells restores normal rates of mucus transport to

[109] Crawford I, Maloney PC, Zeitlin PL, Guggino WB, Hyde SC, Turley H, et al. Immu‐ nocytochemical localization of the cystic fibrosis gene product CFTR. Proc Natl Acad

[110] Trezise AE, Chambers JA, Wardle CJ, Gould S, Harris A. Expression of the cystic fib‐

cystic fibrosis airway epithelial cells. J Clin Invest 1995;95(3):1377-82.

human cystic fibrosis airway epithelium. PLoS Biol 2009;7(7):e1000155.

rosis gene in human foetal tissues. Hum Mol Genet 1993;2(3):213-8.

tial therapy for cystic fibrosis. Proc Natl Acad Sci U S A 2005;102(1):186-91.

cell engineering. Mob DNA 2010;1(1):25.

clinical applications. Gene Ther 2008;15(2):109-16.

enchymal stem cells. Stem Cells 2006;24(2):454-61.

2009;20(11):1329-43.

558 Gene Therapy - Tools and Potential Applications

2007;131(5):861-72.

2:S114-28.

1995;333(2):95-9.

Sci U S A 1991;88(20):9262-6.

2007;318(5858):1917-20.


cavity. The University of North Carolina at Chapel Hill. Hum Gene Ther 1994;5(5): 615-39.

**Chapter 23**

**Gene Therapy for the** *COL7A1* **Gene**

Additional information is available at the end of the chapter

Epidermolysis bullosa (EB) is a genetically and clinically variable disease characterized by blis‐ ter formation and erosions of the skin and mucous membranes after minor trauma [1]. The in‐ heritance of the affected genes can occur in a dominant or recessive way depending on the subform of the disease. In general, epidermolysis bullosa is caused by mutations in genes en‐ coding structural proteins within the basal membrane zone of the skin. Absence or functional loss of one of these proteins results in a lack of stability of the microarchitecture of the connec‐ tion between dermis and epidermis leading to a loss of coherence [1]. The basement membrane between the dermis and the epidermis is a complex membrane produced by basal keratino‐ cytes and dermal fibroblasts that acts as mechanical support for the connection of both skin lay‐ ers. The basal membrane also regulates the metabolic exchange between the two skin compartments [2]. Up to date, there are at least 15 genes associated with EB causing different forms of the disease. Numerous mutations in these genes that encode for structural proteins within keratinocytes or within mucocutaneous basement membranes have been identified up

Mutations in the genes, encoding for the keratins 5 and 14 and plectin, lead to epidermolysis bullosa simplex (EBS) characterized by the cytolysis within basal keratinocytes. Junctional epi‐ dermolysis bullosa (JEB) is caused by the absence or loss of function of laminin-332, type XVII collagen or integrin-β4. JEB is a severe EB form and is characterized by the separation of the skin within the lamina lucida. Mutations in type VII collagen (encoded by *COL7A1*) lead to the dystrophic form of epidermolysis bullosa, characterized by skin separation below the lamina densa. The severity and clinical manifestation of the disease depend on the mutation type (mis‐ sense mutation, nonsense mutation, splice site mutations, deletion or insertion), the mode of inheritance and the localization of the mutation within the gene. Due to this fact, diagnosis,

> © 2013 Mayr et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

distribution, and reproduction in any medium, provided the original work is properly cited.

E. Mayr, U. Koller and J.W. Bauer

http://dx.doi.org/10.5772/51926

**1. Introduction**

to now [1].

**1.1. Epidermolysis bullosa**


**Chapter 23**

### **Gene Therapy for the** *COL7A1* **Gene**

E. Mayr, U. Koller and J.W. Bauer

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/51926

#### **1. Introduction**

cavity. The University of North Carolina at Chapel Hill. Hum Gene Ther 1994;5(5):

[124] Knowles MR, Hohneker KW, Zhou Z, Olsen JC, Noah TL, Hu PC, et al. A controlled study of adenoviral-vector-mediated gene transfer in the nasal epithelium of patients

[125] Hay JG, McElvaney NG, Herena J, Crystal RG. Modification of nasal epithelial poten‐ tial differences of individuals with cystic fibrosis consequent to local administration of a normal CFTR cDNA adenovirus gene transfer vector. Hum Gene Ther

[126] Bellon G, Michel-Calemard L, Thouvenot D, Jagneaux V, Poitevin F, Malcus C, et al. Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibro‐

[127] Noone PG, Hohneker KW, Zhou Z, Johnson LG, Foy C, Gipson C, et al. Safety and biological efficacy of a lipid-CFTR complex for gene transfer in the nasal epithelium

[128] Zuckerman JB, Robinson CB, McCoy KS, Shell R, Sferra TJ, Chirmule N, et al. A phase I study of adenovirus-mediated transfer of the human cystic fibrosis trans‐ membrane conductance regulator gene to a lung segment of individuals with cystic

[129] Joseph PM, O'Sullivan BP, Lapey A, Dorkin H, Oren J, Balfour R, et al. Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications. Hum Gene Ther 2001;12(11):1369-82.

[130] Perricone MA, Morris JE, Pavelka K, Plog MS, O'Sullivan BP, Joseph PM, et al. Aero‐ sol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. II. Transfection efficiency in airway epithelium. Hum Gene Ther 2001;12(11):

sis patients: a phase I clinical trial. Hum Gene Ther 1997;8(1):15-25.

of adult patients with cystic fibrosis. Mol Ther 2000;1(1):105-14.

fibrosis. Hum Gene Ther 1999;10(18):2973-85.

with cystic fibrosis. N Engl J Med 1995;333(13):823-31.

615-39.

560 Gene Therapy - Tools and Potential Applications

1995;6(11):1487-96.

1383-94.

#### **1.1. Epidermolysis bullosa**

Epidermolysis bullosa (EB) is a genetically and clinically variable disease characterized by blis‐ ter formation and erosions of the skin and mucous membranes after minor trauma [1]. The in‐ heritance of the affected genes can occur in a dominant or recessive way depending on the subform of the disease. In general, epidermolysis bullosa is caused by mutations in genes en‐ coding structural proteins within the basal membrane zone of the skin. Absence or functional loss of one of these proteins results in a lack of stability of the microarchitecture of the connec‐ tion between dermis and epidermis leading to a loss of coherence [1]. The basement membrane between the dermis and the epidermis is a complex membrane produced by basal keratino‐ cytes and dermal fibroblasts that acts as mechanical support for the connection of both skin lay‐ ers. The basal membrane also regulates the metabolic exchange between the two skin compartments [2]. Up to date, there are at least 15 genes associated with EB causing different forms of the disease. Numerous mutations in these genes that encode for structural proteins within keratinocytes or within mucocutaneous basement membranes have been identified up to now [1].

Mutations in the genes, encoding for the keratins 5 and 14 and plectin, lead to epidermolysis bullosa simplex (EBS) characterized by the cytolysis within basal keratinocytes. Junctional epi‐ dermolysis bullosa (JEB) is caused by the absence or loss of function of laminin-332, type XVII collagen or integrin-β4. JEB is a severe EB form and is characterized by the separation of the skin within the lamina lucida. Mutations in type VII collagen (encoded by *COL7A1*) lead to the dystrophic form of epidermolysis bullosa, characterized by skin separation below the lamina densa. The severity and clinical manifestation of the disease depend on the mutation type (mis‐ sense mutation, nonsense mutation, splice site mutations, deletion or insertion), the mode of inheritance and the localization of the mutation within the gene. Due to this fact, diagnosis,

© 2013 Mayr et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

course of disease and therapy vary significantly depending on the present EB subform [3]. Blis‐ ter formation can be restricted to the soles of the feet or occur generalized. Severe systemic com‐ plications and extracutaneous manifestations including blistering and erosions of the cornea and mucosal tissues, stenoses or strictures of respiratory, gastrointestinal and urogenital tracts, pylorus atresia, muscular dystrophy and skin cancer are certain complications associated with different EB subtypes [1]. So far over 30 distinctive subtypes have been described and classified in a system, which was recently revised [4]. See Table 1.

JEB, non-Herlitz localized (JEB-nH loc) typeXVII collagen JEB with pyloric atresia (JEB-PA) α6β4 integrin *JEB, inversa (JEB-I)* laminin-332

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 563

*LOC syndrome (laryngo-onycho-cutaneous syndrome)* laminin-332 α3 chain ? α3 integrin

*JEB, late onset (JEB-lo)b*

Major DEB subtype Subtypes Affected protein DDEB DDEB, generalized (DDEB-gen) type VII collagen

RDEB RDEB, severe generalized (RDEB-sev gen)a type VII collagen RDEB, generalized other (RDEB-O)

*DDEB, bullous dermolysis of the newborn (DDEB-BDN)*

*RDEB, bullous dermolysis of the Newborn (RDEB-BDN)*

Mutations in the gene *COL7A1*, encoding for type VII collagen, cause the dystrophic form of epidermolysis bullosa (DEB). Type VII collagen is the major constituent of the basement membrane's anchoring fibrils and belongs to the superfamily of collagens [5]. *COL7A1* com‐ prises 118 exons and mostly short intervening introns resulting in a size of the entire *COL7A1* gene of 32kb encoding an mRNA of over 9kb [6,7]. The remarkable number of *COL7A1* mutations and the variable genotype-phenotype correlation hamper the finding of an optimal therapy for DEB patients. Nevertheless severity of clinical manifestations can of‐ ten be defined by the type of the mutation and its localization within the *COL7A1* gene [3]. DEB is divided into two main subtypes according to the mode of inheritance. Dominant dystrophic EB (DDEB) is inherited in an autosomal dominant way, whereas recessive dys‐ trophic EB (RDEB) is transmitted in an autosomal recessive mode [8]. RDEB is classified in

*DDEB, acral (DDEB-ac) DDEB, pretibial (DDEB-Pt) DDEB, pruriginosa (DDEB-Pr) DDEB, nails only (DDEB-no)*

*RDEB, inversa (RDEB-I) RDEB, pretibial (RDEB-Pt) RDEB pruriginosa (RDEB-Pr) RDEB, centripetalis (RDEB-Ce)*

**Table 1.** Classification system for inherited epidermolysis bullosa. Based on Fine et al. [4].

**2. Dystrophic epidermolysis bullosa (DEB)**

*(rare variants in italics)*

*(rare variants in italics)*

Previously called RDEB, Hallopeau-Siemens

a

b Formerly known as EB progressive

a Formerly known as generalized atrophic benign EB (GABEB)

**D: Classification scheme for all known dystrophic EB subtypes**


JEB, other (JEB-O) JEB, non-Herlitz, generalized (JEB-nH gen)<sup>a</sup> laminin-332, type XVII collagen


**Table 1.** Classification system for inherited epidermolysis bullosa. Based on Fine et al. [4].

#### **2. Dystrophic epidermolysis bullosa (DEB)**

course of disease and therapy vary significantly depending on the present EB subform [3]. Blis‐ ter formation can be restricted to the soles of the feet or occur generalized. Severe systemic com‐ plications and extracutaneous manifestations including blistering and erosions of the cornea and mucosal tissues, stenoses or strictures of respiratory, gastrointestinal and urogenital tracts, pylorus atresia, muscular dystrophy and skin cancer are certain complications associated with different EB subtypes [1]. So far over 30 distinctive subtypes have been described and classified

Basal EBS keratins 5 & 14; plectin,

Recessive DEB (RDEB) type VII collagen

*plakophilin deficiency* plakophilin-1

*EBS superficialis* ?

EBS, Dowling Meara (EBS-DM) K5, K14 EBS, other generalized (EBS,gen-nonDM)<sup>b</sup> K5, K14, BPAG1

*EBS with pylorus atresia (EBS-PA)* plectin, α6β4 integrin

*EBS with mottled pigmentation(EBS-MP)* K5 EBS with muscular dystrophy (EBS-MD) plectin

*EBS, autosomal recessive (EBS-AR)* K14 *EBS, ogna (EBS-Og)* plectin *EBS, migratory circinate (EBS-migr)* K5

JEB, other laminin-332, type XVII collagen

others?

α6β4 integrin, BPAG1

α6β4 integrin, α3 integrin

in a system, which was recently revised [4]. See Table 1.

Major EB type Major EB subtypes Affected proteins EB simplex (EBS) Suprabasal EBS plakophilin-1, desmoplakin;

Junctional EB (JEB) JEB, Herlitz (JEB-H) laminin-332, (laminin-5)

Dystrophic EB (DEB) Dominant DEB (DDEB) type VII collagen

Major types EBS subtypes Affected proteins EBS suprabasal *lethal acantholytic EB* desmoplakin

Major JEB subtype Subtypes Affected proteins JEB, Herlitz (JEB-H) laminin-332

JEB, other (JEB-O) JEB, non-Herlitz, generalized (JEB-nH gen)<sup>a</sup> laminin-332, type XVII collagen

Kindler syndrome kindlin-1

EBS basal EBS, localized (EBS-loc)a K5, K14

**A: Classification scheme for the major EB subtypes**

562 Gene Therapy - Tools and Potential Applications

**B: Classification scheme for all known EB simplex subtypes**

*(rare variants in italics)*

Previously called EBS, Weber-Cockayne

b Includes patients previously classified as EBS-Koebner **C: Classification scheme for all known junctional subtypes**

a

Mutations in the gene *COL7A1*, encoding for type VII collagen, cause the dystrophic form of epidermolysis bullosa (DEB). Type VII collagen is the major constituent of the basement membrane's anchoring fibrils and belongs to the superfamily of collagens [5]. *COL7A1* com‐ prises 118 exons and mostly short intervening introns resulting in a size of the entire *COL7A1* gene of 32kb encoding an mRNA of over 9kb [6,7]. The remarkable number of *COL7A1* mutations and the variable genotype-phenotype correlation hamper the finding of an optimal therapy for DEB patients. Nevertheless severity of clinical manifestations can of‐ ten be defined by the type of the mutation and its localization within the *COL7A1* gene [3]. DEB is divided into two main subtypes according to the mode of inheritance. Dominant dystrophic EB (DDEB) is inherited in an autosomal dominant way, whereas recessive dys‐ trophic EB (RDEB) is transmitted in an autosomal recessive mode [8]. RDEB is classified in severe generalized RDEB (RDEB-sev gen) – formerly called RDEB, Hallopeau Siemens - and RDEB-generalized other (RDEB-O) – formerly called RDEB-non Hallopeau Siemens [3].

ly supported by the so called basement membrane zone (BMZ). Moreover it regulates the metabolic exchange between these two compartments. Up to now more than 20 macromole‐ cules situated in the dermal-epidermal-junction have been detected and characterized at bio‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 565

Three protein-junction complexes stabilize the adherence of the basal keratinocytes to the dermis. See Figure 2. The hemidesmosomes built up by plectin, the bullous pemphigoid an‐ tigen 1 (BPAG1), α6β4 integrin and type XVII collagen (bullous pemphigoid antigen 2 - BPAG2) link the basal keratinocytes with the basement membrane, spanning the lamina lucida and anchored in the lamina densa [2]. Different laminin isoforms are located in the lamina lucida (laminin-332, laminin 6, laminin 10) and contribute along with BPAG2 to the formation of the anchoring filaments. The lamina densa is mainly built up by type VII colla‐ gen anchoring the lamina densa to the underlying dermis by the formation of anchoring fi‐ brils [2]. Some other antigens as uncein (19-DEJ-1 antigen), NU-T2 antigen, KF1 antigen, LDA1 antigen, nidogen, heparin-sulfate, proteoglycan, antigens AF1 and AF2, thrombo‐ spondin, type V collagen and osteonectin/BM-40 have been detected in the lamina densa but

**Figure 2.** Schematic setup of the cutaneous dermal-epidermal junction zone and localization of structural proteins affected in inherited EB (Diagram by R. Hametner). laminin 5 = laminin 332; EBS = epidermolysis bullosa simplex; JEB =

Type VII collagen is classified in the superfamily of collagens [7]. A protein domain in triplehelical conformation, which provides stability and integrity between connective tissues, is a common structural feature of all collagens [7]. Type VII collagen is a minor collagen in human

chemical and genomic level [2].

have not yet been adequately characterized [2].

junctional epidermolysis bullosa; DEB = dystrophic epidermolysis bullosa

**4. Type VII collagen**

The DDEB phenotype is mostly generalized but mild and clinically characterized by recurrent blistering, milia, atrophic scarring, nail dystrophy and eventual loss of nails [3]. See Figure 1A,B. The fact that the defective and wildtype alleles are expressed equally explains the rela‐ tive mild phenotype in comparison to RDEB [3]. Missense mutations or in frame deletions in *COL7A1* causing RDEB disturb the assembly and aggregation of type VII collagen into anchor‐ ing fibrils. As a result, the number of anchoring fibrils and their morphology is altered signifi‐ cantly. The resulting subforms of RDEB are classified as RDEB, generalized other [3]. RDEBsev gen is caused by nonsense mutations in both alleles, resulting in a complete loss of type VII collagen within the basal membrane zone of the skin. Clinical manifestations of RDEB are gen‐ eralized blistering, erosions, crusts, atrophic scarring, onychodystrophy, loss of nails, mutilat‐ ing pseudosyndactyly of hands and feet and functionally disabling contractures in hands, feet, elbows and knees. See Figure 1C-H. Additionally, severe extracutaneous complications as gas‐ trointestinal and urogenital tracts involvement, external eye, chronic anaemia, growth retarda‐ tion and a high risk for the development of aggressive squamous cell carcinoma decrease (Figure 1 I) the life quality of the patient [3,9-14].

**Figure 1.** Clinical phenotype of DEB. **A,B:** DDEB with milia formation and atrophy. **C:** Atrophic scar with crusts and erosions in RDEB. **D:** Boy with severe-generalized RDEB leading to ulcerations and large non healing wounds with atrophic scarring at the back; **E:** Nail dystrophy on both feet. **F,G:** Mitten formation in hands and feet. **H:** Severe caries **I:** Squamous cell carcinoma on the foot (Photos: R. Hametner)

#### **3. The dermal epidermal junction**

The blisters characteristic for EB arise within the dermal-epidermal junction. Having a look at this compartment of the skin helps to understand the cause of blistering in EB. The der‐ mal-epidermal junction is a complex basement membrane synthesized by dermal fibroblasts and basal keratinocytes. Adhesion of the epidermis to the underlying dermis is mechanical‐ ly supported by the so called basement membrane zone (BMZ). Moreover it regulates the metabolic exchange between these two compartments. Up to now more than 20 macromole‐ cules situated in the dermal-epidermal-junction have been detected and characterized at bio‐ chemical and genomic level [2].

Three protein-junction complexes stabilize the adherence of the basal keratinocytes to the dermis. See Figure 2. The hemidesmosomes built up by plectin, the bullous pemphigoid an‐ tigen 1 (BPAG1), α6β4 integrin and type XVII collagen (bullous pemphigoid antigen 2 - BPAG2) link the basal keratinocytes with the basement membrane, spanning the lamina lucida and anchored in the lamina densa [2]. Different laminin isoforms are located in the lamina lucida (laminin-332, laminin 6, laminin 10) and contribute along with BPAG2 to the formation of the anchoring filaments. The lamina densa is mainly built up by type VII colla‐ gen anchoring the lamina densa to the underlying dermis by the formation of anchoring fi‐ brils [2]. Some other antigens as uncein (19-DEJ-1 antigen), NU-T2 antigen, KF1 antigen, LDA1 antigen, nidogen, heparin-sulfate, proteoglycan, antigens AF1 and AF2, thrombo‐ spondin, type V collagen and osteonectin/BM-40 have been detected in the lamina densa but have not yet been adequately characterized [2].

**Figure 2.** Schematic setup of the cutaneous dermal-epidermal junction zone and localization of structural proteins affected in inherited EB (Diagram by R. Hametner). laminin 5 = laminin 332; EBS = epidermolysis bullosa simplex; JEB = junctional epidermolysis bullosa; DEB = dystrophic epidermolysis bullosa

#### **4. Type VII collagen**

severe generalized RDEB (RDEB-sev gen) – formerly called RDEB, Hallopeau Siemens - and RDEB-generalized other (RDEB-O) – formerly called RDEB-non Hallopeau Siemens [3].

The DDEB phenotype is mostly generalized but mild and clinically characterized by recurrent blistering, milia, atrophic scarring, nail dystrophy and eventual loss of nails [3]. See Figure 1A,B. The fact that the defective and wildtype alleles are expressed equally explains the rela‐ tive mild phenotype in comparison to RDEB [3]. Missense mutations or in frame deletions in *COL7A1* causing RDEB disturb the assembly and aggregation of type VII collagen into anchor‐ ing fibrils. As a result, the number of anchoring fibrils and their morphology is altered signifi‐ cantly. The resulting subforms of RDEB are classified as RDEB, generalized other [3]. RDEBsev gen is caused by nonsense mutations in both alleles, resulting in a complete loss of type VII collagen within the basal membrane zone of the skin. Clinical manifestations of RDEB are gen‐ eralized blistering, erosions, crusts, atrophic scarring, onychodystrophy, loss of nails, mutilat‐ ing pseudosyndactyly of hands and feet and functionally disabling contractures in hands, feet, elbows and knees. See Figure 1C-H. Additionally, severe extracutaneous complications as gas‐ trointestinal and urogenital tracts involvement, external eye, chronic anaemia, growth retarda‐ tion and a high risk for the development of aggressive squamous cell carcinoma decrease

**Figure 1.** Clinical phenotype of DEB. **A,B:** DDEB with milia formation and atrophy. **C:** Atrophic scar with crusts and erosions in RDEB. **D:** Boy with severe-generalized RDEB leading to ulcerations and large non healing wounds with atrophic scarring at the back; **E:** Nail dystrophy on both feet. **F,G:** Mitten formation in hands and feet. **H:** Severe caries

The blisters characteristic for EB arise within the dermal-epidermal junction. Having a look at this compartment of the skin helps to understand the cause of blistering in EB. The der‐ mal-epidermal junction is a complex basement membrane synthesized by dermal fibroblasts and basal keratinocytes. Adhesion of the epidermis to the underlying dermis is mechanical‐

(Figure 1 I) the life quality of the patient [3,9-14].

564 Gene Therapy - Tools and Potential Applications

**I:** Squamous cell carcinoma on the foot (Photos: R. Hametner)

**3. The dermal epidermal junction**

Type VII collagen is classified in the superfamily of collagens [7]. A protein domain in triplehelical conformation, which provides stability and integrity between connective tissues, is a common structural feature of all collagens [7]. Type VII collagen is a minor collagen in human skin and demonstrates spatially restricted location but it plays a critical role in providing inte‐ gral stability to the skin because it is the major component of the anchoring fibrils [6,7].

bers of these anti-parallel dimers aggregate laterally to form anchoring fibrils, which then can be identified by their characteristic, centro-symmetric banding patterns in transmission elec‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 567

The affinity of the NC-1 domain to bind the principal components of the cutaneous base‐ ment membrane, laminin-332, laminin-311 and type IV collagen provides stability to the dermo-epidermal adhesion on the dermal site at the lamina lucida/papillary dermis inter‐ face [6,24,25]. Arg-Gly-Asp sequences in the NC-1 domain serve as integrin mediated at‐ tachment sites for cells to adhere to extracellular matrix components such as fibronectin [26].

Mutations in *COL7A1* have clinical consequences in terms of disrupted integrity of the skin, due to the complexity of the *COL7A1* gene, type VII collagen protein structures and the criti‐ cal importance of its distinct domains in macromolecular interactions [7]. At least 324 patho‐ genic mutations have been detected within *COL7A1* in different variants of DEB up to now including 43 nonsense, 127 missense, 65 deletion, 28 insertion, 9 insertion-deletion, 51 splicesite and 1 regulatory mutations [27]. See Figure 3-5. Exon 73 constitutes a region with a high frequency of mutations, what suggests being a region in which mutations commonly affect the function of anchoring fibrils [28]. RDEB is caused by nonsense, splice-site, deletions or insertions, silent glycine substitutions within the triple helix and non-glycine missense mu‐ tations within the triple-helix or non-collagenous NC-2 domain [29]. RDEB-severe general‐ ized originates from nonsense, frameshift or splice-site mutations on both alleles leading to premature termination codons (PTCs) [30], which result in nonsense mediated mRNA decay or truncated proteins, leading to a reduced number of collagen VII monomers, which are unable to assemble into functional anchoring fibrils [29,30]. PTC mutations do not cause a clinical phenotype if they appear in the heterozygous state, but if they are homozygous or combined with another PTC mutation they are causing severe generalized RDEB [27]. Two missense mutations or compound heterozygosity of a missense and a PTC mutation lead to

RDEB, generalized other, the milder phenotype, is mostly caused by PTCs, small deletions, substitutions of glycine residues in the collagenous domain, splice-site mutations within NC-2 [32-35], delayed termination codons [36], in frame exon skipping [29,36], or missense substitution mutations involving amino acids other than glycine [29,37,38], the majority in‐ volving arginine residues resulting either in the loss of an ionic charge or in the introduction of a bulky chain at an external position of the triple helix [27]. Thereby these mutations usu‐ ally concern a critical amino acid and change the conformation of the protein, which then might still be able to assemble into a small number of anchoring fibrils but is likely to be unstable when they laterally aggregate. Anyhow some full length type VII collagen poly‐

DDEB is caused by glycine substitutions within the triple helical domain of *COL7A1* or other missense mutations, deletions or splice site mutations in some cases [5,26,40-44]. Critical ami‐

tron microscopy [7].

**7. Mutations in** *COL7A1*

severe generalized RDEB in very rare cases [31].

peptides can still be built up [39].

#### **5. Biology of type VII collagen**

Type VII collagen molecules are characterized by the two non-collagenous NC-1 and NC-2 domains flanking a central collagenous, triple-helical segment [7]. In contrast to other inter‐ stitial collagens the repeating Gly-X-Y collagenous sequence is interrupted by 19 imperfec‐ tions due to insertions or deletions of amino acids. There is a 39 amino acid non-collagenous hinge region susceptible to proteolytic digestion with pepsin in the middle of the triple-heli‐ cal domain [15]. The amino terminal NC-1 domain (approximately 145kD in size), is built up of sub-modules with homology to known adhesive proteins, including segments with ho‐ mology to cartilage matrix protein (CMP), nine consecutive fibronectin type III-like (FN-III) domains, a segment with homology to the A domain of von Willebrand factor, and a short cysteine and proline-rich region [15]. The C-terminal non-collagenous NC-2 domain is with 30kD in size relatively small, and contains a segment with homology to Kuniz protease in‐ hibitor molecule [16,17].

The 32kb gene encoding a 9,2kb mRNA has been mapped to the short-arm of chromosome 3p21.1 [18]. The encoding primary sequence and the gene structure of type VII collagen are well conserved. The mouse gene shows 90.4% identity at the protein level and 84.7% homology at the nucleotide level, indicating the importance of type VII collagen as a structural protein [19].

The expression pattern of *COL7A1* is tissue specific and restricted. Type VII collagen has been detected by immunomapping to a selected number of epithelia, including the dermalepidermal BMZ of skin, the amniotic epithelial BMZ of the chorioamnion, the corneal epi‐ thelial basement membrane (Bowman's membrane) and the epithelial basement membrane of oral mucosa and cervix. Moreover the presence of type VII collagen correlates with the presence of ultrastructurally detected anchoring fibrils [6]. A number of cytokines modulate type VII collagen expression. Especially transforming growth factor-β is a powerful upregu‐ lator of *COL7A1* at transcription level in fibroblasts and keratinocytes [20,21].

#### **6. Type VII collagen – A major component of the anchoring fibrils**

Type VII collagen is synthesized by two cell types in the skin: keratinocytes and fibroblasts [22]. After synthesis of complete pro-α1 (VII) polypeptides, three polypeptides are associated through their carboxy-terminal ends to a trimer molecule, which is then folded in its collage‐ nous segment into the triple-helical formation. Past to secretion into the extracellular milieu two type VII collagen molecules are aligned into an anti-parallel dimer with the amino-termi‐ nal domains present at both ends of the molecule [6]. During dimer-assembly stabilization by inter-molecular disulfide bond formation and a proteolytic removal of a part of the carboxyterminal ends (NC-2 domain) of both type VII collagen molecules take place [23]. Large num‐ bers of these anti-parallel dimers aggregate laterally to form anchoring fibrils, which then can be identified by their characteristic, centro-symmetric banding patterns in transmission elec‐ tron microscopy [7].

The affinity of the NC-1 domain to bind the principal components of the cutaneous base‐ ment membrane, laminin-332, laminin-311 and type IV collagen provides stability to the dermo-epidermal adhesion on the dermal site at the lamina lucida/papillary dermis inter‐ face [6,24,25]. Arg-Gly-Asp sequences in the NC-1 domain serve as integrin mediated at‐ tachment sites for cells to adhere to extracellular matrix components such as fibronectin [26].

#### **7. Mutations in** *COL7A1*

skin and demonstrates spatially restricted location but it plays a critical role in providing inte‐

Type VII collagen molecules are characterized by the two non-collagenous NC-1 and NC-2 domains flanking a central collagenous, triple-helical segment [7]. In contrast to other inter‐ stitial collagens the repeating Gly-X-Y collagenous sequence is interrupted by 19 imperfec‐ tions due to insertions or deletions of amino acids. There is a 39 amino acid non-collagenous hinge region susceptible to proteolytic digestion with pepsin in the middle of the triple-heli‐ cal domain [15]. The amino terminal NC-1 domain (approximately 145kD in size), is built up of sub-modules with homology to known adhesive proteins, including segments with ho‐ mology to cartilage matrix protein (CMP), nine consecutive fibronectin type III-like (FN-III) domains, a segment with homology to the A domain of von Willebrand factor, and a short cysteine and proline-rich region [15]. The C-terminal non-collagenous NC-2 domain is with 30kD in size relatively small, and contains a segment with homology to Kuniz protease in‐

The 32kb gene encoding a 9,2kb mRNA has been mapped to the short-arm of chromosome 3p21.1 [18]. The encoding primary sequence and the gene structure of type VII collagen are well conserved. The mouse gene shows 90.4% identity at the protein level and 84.7% homology at the nucleotide level, indicating the importance of type VII collagen as a structural protein [19]. The expression pattern of *COL7A1* is tissue specific and restricted. Type VII collagen has been detected by immunomapping to a selected number of epithelia, including the dermalepidermal BMZ of skin, the amniotic epithelial BMZ of the chorioamnion, the corneal epi‐ thelial basement membrane (Bowman's membrane) and the epithelial basement membrane of oral mucosa and cervix. Moreover the presence of type VII collagen correlates with the presence of ultrastructurally detected anchoring fibrils [6]. A number of cytokines modulate type VII collagen expression. Especially transforming growth factor-β is a powerful upregu‐

lator of *COL7A1* at transcription level in fibroblasts and keratinocytes [20,21].

**6. Type VII collagen – A major component of the anchoring fibrils**

Type VII collagen is synthesized by two cell types in the skin: keratinocytes and fibroblasts [22]. After synthesis of complete pro-α1 (VII) polypeptides, three polypeptides are associated through their carboxy-terminal ends to a trimer molecule, which is then folded in its collage‐ nous segment into the triple-helical formation. Past to secretion into the extracellular milieu two type VII collagen molecules are aligned into an anti-parallel dimer with the amino-termi‐ nal domains present at both ends of the molecule [6]. During dimer-assembly stabilization by inter-molecular disulfide bond formation and a proteolytic removal of a part of the carboxyterminal ends (NC-2 domain) of both type VII collagen molecules take place [23]. Large num‐

gral stability to the skin because it is the major component of the anchoring fibrils [6,7].

**5. Biology of type VII collagen**

566 Gene Therapy - Tools and Potential Applications

hibitor molecule [16,17].

Mutations in *COL7A1* have clinical consequences in terms of disrupted integrity of the skin, due to the complexity of the *COL7A1* gene, type VII collagen protein structures and the criti‐ cal importance of its distinct domains in macromolecular interactions [7]. At least 324 patho‐ genic mutations have been detected within *COL7A1* in different variants of DEB up to now including 43 nonsense, 127 missense, 65 deletion, 28 insertion, 9 insertion-deletion, 51 splicesite and 1 regulatory mutations [27]. See Figure 3-5. Exon 73 constitutes a region with a high frequency of mutations, what suggests being a region in which mutations commonly affect the function of anchoring fibrils [28]. RDEB is caused by nonsense, splice-site, deletions or insertions, silent glycine substitutions within the triple helix and non-glycine missense mu‐ tations within the triple-helix or non-collagenous NC-2 domain [29]. RDEB-severe general‐ ized originates from nonsense, frameshift or splice-site mutations on both alleles leading to premature termination codons (PTCs) [30], which result in nonsense mediated mRNA decay or truncated proteins, leading to a reduced number of collagen VII monomers, which are unable to assemble into functional anchoring fibrils [29,30]. PTC mutations do not cause a clinical phenotype if they appear in the heterozygous state, but if they are homozygous or combined with another PTC mutation they are causing severe generalized RDEB [27]. Two missense mutations or compound heterozygosity of a missense and a PTC mutation lead to severe generalized RDEB in very rare cases [31].

RDEB, generalized other, the milder phenotype, is mostly caused by PTCs, small deletions, substitutions of glycine residues in the collagenous domain, splice-site mutations within NC-2 [32-35], delayed termination codons [36], in frame exon skipping [29,36], or missense substitution mutations involving amino acids other than glycine [29,37,38], the majority in‐ volving arginine residues resulting either in the loss of an ionic charge or in the introduction of a bulky chain at an external position of the triple helix [27]. Thereby these mutations usu‐ ally concern a critical amino acid and change the conformation of the protein, which then might still be able to assemble into a small number of anchoring fibrils but is likely to be unstable when they laterally aggregate. Anyhow some full length type VII collagen poly‐ peptides can still be built up [39].

DDEB is caused by glycine substitutions within the triple helical domain of *COL7A1* or other missense mutations, deletions or splice site mutations in some cases [5,26,40-44]. Critical ami‐ no acids in the structure of the triple helix are affected by these mutations and therefore the overall stability of the anchoring fibrils is disturbed. More than 100 missense mutations result‐ ing in a Gly-Xaa substitution have been detected in the collagenous domain of *COL7A1*; half of these are situated in amino acids 1522-2791 and have a dominant negative effect [27].

**Figure 3. Missense and nonsense mutations in DEB patients.** The red lettering signifies dominant and the black signifies recessive inheritance. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

**Figure 5. Glycine substitutions in DEB.** These are all in the triple-helical collagenous domain; the ones above repre‐ sent DDEB, the ones below RDEB. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 569

So far there are only two viable mouse models with defects in the *COL7A1* gene. A transgen‐ ic mouse carrying human *COL7A1* cDNA inclusive the human 7528delG mutation in exon 101, which develops the DEB phenotype gradually [45], and a collagen VII hypomorphic mouse published by Fritsch et al. 2008 [46]. In the collagen VII hypomorphic mouse reduced expression of collagen VII originates from aberrant splicing resulting from the introduction of a phosphoglycerate kinase promoter-driven neomycin phosphotransferase expression cassette (PGK-Neo cassette) in intron 2 of *COL7A1.* One out of three possible splice variants is translated into full-length type VII collagen resulting in a reduction of type VII collagen levels to about 9% of wildtype levels in *COL7A1*flNeo/flNeo mice. Hemorrhagic blisters on the soles of fore and hind paws, ears and mouth are developed by the collagen VII mice within the first 48 hours of life. Blisters of newborn *COL7A1*flNeo/flNeo mice were histopathologically classified as hemorrhagic and subepidermal. Type VII collagen immunofluorescence stain‐ ing revealed weak reactivity in comparison to wildtype littermates. Ultrastructurally normal but reduced in number anchoring fibrils were detected in transmission electron microscopy of the dermal-epidermal junction of the skin. *COL7A1*flNeo/flNeo mice suffer from growth retar‐ dation due to malnutrition and subsequently have a reduced life expectancy. Moreover healing of the initial blistering on the paws with scarring results in the development of mit‐

**8. Mouse model**

ten deformities beginning at 2-3 weeks of age [46].

**Figure 4.** *COL7A1* **deletions, insertions and splice site mutations in DEB patients**. The red lettering signifies dominant and the black signifies recessive inheritance. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

**Figure 5. Glycine substitutions in DEB.** These are all in the triple-helical collagenous domain; the ones above repre‐ sent DDEB, the ones below RDEB. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

#### **8. Mouse model**

no acids in the structure of the triple helix are affected by these mutations and therefore the overall stability of the anchoring fibrils is disturbed. More than 100 missense mutations result‐ ing in a Gly-Xaa substitution have been detected in the collagenous domain of *COL7A1*; half of

**Figure 3. Missense and nonsense mutations in DEB patients.** The red lettering signifies dominant and the black

**Figure 4.** *COL7A1* **deletions, insertions and splice site mutations in DEB patients**. The red lettering signifies dominant and the black signifies recessive inheritance. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

signifies recessive inheritance. (Dang et al. [27] © 2008 Blackwell Munksgaard, Experimental Dermatology)

these are situated in amino acids 1522-2791 and have a dominant negative effect [27].

568 Gene Therapy - Tools and Potential Applications

So far there are only two viable mouse models with defects in the *COL7A1* gene. A transgen‐ ic mouse carrying human *COL7A1* cDNA inclusive the human 7528delG mutation in exon 101, which develops the DEB phenotype gradually [45], and a collagen VII hypomorphic mouse published by Fritsch et al. 2008 [46]. In the collagen VII hypomorphic mouse reduced expression of collagen VII originates from aberrant splicing resulting from the introduction of a phosphoglycerate kinase promoter-driven neomycin phosphotransferase expression cassette (PGK-Neo cassette) in intron 2 of *COL7A1.* One out of three possible splice variants is translated into full-length type VII collagen resulting in a reduction of type VII collagen levels to about 9% of wildtype levels in *COL7A1*flNeo/flNeo mice. Hemorrhagic blisters on the soles of fore and hind paws, ears and mouth are developed by the collagen VII mice within the first 48 hours of life. Blisters of newborn *COL7A1*flNeo/flNeo mice were histopathologically classified as hemorrhagic and subepidermal. Type VII collagen immunofluorescence stain‐ ing revealed weak reactivity in comparison to wildtype littermates. Ultrastructurally normal but reduced in number anchoring fibrils were detected in transmission electron microscopy of the dermal-epidermal junction of the skin. *COL7A1*flNeo/flNeo mice suffer from growth retar‐ dation due to malnutrition and subsequently have a reduced life expectancy. Moreover healing of the initial blistering on the paws with scarring results in the development of mit‐ ten deformities beginning at 2-3 weeks of age [46].

#### **9. Therapy approaches**

Due to the size of the *COL7A1* gene, a causal therapy for dystrophic epidermolysis bullosa is a great challenge. Symptomatic therapy is concentrated on prevention of skin trauma to minimize blister formation, prevention of secondary bacterial infection, treatment of infec‐ tion, measures to improve wound healing, maintenance of good nutrition, treatment of cor‐ rectable complications, and finally rehabilitation [3]. However, several gene and cell therapy strategies showed the potential to revert the disease-associated phenotype. Phenotypic cor‐ rection of recessive DEB forms (RDEB) can be achieved by gene insertion therapy, in which the wildtype sequence of a mutated gene of interest is introduced into the target cells. More‐ over alternative avenues including gene-, cell-, protein- and other systemic- therapy ap‐ proaches have been tested to restore type VII collagen expression. See Table 2.

Woodley et al. used a type VII collagen minigene, which contains the intact noncollage‐ nous domains NC1 and NC2 and part of the central collagenous domain. This approach resulted after transduction into DEB keratinocytes in persistent synthesis and secretion of a 230kDa recombinant minicollagen VII [47]. However deletions in *COL7A1* have been reported to be associated with a pathologic phenotype [5,48,49]. The same group intro‐ duced recombinant human type VII collagen into mouse and human skin equivalents transplanted onto mice, by injection. As a result the injected type VII collagen was de‐ tected within the basal membrane zone leading to a reversion of the disease associated phenotype [50]. Additionally, the group expressed type VII collagen using a self-inacti‐ vating lentiviral vector, which was injected into human skin equivalents, expanded from DEB cells, placed on nude immunodeficient mice. Experiments revealed the synthesis and insertion of the protein into the basal membrane zone [51]. Using a cosmid clone, carrying the entire *COL7A1* gene, was also shown to be a promising way to direct ex‐ pression of type VII collagen in skin in fetal and neonatal mice. The tested neonatal or fetal mice produced type VII collagen within the basal membrane zone of the skin show‐ ing a stable expression of the protein *in vivo* [52]. Microinjection of a P1-derived artificial chromosome (PAC) carrying the entire *COL7A1* locus resulted in production of a procol‐ lagen VII similar to the authentic one by Mecklenbeck et al. [53]. The ΦC31 bacterio‐ phage integrase, facilitating integration only in pseudo attP sites, was used to integrate *COL7A1* stably into DEB primary epidermal progenitor cells by Urda et al. [54]. Balde‐ schi et al. also showed sustained and permanent expression of the transgene after trans‐ duction of canine type VII collagen into human and canine DEB keratinocytes [55]. Based on this study Gache et al. yield a full phenotypic reversion of the disease-associated phe‐ notype of RDEB epidermal clonogenic cells after full-length human *COL7A1* cDNA intro‐ duction using a retroviral system. However, the expression of *COL7A1* was 50 times higher than the levels monitored in wildtype keratinocytes in monolayers, increasing the risk for an ectopic transgene expression and an abnormal accumulation in skin equiva‐ lents [56]. Chen et al. used a minimal lentiviral vector for *COL7A1* expression *in vitro* as an alternative to the retroviral system applied by Gache et al. [57]. Goto et al. showed that *COL7A1* treated fibroblasts of skin grafts provide higher amounts of type VII colla‐ gen for the dermal-epidermal junction than keratinocytes [58]. They have also demon‐ strated an antisense oligoribonucleotide therapy to maintain exon skipping of an exon comprising a premature stop codon. As a result a truncated type VII collagen variant was expressed [59]. Additionally, intradermal injection of untreated normal human or gene-corrected fibroblasts in mice can result in a stable production of human type VII collagen at the basal membrane zone of the skin [60]. Moreover, Wong et al. demonstrat‐ ed an increased source of type VII collagen in the dermal-epidermal junction for at least three months after intradermal injection of allogeneic fibroblasts [61]. In a mouse model Fritsch et al. showed an accumulation of type VII collagen and restoration of a function‐ al dermal-epidermal junction after injection of murine wildtype fibroblasts into a type VII collagen hypomorphic mouse [46]. In 2009 Remington et al. injected human type VII collagen into *COL7A1* -/- mice, also restoring type VII collagen expression and correct generation of anchoring fibrils [62]. Titeux et al. transduced *COL7A1* cDNA under the

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 571


**Table 2.** Therapy approaches to restore type VII collagen expression

Woodley et al. used a type VII collagen minigene, which contains the intact noncollage‐ nous domains NC1 and NC2 and part of the central collagenous domain. This approach resulted after transduction into DEB keratinocytes in persistent synthesis and secretion of a 230kDa recombinant minicollagen VII [47]. However deletions in *COL7A1* have been reported to be associated with a pathologic phenotype [5,48,49]. The same group intro‐ duced recombinant human type VII collagen into mouse and human skin equivalents transplanted onto mice, by injection. As a result the injected type VII collagen was de‐ tected within the basal membrane zone leading to a reversion of the disease associated phenotype [50]. Additionally, the group expressed type VII collagen using a self-inacti‐ vating lentiviral vector, which was injected into human skin equivalents, expanded from DEB cells, placed on nude immunodeficient mice. Experiments revealed the synthesis and insertion of the protein into the basal membrane zone [51]. Using a cosmid clone, carrying the entire *COL7A1* gene, was also shown to be a promising way to direct ex‐ pression of type VII collagen in skin in fetal and neonatal mice. The tested neonatal or fetal mice produced type VII collagen within the basal membrane zone of the skin show‐ ing a stable expression of the protein *in vivo* [52]. Microinjection of a P1-derived artificial chromosome (PAC) carrying the entire *COL7A1* locus resulted in production of a procol‐ lagen VII similar to the authentic one by Mecklenbeck et al. [53]. The ΦC31 bacterio‐ phage integrase, facilitating integration only in pseudo attP sites, was used to integrate *COL7A1* stably into DEB primary epidermal progenitor cells by Urda et al. [54]. Balde‐ schi et al. also showed sustained and permanent expression of the transgene after trans‐ duction of canine type VII collagen into human and canine DEB keratinocytes [55]. Based on this study Gache et al. yield a full phenotypic reversion of the disease-associated phe‐ notype of RDEB epidermal clonogenic cells after full-length human *COL7A1* cDNA intro‐ duction using a retroviral system. However, the expression of *COL7A1* was 50 times higher than the levels monitored in wildtype keratinocytes in monolayers, increasing the risk for an ectopic transgene expression and an abnormal accumulation in skin equiva‐ lents [56]. Chen et al. used a minimal lentiviral vector for *COL7A1* expression *in vitro* as an alternative to the retroviral system applied by Gache et al. [57]. Goto et al. showed that *COL7A1* treated fibroblasts of skin grafts provide higher amounts of type VII colla‐ gen for the dermal-epidermal junction than keratinocytes [58]. They have also demon‐ strated an antisense oligoribonucleotide therapy to maintain exon skipping of an exon comprising a premature stop codon. As a result a truncated type VII collagen variant was expressed [59]. Additionally, intradermal injection of untreated normal human or gene-corrected fibroblasts in mice can result in a stable production of human type VII collagen at the basal membrane zone of the skin [60]. Moreover, Wong et al. demonstrat‐ ed an increased source of type VII collagen in the dermal-epidermal junction for at least three months after intradermal injection of allogeneic fibroblasts [61]. In a mouse model Fritsch et al. showed an accumulation of type VII collagen and restoration of a function‐ al dermal-epidermal junction after injection of murine wildtype fibroblasts into a type VII collagen hypomorphic mouse [46]. In 2009 Remington et al. injected human type VII collagen into *COL7A1* -/- mice, also restoring type VII collagen expression and correct generation of anchoring fibrils [62]. Titeux et al. transduced *COL7A1* cDNA under the

**9. Therapy approaches**

570 Gene Therapy - Tools and Potential Applications

Due to the size of the *COL7A1* gene, a causal therapy for dystrophic epidermolysis bullosa is a great challenge. Symptomatic therapy is concentrated on prevention of skin trauma to minimize blister formation, prevention of secondary bacterial infection, treatment of infec‐ tion, measures to improve wound healing, maintenance of good nutrition, treatment of cor‐ rectable complications, and finally rehabilitation [3]. However, several gene and cell therapy strategies showed the potential to revert the disease-associated phenotype. Phenotypic cor‐ rection of recessive DEB forms (RDEB) can be achieved by gene insertion therapy, in which the wildtype sequence of a mutated gene of interest is introduced into the target cells. More‐ over alternative avenues including gene-, cell-, protein- and other systemic- therapy ap‐

proaches have been tested to restore type VII collagen expression. See Table 2.

**Author Approach Year** Woodley et al. Type VII collagen minigene 2000 Sat et al. Cosmid clone containing the entire *COL7A1* gene 2000 Mecklenbeck et al. Microinjection of a *COL7A1*-PAC vector 2002 Urda et al. ФC31 bacteriophage integrase 2002 Chen et al. Minimal lentiviral vectors 2002 Baldeschi et al Canine type VII collagen 2003 Woodley et al Targeting fibroblasts instead of keratinocytes (lentivirally) 2003 Gache et al. Full-length cDNA (retrovirally) 2004 Woodley et al. Intradermal injection of recombinant type VII collagen 2004 Woodley et al. Intradermal injection of lentiviral vectors in vivo 2006 Goto et al. Targeting fibroblasts instead of keratinocytes (retrovirally) 2006 Goto et al. Targeted exon skipping using antisense 2006 Wong et al. Intradermal injection of allogenic wildtype fibroblasts into a patient 2007 Fritsch et al. Intradermal injection of murine wildtype fibroblasts in a DEB mouse model 2008 Remington et al. Intradermal injection of human type VII collagen in mice 2009

Titeux et al. Minimal self-inactivating retroviral vectors harbouring the full length human

Wagner et al. Allogeneic bone marrow transplantation 2010 Siprashvili et al. Full-length cDNA (retrovirally) 2010 Murauer et al. 3´ Trans-splicing of *COL7A1* 2011

2010

*COL7A1* gene

**Table 2.** Therapy approaches to restore type VII collagen expression

control of a human promoter using a minimal self-inactivating retroviral vector into RDEB keratinocytes and fibroblasts leading to cell correction and long lasting expression of type VII collagen. The dermal-epidermal junction in generated skin equivalents was restored [63]. A similar strategy was shown by Siprashvili et al. using an epitope-tagged *COL7A1* cDNA, providing a long term expression of the protein in skin equivalents [64]. In a clinical trial executing a bone marrow transplantation 6 RDEB patients received allo‐ geneic stem cells to milder the RDEB phenotype. As a result, 5 patients showed an im‐ proved wound healing, but one patient died [65].

splicing event between the target pre-mRNA and the RTM which is mediated by the spliceosome. An RTM carries three domains; i) a binding domain complementary to the target intron to localize the RTM to the target pre-mRNA; ii) a splicing domain contain‐ ing splicing elements for efficient *trans*-splicing; and iii) a coding domain comprising one or more wildtype exons that are *trans*-spliced to the target. The *cis*-splicing elements and the binding domain are not retained in the modified RNA product [71]. Depending on the gene portion to replace, SMaRT can be divided into 3´, 5´ or internal exon replace‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 573

**Figure 6.** Schematic overview on different applications of SMaRT. **A:3`***Trans***-splicing:** If there is a mutation in the 3´ part of the target gene a wildtype mRNA can be obtained by 3´splicing. Therefore, a 3´RTM with a binding domain situated in the intron 5´ to the first exon to be exchanged is necessary. E.g. if the mutation to be corrected is in exon 15, a binding domain for intron 14 is designed. This RTM can correct mutations more 3´ as well. After binding of the RTM the two mRNAs are *trans-*spliced and combined into a wildtype mRNA. **B: 5`***Trans***-splicing:** If correction of a mutation in the 5´ part of a gene is desired a 5´ RTM with a binding domain located in the intron 3´ to the exon to be exchanged is created. If the mutation to be corrected with 5´splicing is in exon 15, a RTM with a binding domain in intron 15 is required. This RTM can repair mutations more 5´ than exon 15 as well. **C: Internal** *Trans***-splicing:** There is also a method to exchange only one exon, called internal *trans*-splicing or internal exon replacement (IER). Here an

The efficiency of *trans*-splicing to correct genetic defects and acquired disorders at premRNA level has already been demonstrated for 3´ as well as for 5´ *trans*-splicing in different

RTM with two binding domains and 5´ and 3´ splice elements is applied. Arrowheads indicate mutations.

ment [69]. See Figure 6.

**10.3. Efficiency of SMaRT**

diseases *in vitro* and *in vivo*. See Table 3.

Until now, no *ex vivo* gene therapy approach passed through a phase I/II gene therapy trial. Most of these applications are focusing on the transfer of full-length *COL7A1* cDNA into the affected patient cells. The drawbacks of the insertion of the full-length 9kb cDNA of *COL7A1* are the cloning and packaging limitations of commonly used vector systems to transduce keratinocytes or fibroblasts and the instability of the *COL7A1* gene due to possible genetic rearrangements of the large repetitive cDNA sequence [47]. Additionally, the influence of *COL7A1* over- or ectopic expression in treated cells has to be clarified for a clinical applica‐ tion. Using the methodology of spliceosome mediated RNA *Trans*-splicing (SMaRT) can be a promising alternative to the mentioned approaches to cope with some of the suspected is‐ sues present in full-length *COL7A1* replacement strategies. Murauer et al. demonstrated the exchange of the 3´ coding *COL7A1* cDNA region spanning from exon 65 to the last exon 118 by SMaRT [66]. Thereby the risk of genetic rearrangements of the *COL7A1* cDNA sequence should be reduced significantly. Alternatively to this, we will present in this work a 5´ exon replacement strategy using SMaRT, providing the possibility to repair also relevant muta‐ tions 5´ within the *COL7A1* gene.

#### **10. Spliceosome mediated mRNA** *Trans***-splicing**

#### **10.1. General aspects**

RNA *trans*-splicing is a naturally occurring event to recombine two or more mRNA mole‐ cules to a new chimeric gene product [67]. For therapeutic purposes such products can be generated by *trans*-splicing a second RNA species from a RNA *trans*-splicing molecule (RTM) into the 3´, 5´ or internal sequence of an endogenously expressed target. See Figure 6. The main advantages of this methodology are the possibility to reduce the size of the trans‐ gene, the maintained endogenous regulation of transgene expression and the feasibility to treat dominant negative diseases [68]. Undesired gene expression due to unintended deliv‐ ery or misregulation is minimized as *trans*-splicing should only occur in cells expressing the target pre-mRNA [69]. Furthermore, SMaRT offers the potential for correction of dominant negative mutations into wildtype gene products [70].

#### **10.2. Methodology of spliceosome mediated mRNA** *trans***-splicing (SMaRT)**

In SMaRT constructs that are engineered to bind the introns of specific pre-mRNAs – RNA *trans*-splicing molecules (RTMs) - are the key players. These RTMs effect a *trans*- splicing event between the target pre-mRNA and the RTM which is mediated by the spliceosome. An RTM carries three domains; i) a binding domain complementary to the target intron to localize the RTM to the target pre-mRNA; ii) a splicing domain contain‐ ing splicing elements for efficient *trans*-splicing; and iii) a coding domain comprising one or more wildtype exons that are *trans*-spliced to the target. The *cis*-splicing elements and the binding domain are not retained in the modified RNA product [71]. Depending on the gene portion to replace, SMaRT can be divided into 3´, 5´ or internal exon replace‐ ment [69]. See Figure 6.

**Figure 6.** Schematic overview on different applications of SMaRT. **A:3`***Trans***-splicing:** If there is a mutation in the 3´ part of the target gene a wildtype mRNA can be obtained by 3´splicing. Therefore, a 3´RTM with a binding domain situated in the intron 5´ to the first exon to be exchanged is necessary. E.g. if the mutation to be corrected is in exon 15, a binding domain for intron 14 is designed. This RTM can correct mutations more 3´ as well. After binding of the RTM the two mRNAs are *trans-*spliced and combined into a wildtype mRNA. **B: 5`***Trans***-splicing:** If correction of a mutation in the 5´ part of a gene is desired a 5´ RTM with a binding domain located in the intron 3´ to the exon to be exchanged is created. If the mutation to be corrected with 5´splicing is in exon 15, a RTM with a binding domain in intron 15 is required. This RTM can repair mutations more 5´ than exon 15 as well. **C: Internal** *Trans***-splicing:** There is also a method to exchange only one exon, called internal *trans*-splicing or internal exon replacement (IER). Here an RTM with two binding domains and 5´ and 3´ splice elements is applied. Arrowheads indicate mutations.

#### **10.3. Efficiency of SMaRT**

control of a human promoter using a minimal self-inactivating retroviral vector into RDEB keratinocytes and fibroblasts leading to cell correction and long lasting expression of type VII collagen. The dermal-epidermal junction in generated skin equivalents was restored [63]. A similar strategy was shown by Siprashvili et al. using an epitope-tagged *COL7A1* cDNA, providing a long term expression of the protein in skin equivalents [64]. In a clinical trial executing a bone marrow transplantation 6 RDEB patients received allo‐ geneic stem cells to milder the RDEB phenotype. As a result, 5 patients showed an im‐

Until now, no *ex vivo* gene therapy approach passed through a phase I/II gene therapy trial. Most of these applications are focusing on the transfer of full-length *COL7A1* cDNA into the affected patient cells. The drawbacks of the insertion of the full-length 9kb cDNA of *COL7A1* are the cloning and packaging limitations of commonly used vector systems to transduce keratinocytes or fibroblasts and the instability of the *COL7A1* gene due to possible genetic rearrangements of the large repetitive cDNA sequence [47]. Additionally, the influence of *COL7A1* over- or ectopic expression in treated cells has to be clarified for a clinical applica‐ tion. Using the methodology of spliceosome mediated RNA *Trans*-splicing (SMaRT) can be a promising alternative to the mentioned approaches to cope with some of the suspected is‐ sues present in full-length *COL7A1* replacement strategies. Murauer et al. demonstrated the exchange of the 3´ coding *COL7A1* cDNA region spanning from exon 65 to the last exon 118 by SMaRT [66]. Thereby the risk of genetic rearrangements of the *COL7A1* cDNA sequence should be reduced significantly. Alternatively to this, we will present in this work a 5´ exon replacement strategy using SMaRT, providing the possibility to repair also relevant muta‐

RNA *trans*-splicing is a naturally occurring event to recombine two or more mRNA mole‐ cules to a new chimeric gene product [67]. For therapeutic purposes such products can be generated by *trans*-splicing a second RNA species from a RNA *trans*-splicing molecule (RTM) into the 3´, 5´ or internal sequence of an endogenously expressed target. See Figure 6. The main advantages of this methodology are the possibility to reduce the size of the trans‐ gene, the maintained endogenous regulation of transgene expression and the feasibility to treat dominant negative diseases [68]. Undesired gene expression due to unintended deliv‐ ery or misregulation is minimized as *trans*-splicing should only occur in cells expressing the target pre-mRNA [69]. Furthermore, SMaRT offers the potential for correction of dominant

In SMaRT constructs that are engineered to bind the introns of specific pre-mRNAs – RNA *trans*-splicing molecules (RTMs) - are the key players. These RTMs effect a *trans*-

proved wound healing, but one patient died [65].

572 Gene Therapy - Tools and Potential Applications

tions 5´ within the *COL7A1* gene.

**10.1. General aspects**

**10. Spliceosome mediated mRNA** *Trans***-splicing**

negative mutations into wildtype gene products [70].

**10.2. Methodology of spliceosome mediated mRNA** *trans***-splicing (SMaRT)**

The efficiency of *trans*-splicing to correct genetic defects and acquired disorders at premRNA level has already been demonstrated for 3´ as well as for 5´ *trans*-splicing in different diseases *in vitro* and *in vivo*. See Table 3.


RNA *trans*-splicing for gene correction is usually performed by 3' RNA *trans*-splicing to ex‐ change 3' coding parts of a gene of interest. 3´ RNA *trans*-splicing was successfully applied to restore wildtype gene expression pattern amongst others in patient cells or in animal models of epidermolysis bullosa, cystic fibrosis, X-linked immunodeficiency and hemophilia A [66,72,73]. Primarily co-transfection experiments with RTMs and artificial targets were

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 575

So a tractable lacZ model repair system, in which user defined target introns can be *trans*-spliced into a mutated lacZ gene to test target specific 3´ RTMs by double transfec‐ tion in 293T cells was developed by Puttaraju et al.. Functional lacZ correction was de‐ tected on mRNA and protein level by qRT-PCR and western blotting for one CFTR intron [74]. Chao et al. showed that the hemophilia A phenotype in factor VIII (FVIII) knockout mice can be repaired by the introduction of a 3' RTM. After delivery of the DNA through the portal vein, the FVIII protein was detected by western blot analysis of cryoprecipitated murine plasma. Long-term correction was shown via adenoviral tail vein transduction of the specific RTM. In the classical tail-clip test all naive knockout mice died, whereas eight out of ten treated mice survived, indicating that 3´ *trans*-splic‐ ing is suitable to correct the bleeding disorder in hemophilia A [73]. Liu et al. used a re‐ combinant adeno-associated viral vector system to target the human cystic fibrosis (CF) polarized airway epithelia from the apical membrane. The measurement of the cAMPsensitive short circuit currents levels confirmed the CFTR correction by SMaRT [75]. Dal‐ linger et al. showed as a proof of principle in the skin the correction of the EB-associated gene *COL17A1* by 3' *trans*-splicing. Using a lacZ model repair system, an intron specific target molecule and a rationally designed RTM, the feasibility of SMaRT was shown by co-transfection experiments in keratinocytes [76]. Using a minigene Rodriguez-Martin et al. published functional 3´ *trans*-splicing on mRNA level after double transfection of the minigene and specific 3´RTM in COS-7 and SH-SY5Y cells for tau mRNA [77]. Zayed et al. demonstrated 3´ correction of the DNA protein kinase catalytic subunit (DNA-PKcs) gene, which is responsible for severe combined immune deficiency (SCID). Specific 3 ´RTMs were transfected into scid.adh cells using the Sleeping Beauty transposon system. After this treatment irradiated cells showed an 4.3 fold increase of surviving cells over irradiated untreated scid.adh cells. Correction of the mutation was shown via QRT-PCR and sequencing on mRNA level. Additionally, functional 3´*trans*-splicing was detected on mRNA level via sequencing and on protein level via western blotting in SCID multipo‐ tent adult progenitor cells [78]. Chen et al. corrected the dystrophia myotica protein kin‐ ase gene responsible for the most common muscular dystrophy in adults by 3´ *trans*splicing on mRNA level [79]. Coady et al. showed *in vivo* correction of spinal muscular atrophy (SMA) by 3´*trans*-splicing in mice recently. A single injection of a repair con‐ struct *trans*-splicing *SMN2* carried by a PMU3 vector into the intracerebral-ventricular space of SMA neonates lessens the severity of the SMA phenotype in a severe mouse model and extends survival by around 70% [80]. Murauer et al. corrected mutations in *COL7A1* by 3´*trans*-splicing. RDEB keratinocytes retrovirally transduced with a 3´*trans*splicing molecule showed an increase of *COL7A1* mRNA sqRT-PCR and recovery of fulllength type VII collagen expression on protein level in western blot and

used to give proof of principle of functionality of the *trans*-splicing process.

**Table 3.** Overview on functional *trans*-splicing approaches so far.

RNA *trans*-splicing for gene correction is usually performed by 3' RNA *trans*-splicing to ex‐ change 3' coding parts of a gene of interest. 3´ RNA *trans*-splicing was successfully applied to restore wildtype gene expression pattern amongst others in patient cells or in animal models of epidermolysis bullosa, cystic fibrosis, X-linked immunodeficiency and hemophilia A [66,72,73]. Primarily co-transfection experiments with RTMs and artificial targets were used to give proof of principle of functionality of the *trans*-splicing process.

**Author Approach Year**

Puttaraju et al. 3´ repair of lacZ in a tractable system 2001

Chao et al. 3´ repair of haemophilia A mice in vivo 2003

Dallinger et al. 3´ repair in a lacZ model system in a keratinocyte specific background 2003

Liu et al. 3´ repair of CFTR mRNA (adenovirally) 2005

Rodriguez-Martin et al. 3´ reprogramming of tau alternative splicing in a model system 2005

Zayed et al. 3´ repair of DNA-PKcs in SCID (delivery via sleeping beauty) 2007

Chen et al. 3´ repair dystrophia myotonica type 1 pre-mRNA 2008

Murauer et al. Functional 3´ repair of the COL7A1 gene 2010

Gruber et al. 3´ reprogramming of tumor marker genes to introduce suicide genes 2011

Mansfield et al. 5´ repair of CFTR mRNA 2000

Kierlin-Duncan et al. 5´ repair of β-globin mRNA 2007

Wally et al. 5´ repair of the PLEC1 gene 2007

Wally et al. 5´ K14 mRNA reprogramming 2010

Rindt et al. 5´ trans-splicing repair of huntingtin at mRNA level 2012

Koller et al. A screening system for IER molecules 2011

Lorain et al. Exon exchange approach to repair Duchenne dystrophin transcripts in a

minigene

**Table 3.** Overview on functional *trans*-splicing approaches so far.

2010

2009

2010

Coady et al. 3´ SMN2 trans-splicing in combination with blocking an cis-splice sit in mice

Wang et al. 3´ introduction of therapeutic proteins in highly abundant albumin

transcripts in mice in vivo

in vivo

**3´ trans-splicing**

574 Gene Therapy - Tools and Potential Applications

**5´trans-splicing**

**Internal trans-splicing**

So a tractable lacZ model repair system, in which user defined target introns can be *trans*-spliced into a mutated lacZ gene to test target specific 3´ RTMs by double transfec‐ tion in 293T cells was developed by Puttaraju et al.. Functional lacZ correction was de‐ tected on mRNA and protein level by qRT-PCR and western blotting for one CFTR intron [74]. Chao et al. showed that the hemophilia A phenotype in factor VIII (FVIII) knockout mice can be repaired by the introduction of a 3' RTM. After delivery of the DNA through the portal vein, the FVIII protein was detected by western blot analysis of cryoprecipitated murine plasma. Long-term correction was shown via adenoviral tail vein transduction of the specific RTM. In the classical tail-clip test all naive knockout mice died, whereas eight out of ten treated mice survived, indicating that 3´ *trans*-splic‐ ing is suitable to correct the bleeding disorder in hemophilia A [73]. Liu et al. used a re‐ combinant adeno-associated viral vector system to target the human cystic fibrosis (CF) polarized airway epithelia from the apical membrane. The measurement of the cAMPsensitive short circuit currents levels confirmed the CFTR correction by SMaRT [75]. Dal‐ linger et al. showed as a proof of principle in the skin the correction of the EB-associated gene *COL17A1* by 3' *trans*-splicing. Using a lacZ model repair system, an intron specific target molecule and a rationally designed RTM, the feasibility of SMaRT was shown by co-transfection experiments in keratinocytes [76]. Using a minigene Rodriguez-Martin et al. published functional 3´ *trans*-splicing on mRNA level after double transfection of the minigene and specific 3´RTM in COS-7 and SH-SY5Y cells for tau mRNA [77]. Zayed et al. demonstrated 3´ correction of the DNA protein kinase catalytic subunit (DNA-PKcs) gene, which is responsible for severe combined immune deficiency (SCID). Specific 3 ´RTMs were transfected into scid.adh cells using the Sleeping Beauty transposon system. After this treatment irradiated cells showed an 4.3 fold increase of surviving cells over irradiated untreated scid.adh cells. Correction of the mutation was shown via QRT-PCR and sequencing on mRNA level. Additionally, functional 3´*trans*-splicing was detected on mRNA level via sequencing and on protein level via western blotting in SCID multipo‐ tent adult progenitor cells [78]. Chen et al. corrected the dystrophia myotica protein kin‐ ase gene responsible for the most common muscular dystrophy in adults by 3´ *trans*splicing on mRNA level [79]. Coady et al. showed *in vivo* correction of spinal muscular atrophy (SMA) by 3´*trans*-splicing in mice recently. A single injection of a repair con‐ struct *trans*-splicing *SMN2* carried by a PMU3 vector into the intracerebral-ventricular space of SMA neonates lessens the severity of the SMA phenotype in a severe mouse model and extends survival by around 70% [80]. Murauer et al. corrected mutations in *COL7A1* by 3´*trans*-splicing. RDEB keratinocytes retrovirally transduced with a 3´*trans*splicing molecule showed an increase of *COL7A1* mRNA sqRT-PCR and recovery of fulllength type VII collagen expression on protein level in western blot and immunofluorescent staining. Moreover normal morphology and reduced invasive capaci‐ ty was achieved in transduced cells. Correct localization of type VII collagen at the base‐ ment membrane zone in skin equivalents, where it assembles into anchoring fibril like structures, showed the potential of *trans*-splicing to correct an RDEB phenotype *in vitro* [66]. There are also alternative approaches in which therapeutic proteins are produced af‐ ter specific 3´*trans*-splicing events into highly abundant albumin transcripts using 3 ´RTMs [81]. Another area of application of SMaRT was performed by Gruber et al. to treat malignant SCC tumors, which are life-threatening issues for RDEB patients. The transfection of RDEB SCC cells with a designed 3' RTM lead to the fusion of the toxin streptolysin O, carried by a 3' RTM, to MMP-9 pre-mRNA molecules, resulting in the ex‐ pression of the toxin and subsequently to the cell death of transfected tumor cells [82].

of a fluorescence reporter, divided into two (5' or 3' *trans*-splicing) or three parts (inter‐ nal exon replacement) and distributed to both screening molecules (target molecule and RTM). The RTM library is composed of individual RTMs with various binding domains. Their efficiency can be evaluated by fluorescence microscopy and flow cytometry. For flow cytometric analysis, individual selected RTMs of the RTM library are co-transfected with the designed target molecule, including the full-length target binding region, and the missing sequence of the split fluorescence reporter into HEK293FT cells. Co-transfec‐ tion of RTM and target molecule into HEK293FT cells results in the restoration of expres‐ sion of the fluorescence reporter. The intensity of the fluorescence signal of the reporter molecule gives information on the functionality of the binding domain. The most effi‐ cient BDs can be tested for endogenous experiments in patient cells. After transfection of the screening-RTM, the fusion of the splitted *trans*-splicing reporter and the endogenous target is detected by RT-PCR. To develop an mRNA based gene therapy an RTM, carry‐ ing the wildtype sequence instead of the coding sequence of the fluorescence molecule, is constructed. After RTM treatment of patient cells a mutated gene part is exchanged by

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 577

We started to establish 5´ *trans*-splicing for murine *COL7A1* in order to analyze the function‐ ality of RNA *trans*-splicing *in viv*o, due to the existence of a mouse model carrying a neo cas‐ sette in intron 2 of *COL7A1* generating aberrant splice variants, which lead to a reduction of type VII collagen expression [46]. By close similarity of this mouse model to the human RDEB phenotype and location of the defect in the 5´ part of *COL7A1*, this mouse model ex‐ hibits obviously an ideal system to test our 5´ repair molecules and investigate different ap‐

Intron 15 was chosen as target intron because its size of about 1,5kb allows to create a large number of different binding domains. To generate a large amount of different RTMs, con‐ taining binding domains with different binding affinities to the target intron, the target exon/intron was digested out of the artificial target used in the screen with HindIII and BamHI and digested with CviJI\*. The resulting fragments with a length of 50-750bp were cloned into the RTM backbone. Binding domains were identified by colony PCR using a for‐ ward primer situated in the 5´ half of the split GFP and a vector specific reverse primer. Pos‐ sible binding domains with different lengths were detected on a 2% agarose gel after gel electrophoresis. To check orientation and location of the binding domain, clones with inserts were sequenced. To evaluate the created RTM library the artificial target containing the tar‐ get intron (intron15) and the 3´ half of the split AcGFP instead of the 3´ part of murine *COL7A1* was cotransfected with the RTM library respectively individual RTMs into HEK293FT. The RTMs contain a transfection reporter (DsRED), the 5´ half of the split AcGFP instead of the first 15 exons of *COL7A1* and variable binding domains. The cotrans‐ fected cells were analyzed concerning their AcGFP/DsRED expression ratios by fluorescence

*trans*-splicing and wildtype transcripts are restored.

**11. RTMs for the murine COL7A1 gene**

plication strategies.

5' *trans*-splicing to correct upstream coding sequences of an mRNA of interest was first shown by a double transfection model to repair mutations in the cystic fibrosis trans‐ membrane receptor (CFTR) pre-mRNA. Functionality tests were performed by anion ef‐ flux transport measurements. RTMs were designed capable to repair the 5' portion of CFTR transcripts [83]. 5' *trans*-splicing was also applied for the substitution of exon 1 of b-globin in cells co-transfected with a target molecule and an RTM in 293T cells and lead specific *trans*-splicing detected by one step RT-PCR [84]. Endogenous 5' *trans*-splicing in‐ duced gene correction was first demonstrated by Wally et al. on the basis of the *PLEC* gene involved in the disease epidermolysis bullosa simplex (EBS). Restoration of wild‐ type plectin expression patterns was shown by immunofluorescence microscopy of pa‐ tient fibroblasts after RTM treatment [61]. Additionally, exons 1–7 of the keratin 14 gene (*KRT14*) were replaced in an autosomal dominant model of EBS resulting in recovery of K14 on RNA and protein level, detected by SQRT-PCR, western blotting and immuno‐ fluorescence staining by transient transfection of specific 5´ RTMs chosen in a screening procedure [85]. Recently 5´*trans*-splicing correction of a disease causing huntingtin allele on mRNA level was reported by Rindt et al. [86].

Lorain et al. primarily published the methodology of internal exon replacement (IER) to cor‐ rect a dystrophin minigene on mRNA level [87]. Recently, Koller et al. developed a new RTM screening system to improve double RNA *trans*-splicing for the correction of the EB as‐ sociated gene *COL17A1* [88].

#### **10.4. RTM screening systems**

So far, there are no general rules for the design of highly efficient *trans*-splicing RTMs. However, recent studies revealed the influence of minor differences in length, composi‐ tion and localization of the binding domain (BD) on RTM efficiency and specificity [85,88]. Due to the fact that an RTM can't be predicted rationally, we established a fluo‐ rescence-based screening system to select an efficient RTM from a pool of randomly de‐ signed RTMs. This screening system is composed of fluorescence-based RTM backbones, in which randomly created binding domains are cloned, and a gene specific target mole‐ cule. The target binding region (exon/intron sequence of a gene of interest) is PCR ampli‐ fied, randomly fragmented and cloned into the RTM vector. The coding region consists of a fluorescence reporter, divided into two (5' or 3' *trans*-splicing) or three parts (inter‐ nal exon replacement) and distributed to both screening molecules (target molecule and RTM). The RTM library is composed of individual RTMs with various binding domains. Their efficiency can be evaluated by fluorescence microscopy and flow cytometry. For flow cytometric analysis, individual selected RTMs of the RTM library are co-transfected with the designed target molecule, including the full-length target binding region, and the missing sequence of the split fluorescence reporter into HEK293FT cells. Co-transfec‐ tion of RTM and target molecule into HEK293FT cells results in the restoration of expres‐ sion of the fluorescence reporter. The intensity of the fluorescence signal of the reporter molecule gives information on the functionality of the binding domain. The most effi‐ cient BDs can be tested for endogenous experiments in patient cells. After transfection of the screening-RTM, the fusion of the splitted *trans*-splicing reporter and the endogenous target is detected by RT-PCR. To develop an mRNA based gene therapy an RTM, carry‐ ing the wildtype sequence instead of the coding sequence of the fluorescence molecule, is constructed. After RTM treatment of patient cells a mutated gene part is exchanged by *trans*-splicing and wildtype transcripts are restored.

#### **11. RTMs for the murine COL7A1 gene**

immunofluorescent staining. Moreover normal morphology and reduced invasive capaci‐ ty was achieved in transduced cells. Correct localization of type VII collagen at the base‐ ment membrane zone in skin equivalents, where it assembles into anchoring fibril like structures, showed the potential of *trans*-splicing to correct an RDEB phenotype *in vitro* [66]. There are also alternative approaches in which therapeutic proteins are produced af‐ ter specific 3´*trans*-splicing events into highly abundant albumin transcripts using 3 ´RTMs [81]. Another area of application of SMaRT was performed by Gruber et al. to treat malignant SCC tumors, which are life-threatening issues for RDEB patients. The transfection of RDEB SCC cells with a designed 3' RTM lead to the fusion of the toxin streptolysin O, carried by a 3' RTM, to MMP-9 pre-mRNA molecules, resulting in the ex‐ pression of the toxin and subsequently to the cell death of transfected tumor cells [82].

5' *trans*-splicing to correct upstream coding sequences of an mRNA of interest was first shown by a double transfection model to repair mutations in the cystic fibrosis trans‐ membrane receptor (CFTR) pre-mRNA. Functionality tests were performed by anion ef‐ flux transport measurements. RTMs were designed capable to repair the 5' portion of CFTR transcripts [83]. 5' *trans*-splicing was also applied for the substitution of exon 1 of b-globin in cells co-transfected with a target molecule and an RTM in 293T cells and lead specific *trans*-splicing detected by one step RT-PCR [84]. Endogenous 5' *trans*-splicing in‐ duced gene correction was first demonstrated by Wally et al. on the basis of the *PLEC* gene involved in the disease epidermolysis bullosa simplex (EBS). Restoration of wild‐ type plectin expression patterns was shown by immunofluorescence microscopy of pa‐ tient fibroblasts after RTM treatment [61]. Additionally, exons 1–7 of the keratin 14 gene (*KRT14*) were replaced in an autosomal dominant model of EBS resulting in recovery of K14 on RNA and protein level, detected by SQRT-PCR, western blotting and immuno‐ fluorescence staining by transient transfection of specific 5´ RTMs chosen in a screening procedure [85]. Recently 5´*trans*-splicing correction of a disease causing huntingtin allele

Lorain et al. primarily published the methodology of internal exon replacement (IER) to cor‐ rect a dystrophin minigene on mRNA level [87]. Recently, Koller et al. developed a new RTM screening system to improve double RNA *trans*-splicing for the correction of the EB as‐

So far, there are no general rules for the design of highly efficient *trans*-splicing RTMs. However, recent studies revealed the influence of minor differences in length, composi‐ tion and localization of the binding domain (BD) on RTM efficiency and specificity [85,88]. Due to the fact that an RTM can't be predicted rationally, we established a fluo‐ rescence-based screening system to select an efficient RTM from a pool of randomly de‐ signed RTMs. This screening system is composed of fluorescence-based RTM backbones, in which randomly created binding domains are cloned, and a gene specific target mole‐ cule. The target binding region (exon/intron sequence of a gene of interest) is PCR ampli‐ fied, randomly fragmented and cloned into the RTM vector. The coding region consists

on mRNA level was reported by Rindt et al. [86].

sociated gene *COL17A1* [88].

576 Gene Therapy - Tools and Potential Applications

**10.4. RTM screening systems**

We started to establish 5´ *trans*-splicing for murine *COL7A1* in order to analyze the function‐ ality of RNA *trans*-splicing *in viv*o, due to the existence of a mouse model carrying a neo cas‐ sette in intron 2 of *COL7A1* generating aberrant splice variants, which lead to a reduction of type VII collagen expression [46]. By close similarity of this mouse model to the human RDEB phenotype and location of the defect in the 5´ part of *COL7A1*, this mouse model ex‐ hibits obviously an ideal system to test our 5´ repair molecules and investigate different ap‐ plication strategies.

Intron 15 was chosen as target intron because its size of about 1,5kb allows to create a large number of different binding domains. To generate a large amount of different RTMs, con‐ taining binding domains with different binding affinities to the target intron, the target exon/intron was digested out of the artificial target used in the screen with HindIII and BamHI and digested with CviJI\*. The resulting fragments with a length of 50-750bp were cloned into the RTM backbone. Binding domains were identified by colony PCR using a for‐ ward primer situated in the 5´ half of the split GFP and a vector specific reverse primer. Pos‐ sible binding domains with different lengths were detected on a 2% agarose gel after gel electrophoresis. To check orientation and location of the binding domain, clones with inserts were sequenced. To evaluate the created RTM library the artificial target containing the tar‐ get intron (intron15) and the 3´ half of the split AcGFP instead of the 3´ part of murine *COL7A1* was cotransfected with the RTM library respectively individual RTMs into HEK293FT. The RTMs contain a transfection reporter (DsRED), the 5´ half of the split AcGFP instead of the first 15 exons of *COL7A1* and variable binding domains. The cotrans‐ fected cells were analyzed concerning their AcGFP/DsRED expression ratios by fluorescence microscopy and flow cytometry, whereby a higher ratio indicates the presence of a more functional binding domain in the RTM. See Figures 7+8.

**Figure 8.** Flow cytometric analysis: 5´ screen for murine *COL7A1* exon/intron 15. Red fluorescence (shown on Y-axis) indicates transfection of RTMs in the cells; green fluorescence (shown on X-axis) indicates specific *trans*-splicing. **A:** The positive control is a vector containing a DsRED linker AcGFP construct. This FACS plot mimics the AcGFP and DsRED ratios expected from the product of optimal *trans*-splicing. **B:** The RTM library shows a *trans*-splicing efficiency (AcGFP/DsRED ratio) of 48,94% calculated from *trans*-splicing positive cells/all transfected cells.(B2 1,8% + B4 0,5%)/ (B1 2,4% + B2 1,8% + B4 0,5%) The fact, that several cells seem to be exclusively green can be explained by the in‐ tense AcGFP fluorescence, which tends to override the weaker DsRED fluorescence. **C**: The most efficient RTM ana‐ lyzed, with a *trans*-splicing efficiency of 96,55%, (B2 4,6% + B4 3,8%)/( B1 0,3% + B2 4,6% + B4 3,8%) shows a dot plot pattern similar to the positive control. Therefore the binding domain of RTM +3A was chosen to be used in fur‐ ther endogenous experiments. **D:** A Comparison of AcGFP/DsRED ratios of single RTMs containing different binding

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 579

The RTM with the highest AcGFP/DsRED ratio (RTM+3A) was chosen for further endoge‐ nous experiments. To check endogenous functionality of the RTM was transiently transfect‐ ed into an immortalized murine keratinocyte cell line [46]. The 5´ part of the split AcGFP contained by the screening RTM was specifically *trans*-spliced with its endogenous target – the murine *COL7A1* mRNA – resulting in a AcGFP-*COL7A1* fusion mRNA detected by RT-

domains, shows a wide variability of AcGFP/DsRED ration spanning less than 10% to nearly 100%.

PCR analysis and subsequent sequencing. See Figure 9.

**Figure 7.** Fluorescence microscopy of with RTM library and target double transfected HEK293 cells. **A:** Functional binding domains lead to specific *trans*-splicing of the two pre mRNAs which are then combined into one mRNA con‐ taining DsRED and full-length AcGFP. Red fluorescence indicates the transfection of a RTM in the cells whereas red and green fluorescence indicates functional *trans*-splicing. **B:** Double transfection of an artificial target containing exon/intron 15 of murine *COL7A1* and an RTM library for this exon/intron in HEK293FT. **C:** Double transfection of an artificial target containing exon/intron 15 of murine *COL7A1* and the best RTM for this intron.

microscopy and flow cytometry, whereby a higher ratio indicates the presence of a more

**Figure 7.** Fluorescence microscopy of with RTM library and target double transfected HEK293 cells. **A:** Functional binding domains lead to specific *trans*-splicing of the two pre mRNAs which are then combined into one mRNA con‐ taining DsRED and full-length AcGFP. Red fluorescence indicates the transfection of a RTM in the cells whereas red and green fluorescence indicates functional *trans*-splicing. **B:** Double transfection of an artificial target containing exon/intron 15 of murine *COL7A1* and an RTM library for this exon/intron in HEK293FT. **C:** Double transfection of an

artificial target containing exon/intron 15 of murine *COL7A1* and the best RTM for this intron.

functional binding domain in the RTM. See Figures 7+8.

578 Gene Therapy - Tools and Potential Applications

**Figure 8.** Flow cytometric analysis: 5´ screen for murine *COL7A1* exon/intron 15. Red fluorescence (shown on Y-axis) indicates transfection of RTMs in the cells; green fluorescence (shown on X-axis) indicates specific *trans*-splicing. **A:** The positive control is a vector containing a DsRED linker AcGFP construct. This FACS plot mimics the AcGFP and DsRED ratios expected from the product of optimal *trans*-splicing. **B:** The RTM library shows a *trans*-splicing efficiency (AcGFP/DsRED ratio) of 48,94% calculated from *trans*-splicing positive cells/all transfected cells.(B2 1,8% + B4 0,5%)/ (B1 2,4% + B2 1,8% + B4 0,5%) The fact, that several cells seem to be exclusively green can be explained by the in‐ tense AcGFP fluorescence, which tends to override the weaker DsRED fluorescence. **C**: The most efficient RTM ana‐ lyzed, with a *trans*-splicing efficiency of 96,55%, (B2 4,6% + B4 3,8%)/( B1 0,3% + B2 4,6% + B4 3,8%) shows a dot plot pattern similar to the positive control. Therefore the binding domain of RTM +3A was chosen to be used in fur‐ ther endogenous experiments. **D:** A Comparison of AcGFP/DsRED ratios of single RTMs containing different binding domains, shows a wide variability of AcGFP/DsRED ration spanning less than 10% to nearly 100%.

The RTM with the highest AcGFP/DsRED ratio (RTM+3A) was chosen for further endoge‐ nous experiments. To check endogenous functionality of the RTM was transiently transfect‐ ed into an immortalized murine keratinocyte cell line [46]. The 5´ part of the split AcGFP contained by the screening RTM was specifically *trans*-spliced with its endogenous target – the murine *COL7A1* mRNA – resulting in a AcGFP-*COL7A1* fusion mRNA detected by RT-PCR analysis and subsequent sequencing. See Figure 9.

The development of a gene therapy for type VII collagen deficiency would increase the chance to find a cure for dystrophic EB. Additionally, the improvement of the methodology of 5' RNA *trans*-splicing will help us to move closer to the treatment of other genetic diseas‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 581

We want to thank Prof. Leena Bruckner-Tuderman for providing the murine keratinocytes. Moreover we thank the Austrian Science Fund (FWF) for financing the project "Develop‐ ment of a 5´*trans*-splicing Gene Therapy" [P22039-B12] and DEBRA Austria, DEBRA Alto Adige and the Paracelsus Private Medical University for additional financial support.

[1] Laimer M, Lanschuetzer CM, B, Pohla-Gubo G, Klausegger A, Diem A, Riedl R, Ba‐ uer JW, and Hintner H. Epidermolysis Bullosa. Pädiatrie&Pädologie 2006;6 30-38. [2] Kanitakis J. Anatomy, Histology and Immunohistochemistry of Normal Human

[4] Fine JD, Eady RA, Bauer EA, Bauer JW, Bruckner-Tuderman L, Heagerty A, Hintner H, Hovnanian A, Jonkman MF, Leigh I, McGrath JA, Mellerio JE, Murrell DF, Shimi‐ zu H, Uitto J, Vahlquist A, Woodley D, and Zambruno G. The Classification of Inher‐ ited Epidermolysis Bullosa (EB): Report of the Third International Consensus Meeting on Diagnosis and Classification of EB. J Am Acad Dermatol 2008;58(6)

[5] Pulkkinen L and Uitto J. Mutation Analysis and Molecular Genetics of Epidermolysis

[6] Sakai LY, Keene DR, Morris NP, and Burgeson RE. Type VII Collagen Is a Major Structural Component of Anchoring Fibrils. J Cell Biol 1986;103(4) 1577-1586.

[3] Fine JD and Hintner H. Life With Epidermolysis Bullosa .Wien: Springer Wien;

es caused by mutations in especially large genes.

, U. Koller and J.W. Bauer

\*Address all correspondence to: el.mayr@salk.at

Bullosa. Matrix Biol 1999;18(1) 29-42.

EB House Austria, Dept Dermatol, Paracelsus Med Univ, Salzburg, Austria

Skin. European Journal of Dermatology 2002;12(4) 390-400.

**Acknowledgements**

**Author details**

E. Mayr\*

**References**

931-950.

**Figure 9.** Endogenous *trans*-splicing into exon/intron 15 of murine *COL7A1*. **A:** RT-PCR analysis of transiently RTM+3A transfected spontaneously immortalized mouse wildtype keratinocytes [46] using primers in the 5´ part of split AcGFP and in exon 18 of *COL7A1* resulted in detection of a 365bp band after agarose gel-electrophoresis. B: The fragment was verified to be an AcGFP-*COL7A1* fusion by sequencing. **+3A** cDNA analysis of spontaneously immortalized mouse wildtype keratinocytes transiently transfected with RTM +3A **L** Ladder Mix DNA marker

#### **12. Conclusion**

RNA *trans*-splicing is a useful methodology to reprogram genes for diagnostic and thera‐ peutic purposes. Due to a variety of advantages over traditional gene-replacement strat‐ egies, RNA *trans*-splicing is used to correct the phenotype of many genetic diseases *in vitro*, ranging from epidermolysis bullosa to neurodegenerative diseases. *In vivo* studies are in progress to accelerate the way to the medical use of this RNA-based application.

We have established all three modes of *trans*-splicing (5', 3' and internal exon replacement) in our laboratory on the basis of several EB-associated genes (*KRT14, PLEC, COL7A1, COL17A1*). In this work we focused on the methodology of 5´ RNA *trans*-splicing to correct mutations localized within the first 15 exons of the murine *COL7A1* gene encoding for type VII collagen. *COL7A1* is a large gene with over 9kb and is therefore suitable for this ap‐ proach, in which only a short RTM has to be designed, harbouring only the first 15 exons of the gene. Using an RTM screening system, described by Wally et al 2011 [89], it should be possible to increase the *trans*-splicing efficiency of designed RTMs to a level where the phe‐ notype of *COL7A1* deficient cells can be converted into wildtype. We analyzed the binding properties of randomly designed RTMs specific for intron 15 of murine *COL7A1* and tested the most efficient RTM in *COL7A1* deficient keratinocytes for endogenous functionality. The RTM was able to induce endogenous 5´*trans*-splicing into murine *COL7A1* pre-mRNA mole‐ cules, manifested in the fusion of the 5' GFP part of the RTM with exon 16 of *COL7A1*. Next steps are the exchange of the 5' GFP part by the 5´sequence of murine *COL7A1* (exons1-15) and to investigate if our RTMs are able to increase the level of full-length *COL7A1* mRNA leading to the recovery of functional type VII collagen in *COL7A1* deficient cells and in skin equivalents. In summary we demonstrated a novel RNA-based strategy to correct diseaseassociated mutations within *COL7A1,* thereby avoiding or minimizing many problems present in standard cDNA gene therapies including fragmentation of the large *COL7A1* gene, the size limitation of the transgene and over- and ectopic expression of the transgene. The development of a gene therapy for type VII collagen deficiency would increase the chance to find a cure for dystrophic EB. Additionally, the improvement of the methodology of 5' RNA *trans*-splicing will help us to move closer to the treatment of other genetic diseas‐ es caused by mutations in especially large genes.

#### **Acknowledgements**

We want to thank Prof. Leena Bruckner-Tuderman for providing the murine keratinocytes. Moreover we thank the Austrian Science Fund (FWF) for financing the project "Develop‐ ment of a 5´*trans*-splicing Gene Therapy" [P22039-B12] and DEBRA Austria, DEBRA Alto Adige and the Paracelsus Private Medical University for additional financial support.

#### **Author details**

**Figure 9.** Endogenous *trans*-splicing into exon/intron 15 of murine *COL7A1*. **A:** RT-PCR analysis of transiently RTM+3A transfected spontaneously immortalized mouse wildtype keratinocytes [46] using primers in the 5´ part of split AcGFP and in exon 18 of *COL7A1* resulted in detection of a 365bp band after agarose gel-electrophoresis. B: The fragment was verified to be an AcGFP-*COL7A1* fusion by sequencing. **+3A** cDNA analysis of spontaneously immortalized mouse

RNA *trans*-splicing is a useful methodology to reprogram genes for diagnostic and thera‐ peutic purposes. Due to a variety of advantages over traditional gene-replacement strat‐ egies, RNA *trans*-splicing is used to correct the phenotype of many genetic diseases *in vitro*, ranging from epidermolysis bullosa to neurodegenerative diseases. *In vivo* studies are in

We have established all three modes of *trans*-splicing (5', 3' and internal exon replacement) in our laboratory on the basis of several EB-associated genes (*KRT14, PLEC, COL7A1, COL17A1*). In this work we focused on the methodology of 5´ RNA *trans*-splicing to correct mutations localized within the first 15 exons of the murine *COL7A1* gene encoding for type VII collagen. *COL7A1* is a large gene with over 9kb and is therefore suitable for this ap‐ proach, in which only a short RTM has to be designed, harbouring only the first 15 exons of the gene. Using an RTM screening system, described by Wally et al 2011 [89], it should be possible to increase the *trans*-splicing efficiency of designed RTMs to a level where the phe‐ notype of *COL7A1* deficient cells can be converted into wildtype. We analyzed the binding properties of randomly designed RTMs specific for intron 15 of murine *COL7A1* and tested the most efficient RTM in *COL7A1* deficient keratinocytes for endogenous functionality. The RTM was able to induce endogenous 5´*trans*-splicing into murine *COL7A1* pre-mRNA mole‐ cules, manifested in the fusion of the 5' GFP part of the RTM with exon 16 of *COL7A1*. Next steps are the exchange of the 5' GFP part by the 5´sequence of murine *COL7A1* (exons1-15) and to investigate if our RTMs are able to increase the level of full-length *COL7A1* mRNA leading to the recovery of functional type VII collagen in *COL7A1* deficient cells and in skin equivalents. In summary we demonstrated a novel RNA-based strategy to correct diseaseassociated mutations within *COL7A1,* thereby avoiding or minimizing many problems present in standard cDNA gene therapies including fragmentation of the large *COL7A1* gene, the size limitation of the transgene and over- and ectopic expression of the transgene.

progress to accelerate the way to the medical use of this RNA-based application.

wildtype keratinocytes transiently transfected with RTM +3A **L** Ladder Mix DNA marker

**12. Conclusion**

580 Gene Therapy - Tools and Potential Applications

E. Mayr\* , U. Koller and J.W. Bauer

\*Address all correspondence to: el.mayr@salk.at

EB House Austria, Dept Dermatol, Paracelsus Med Univ, Salzburg, Austria

#### **References**


[7] Chung HJ and Uitto J. Type VII Collagen: the Anchoring Fibril Protein at Fault in Dystrophic Epidermolysis Bullosa. Dermatol Clin 2010;28(1) 93-105.

[18] Parente MG, Chung LC, Ryynanen J, Woodley DT, Wynn KC, Bauer EA, Mattei MG, Chu ML, and Uitto J. Human Type VII Collagen: CDNA Cloning and Chromosomal

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 583

[19] Kivirikko S, Li K, Christiano AM, and Uitto J. Cloning of Mouse Type VII Collagen Reveals Evolutionary Conservation of Functional Protein Domains and Genomic Or‐

[20] Ryynanen J, Sollberg S, Olsen DR, and Uitto J. Transforming Growth Factor-Beta Up-Regulates Type VII Collagen Gene Expression in Normal and Transformed Epider‐ mal Keratinocytes in Culture. Biochem Biophys Res Commun 1991;180(2) 673-680.

[21] Vindevoghel L, Kon A, Lechleider RJ, Uitto J, Roberts AB, and Mauviel A. Smad-De‐ pendent Transcriptional Activation of Human Type VII Collagen Gene (COL7A1) Promoter by Transforming Growth Factor-Beta. J Biol Chem 1998;273(21)

[22] Ryynanen J, Sollberg S, Parente MG, Chung LC, Christiano AM, and Uitto J. Type VII Collagen Gene Expression by Cultured Human Cells and in Fetal Skin. Abundant MRNA and Protein Levels in Epidermal Keratinocytes. J Clin Invest 1992;89(1)

[23] Colombo M, Brittingham RJ, Klement JF, Majsterek I, Birk DE, Uitto J, and Fertala A. Procollagen VII Self-Assembly Depends on Site-Specific Interactions and Is Promoted by Cleavage of the NC2 Domain With Procollagen C-Proteinase. Biochemistry

[24] Chen M, Marinkovich MP, Veis A, Cai X, Rao CN, O'toole EA, and Woodley DT. In‐ teractions of the Amino-Terminal Noncollagenous (NC1) Domain of Type VII Colla‐ gen With Extracellular Matrix Components. A Potential Role in Epidermal-Dermal

[25] Brittingham R, Uitto J, and Fertala A. High-Affinity Binding of the NC1 Domain of Collagen VII to Laminin 5 and Collagen IV. Biochem Biophys Res Commun

[26] Ruoslahti E and Pierschbacher MD. New Perspectives in Cell Adhesion: RGD and In‐

[27] Dang N and Murrell DF. Mutation Analysis and Characterization of COL7A1 Muta‐ tions in Dystrophic Epidermolysis Bullosa. Exp Dermatol 2008;17(7) 553-568.

[28] Mecklenbeck S, Hammami-Hauasli N, Hopfner B, Schumann H, Kramer A, Kuster W, and Bruckner-Tuderman L. Clustering of COL7A1 Mutations in Exon 73: Implica‐ tions for Mutation Analysis in Dystrophic Epidermolysis Bullosa. J Invest Dermatol

[29] Whittock NV, Ashton GH, Mohammedi R, Mellerio JE, Mathew CG, Abbs SJ, Eady RA, and McGrath JA. Comparative Mutation Detection Screening of the Type VII

Adherence in Human Skin. J Biol Chem 1997;272(23) 14516-14522.

Mapping of the Gene. Proc Natl Acad Sci U S A 1991;88(16) 6931-6935.

ganization. J Invest Dermatol 1996;106(6) 1300-1306.

13053-13057.

163-168.

2003;42(39) 11434-11442.

2006;343(3) 692-699.

1999;112(3) 398-400.

tegrins. Science 1987;238(4826) 491-497.


[18] Parente MG, Chung LC, Ryynanen J, Woodley DT, Wynn KC, Bauer EA, Mattei MG, Chu ML, and Uitto J. Human Type VII Collagen: CDNA Cloning and Chromosomal Mapping of the Gene. Proc Natl Acad Sci U S A 1991;88(16) 6931-6935.

[7] Chung HJ and Uitto J. Type VII Collagen: the Anchoring Fibril Protein at Fault in

[8] Fine JD, Eady RA, Bauer EA, Briggaman RA, Bruckner-Tuderman L, Christiano A, Heagerty A, Hintner H, Jonkman MF, McGrath J, McGuire J, Moshell A, Shimizu H, Tadini G, and Uitto J. Revised Classification System for Inherited Epidermolysis Bul‐ losa: Report of the Second International Consensus Meeting on Diagnosis and Classi‐

fication of Epidermolysis Bullosa. J Am Acad Dermatol 2000;42(6) 1051-1066.

ysis Bullosa Registry. J Pediatr Gastroenterol Nutr 2008;46(2) 147-158.

molysis Bullosa Registry. Laryngoscope 2007;117(9) 1652-1660.

1986-2002. J Hand Surg Br 2005;30(1) 14-22.

2004;172(5 Pt 1) 2040-2044.

togenet Cell Genet 1993;62(1) 35-36.

2004;44(4) 651-660.

582 Gene Therapy - Tools and Potential Applications

[9] Fine JD, Johnson LB, Weiner M, and Suchindran C. Gastrointestinal Complications of Inherited Epidermolysis Bullosa: Cumulative Experience of the National Epidermol‐

[10] Fine JD, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J, Devries DT, and Suchin‐ dran C. Eye Involvement in Inherited Epidermolysis Bullosa: Experience of the Na‐

[11] Fine JD, Johnson LB, Weiner M, and Suchindran C. Tracheolaryngeal Complications of Inherited Epidermolysis Bullosa: Cumulative Experience of the National Epider‐

[12] Fine JD, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J, Devries DT, and Suchin‐ dran C. Pseudosyndactyly and Musculoskeletal Contractures in Inherited Epider‐ molysis Bullosa: Experience of the National Epidermolysis Bullosa Registry,

[13] Fine JD, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J, Devries DT, and Suchin‐ dran C. Genitourinary Complications of Inherited Epidermolysis Bullosa: Experience of the National Epidermylosis Bullosa Registry and Review of the Literature. J Urol

[14] Fine JD, Johnson LB, Weiner M, Stein A, Cash S, Deleoz J, Devries DT, and Suchin‐ dran C. Inherited Epidermolysis Bullosa and the Risk of Death From Renal Disease: Experience of the National Epidermolysis Bullosa Registry. Am J Kidney Dis

[15] Christiano AM, Rosenbaum LM, Chung-Honet LC, Parente MG, Woodley DT, Pan TC, Zhang RZ, Chu ML, Burgeson RE, and Uitto J. The Large Non-Collagenous Do‐ main (NC-1) of Type VII Collagen Is Amino-Terminal and Chimeric. Homology to Cartilage Matrix Protein, the Type III Domains of Fibronectin and the A Domains of

[16] Christiano AM, Greenspan DS, Lee S, and Uitto J. Cloning of Human Type VII Colla‐ gen. Complete Primary Sequence of the Alpha 1(VII) Chain and Identification of In‐

[17] Greenspan DS, Byers MG, Eddy RL, Hoffman GG, and Shows TB. Localization of the Human Collagen Gene COL7A1 to 3p21.3 by Fluorescence in Situ Hybridization. Cy‐

Von Willebrand Factor. Hum Mol Genet 1992;1(7) 475-481.

tragenic Polymorphisms. J Biol Chem 1994;269(32) 20256-20262.

tional Epidermolysis Bullosa Registry. Am J Ophthalmol 2004;138(2) 254-262.

Dystrophic Epidermolysis Bullosa. Dermatol Clin 2010;28(1) 93-105.


Collagen Gene (COL7A1) Using the Protein Truncation Test, Fluorescent Chemical Cleavage of Mismatch, and Conformation Sensitive Gel Electrophoresis. J Invest Der‐ matol 1999;113(4) 673-686.

[40] Sakuntabhai A, Hammami-Hauasli N, Bodemer C, Rochat A, Prost C, Barrandon Y, de Prost Y, Lathrop M, Wojnarowska F, Bruckner-Tuderman L, and Hovnanian A. Deletions Within COL7A1 Exons Distant From Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis Bullo‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 585

[41] Cserhalmi-Friedman PB, McGrath JA, Mellerio JE, Romero R, Salas-Alanis JC, Paller AS, Dietz HC, and Christiano AM. Restoration of Open Reading Frame Resulting From Skipping of an Exon With an Internal Deletion in the COL7A1 Gene. Lab Invest

[42] Posteraro P, Pascucci M, Colombi M, Barlati S, Giannetti A, Paradisi M, Mustonen A, Zambruno G, and Castiglia D. Denaturing HPLC-Based Approach for Detection of COL7A1 Gene Mutations Causing Dystrophic Epidermolysis Bullosa. Biochem Bio‐

[43] Christiano AM, Fine JD, and Uitto J. Genetic Basis of Dominantly Inherited Transient Bullous Dermolysis of the Newborn: a Splice Site Mutation in the Type VII Collagen

[44] Jiang W, Bu D, Yang Y, and Zhu X. A Novel Splice Site Mutation in Collagen Type VII Gene in a Chinese Family With Dominant Dystrophic Epidermolysis Bullosa Pru‐

[45] Ito K, Sawamura D, Goto M, Nakamura H, Nishie W, Sakai K, Natsuga K, Shinkuma S, Shibaki A, Uitto J, Denton CP, Nakajima O, Akiyama M, and Shimizu H. Keratino‐ cyte-/Fibroblast-Targeted Rescue of Col7a1-Disrupted Mice and Generation of an Ex‐ act Dystrophic Epidermolysis Bullosa Model Using a Human COL7A1 Mutation. Am

[46] Fritsch A, Loeckermann S, Kern JS, Braun A, Bosl MR, Bley TA, Schumann H, von Elverfeldt D, Paul D, Erlacher M, von Rautenfeld DB, Hausser I, Fassler R, and Bruckner-Tuderman L. A Hypomorphic Mouse Model of Dystrophic Epidermolysis Bullosa Reveals Mechanisms of Disease and Response to Fibroblast Therapy. Journal

[47] Chen M, O'Toole EA, Muellenhoff M, Medina E, Kasahara N, and Woodley DT. De‐ velopment and Characterization of a Recombinant Truncated Type VII Collagen "Minigene". Implication for Gene Therapy of Dystrophic Epidermolysis Bullosa. J Bi‐

[48] Bruckner-Tuderman L. Hereditary Skin Diseases of Anchoring Fibrils. J Dermatol Sci

[49] Bruckner-Tuderman L, Hopfner B, and Hammami-Hauasli N. Biology of Anchoring Fibrils: Lessons From Dystrophic Epidermolysis Bullosa. Matrix Biol 1999;18(1) 43-54.

sa. Am J Hum Genet 1998;63(3) 737-748.

phys Res Commun 2005;338(3) 1391-1401.

Gene. J Invest Dermatol 1997;109(6) 811-814.

J Pathol 2009;175(6) 2508-2517.

ol Chem 2000;275(32) 24429-24435.

1999;20(2) 122-133.

riginosa. Acta Derm Venereol 2002;82(3) 187-191.

of Clinical Investigation 2008;118(5) 1669-1679.

1998;78(12) 1483-1492.


[40] Sakuntabhai A, Hammami-Hauasli N, Bodemer C, Rochat A, Prost C, Barrandon Y, de Prost Y, Lathrop M, Wojnarowska F, Bruckner-Tuderman L, and Hovnanian A. Deletions Within COL7A1 Exons Distant From Consensus Splice Sites Alter Splicing and Produce Shortened Polypeptides in Dominant Dystrophic Epidermolysis Bullo‐ sa. Am J Hum Genet 1998;63(3) 737-748.

Collagen Gene (COL7A1) Using the Protein Truncation Test, Fluorescent Chemical Cleavage of Mismatch, and Conformation Sensitive Gel Electrophoresis. J Invest Der‐

[30] Christiano AM, Anhalt G, Gibbons S, Bauer EA, and Uitto J. Premature Termination Codons in the Type VII Collagen Gene (COL7A1) Underlie Severe, Mutilating Reces‐

[31] Christiano AM, McGrath JA, Tan KC, and Uitto J. Glycine Substitutions in the Triple-Helical Region of Type VII Collagen Result in a Spectrum of Dystrophic Epidermoly‐ sis Bullosa Phenotypes and Patterns of Inheritance. Am J Hum Genet 1996;58(4)

[32] Bruckner-Tuderman L, Nilssen O, Zimmermann DR, Dours-Zimmermann MT, Ka‐ linke DU, Gedde-Dahl T, Jr., and Winberg JO. Immunohistochemical and Mutation Analyses Demonstrate That Procollagen VII Is Processed to Collagen VII Through

[33] Sawamura D, Goto M, Yasukawa K, Sato-Matsumura K, Nakamura H, Ito K, Naka‐ mura H, Tomita Y, and Shimizu H. Genetic Studies of 20 Japanese Families of Dys‐

[34] Dunnill MG, McGrath JA, Richards AJ, Christiano AM, Uitto J, Pope FM, and Eady RA. Clinicopathological Correlations of Compound Heterozygous COL7A1 Muta‐ tions in Recessive Dystrophic Epidermolysis Bullosa. J Invest Dermatol 1996;107(2)

[35] Kern JS, Kohlhase J, Bruckner-Tuderman L, and Has C. Expanding the COL7A1 Mu‐ tation Database: Novel and Recurrent Mutations and Unusual Genotype-Phenotype Constellations in 41 Patients With Dystrophic Epidermolysis Bullosa. J Invest Derma‐

[36] Christiano AM, McGrath JA, and Uitto J. Influence of the Second COL7A1 Mutation in Determining the Phenotypic Severity of Recessive Dystrophic Epidermolysis Bul‐

[37] Hovnanian A, Rochat A, Bodemer C, Petit E, Rivers CA, Prost C, Fraitag S, Christi‐ ano AM, Uitto J, Lathrop M, Barrandon Y, and de Prost Y. Characterization of 18 New Mutations in COL7A1 in Recessive Dystrophic Epidermolysis Bullosa Provides Evidence for Distinct Molecular Mechanisms Underlying Defective Anchoring Fibril

[38] Ryoo YW, Kim BC, and Lee KS. Characterization of Mutations of the Type VII Colla‐ gen Gene (COL7A1) in Recessive Dystrophic Epidermolysis Bullosa Mitis (M-RDEB)

[39] Jarvikallio A, Pulkkinen L, and Uitto J. Molecular Basis of Dystrophic Epidermolysis Bullosa: Mutations in the Type VII Collagen Gene (COL7A1). Hum Mutat 1997;10(5)

sive Dystrophic Epidermolysis Bullosa. Genomics 1994;21(1) 160-168.

Removal of the NC-2 Domain. J Cell Biol 1995;131(2) 551-559.

trophic Epidermolysis Bullosa. J Hum Genet 2005;50(10) 543-546.

matol 1999;113(4) 673-686.

584 Gene Therapy - Tools and Potential Applications

671-681.

171-177.

338-347.

tol 2006;126(5) 1006-1012.

losa. J Invest Dermatol 1996;106(4) 766-770.

Formation. Am J Hum Genet 1997;61(3) 599-610.

From Three Korean Patients. J Dermatol Sci 2001;26(2) 125-132.


[50] Woodley DT, Keene DR, Atha T, Huang Y, Lipman K, Li W, and Chen M. Injection of Recombinant Human Type VII Collagen Restores Collagen Function in Dystrophic Epidermolysis Bullosa. Nat Med 2004;10(7) 693-695.

[61] Wally V, Klausegger A, Koller U, Lochmuller H, Krause S, Wiche G, Mitchell LG, Hintner H, and Bauer JW. 5' Trans-Splicing Repair of the PLEC1 Gene. J Invest Der‐

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 587

[62] Remington J, Wang XY, Hou YP, Zhou H, Burnett J, Muirhead T, Uitto J, Keene DR, Woodley DT, and Chen M. Injection of Recombinant Human Type VII Collagen Cor‐ rects the Disease Phenotype in a Murine Model of Dystrophic Epidermolysis Bullosa.

[63] Titeux M, Pendaries V, Zanta-Boussif MA, Decha A, Pironon N, Tonasso L, Mejia JE, Brice A, Danos O, and Hovnanian A. SIN Retroviral Vectors Expressing COL7A1 Un‐ der Human Promoters for Ex Vivo Gene Therapy of Recessive Dystrophic Epider‐

[64] Siprashvili Z, Nguyen NT, Bezchinsky MY, Marinkovich PM, Lane AT, and Khavari PA. Long-Term Type VII Collagen Restoration to Human Epidermolysis Bullosa Skin

[65] Wagner JE, Ishida-Yamamoto A, McGrath JA, Hordinsky M, Keene DR, Woodley DT, Chen M, Riddle MJ, Osborn MJ, Lund T, Dolan M, Blazar BR, and Tolar J. Bone Marrow Transplantation for Recessive Dystrophic Epidermolysis Bullosa. N Engl J

[66] Murauer EM, Gache Y, Gratz IK, Klausegger A, Muss W, Gruber C, Meneguzzi G, Hintner H, and Bauer JW. Functional Correction of Type VII Collagen Expression in

[67] Horiuchi T and Aigaki T. Alternative Trans-Splicing: a Novel Mode of Pre-MRNA

[68] Mitchell LG and McGarrity GJ. Gene Therapy Progress and Prospects: Reprograming

[69] Wally V, Murauer EM, and Bauer JW. Spliceosome-Mediated Trans-Splicing: The

[70] Wally V, Koller U, Murauer EM, Mayr E, Klausegger A, Hintner H, and Bauer JW. Gene Therapy for Autosomal Dominant Diseases. Experimental Dermatology

[71] Mansfield SG, Chao H, and Walsh CE. RNA Repair Using Spliceosome-Mediated

[72] Tahara M, Pergolizzi RG, Kobayashi H, Krause A, Luettich K, Lesser ML, and Crystal RG. Trans-Splicing Repair of CD40 Ligand Deficiency Results in Naturally Regulated Correction of a Mouse Model of Hyper-IgM X-Linked Immunodeficiency. Nat Med

Dystrophic Epidermolysis Bullosa. J Invest Dermatol 2011;131(1) 74-83.

Gene Expression by Trans-Splicing. Gene Ther 2005;12(20) 1477-1485.

matol 2008;128(3) 568-574.

Molecular Therapy 2009;17(1) 26-33.

molysis Bullosa. Mol Ther 2010

Tissue. Hum Gene Ther 2010

Med 2010;363(7) 629-639.

2009;18(3) 283-283.

2004;10(8) 835-841.

Processing. Biol Cell 2006;98(2) 135-140.

Therapeutic Cut and Paste. J Invest Dermatol 2012

RNA Trans-Splicing. Trends Mol Med 2004;10(6) 263-268.


[61] Wally V, Klausegger A, Koller U, Lochmuller H, Krause S, Wiche G, Mitchell LG, Hintner H, and Bauer JW. 5' Trans-Splicing Repair of the PLEC1 Gene. J Invest Der‐ matol 2008;128(3) 568-574.

[50] Woodley DT, Keene DR, Atha T, Huang Y, Lipman K, Li W, and Chen M. Injection of Recombinant Human Type VII Collagen Restores Collagen Function in Dystrophic

[51] Woodley DT, Keene DR, Atha T, Huang Y, Ram R, Kasahara N, and Chen M. Intra‐ dermal Injection of Lentiviral Vectors Corrects Regenerated Human Dystrophic Epi‐ dermolysis Bullosa Skin Tissue in Vivo. Molecular Therapy 2004;10(2) 318-326.

[52] Sat E, Leung KH, Bruckner-Tuderman L, and Cheah KS. Tissue-Specific Expression and Long-Term Deposition of Human Collagen VII in the Skin of Transgenic Mice:

[53] Mecklenbeck S, Compton SH, Mejia JE, Cervini R, Hovnanian A, Bruckner-Tuder‐ man L, and Barrandon Y. A Microinjected COL7A1-PAC Vector Restores Synthesis of Intact Procollagen VII in a Dystrophic Epidermolysis Bullosa Keratinocyte Cell

[54] Ortiz-Urda S, Thyagarajan B, Keene DR, Lin Q, Fang M, Calos MP, and Khavari PA. Stable Nonviral Genetic Correction of Inherited Human Skin Disease. Nat Med

[55] Baldeschi C, Gache Y, Rattenholl A, Bouille P, Danos O, Ortonne JP, Bruckner-Tuder‐ man L, and Meneguzzi G. Genetic Correction of Canine Dystrophic Epidermolysis Bullosa Mediated by Retroviral Vectors. Hum Mol Genet 2003;12(15) 1897-1905.

[56] Gache Y, Baldeschi C, Del Rio M, Gagnoux-Palacios L, Larcher F, Lacour JP, and Me‐ neguzzi G. Construction of Skin Equivalents for Gene Therapy of Recessive Dystro‐

[57] Chen M, Kasahara N, Keene DR, Chan L, Hoeffler WK, Finlay D, Barcova M, Cannon PM, Mazurek C, and Woodley DT. Restoration of Type VII Collagen Expression and

[58] Goto M, Sawamura D, Ito K, Abe M, Nishie W, Sakai K, Shibaki A, Akiyama M, and Shimizu H. Fibroblasts Show More Potential As Target Cells Than Keratinocytes in COL7A1 Gene Therapy of Dystrophic Epidermolysis Bullosa. J Invest Dermatol

[59] Goto M, Sawamura D, Nishie W, Sakai K, McMillan JR, Akiyama M, and Shimizu H. Targeted Skipping of a Single Exon Harboring a Premature Termination Codon Mu‐ tation: Implications and Potential for Gene Correction Therapy for Selective Dystro‐ phic Epidermolysis Bullosa Patients. Journal of Investigative Dermatology

[60] Woodley DT, Krueger GG, Jorgensen CM, Fairley JA, Atha T, Huang Y, Chan L, Keene DR, and Chen M. Normal and Gene-Corrected Dystrophic Epidermolysis Bul‐ losa Fibroblasts Alone Can Produce Type VII Collagen at the Basement Membrane

Function in Dystrophic Epidermolysis Bullosa. Nat Genet 2002;32(4) 670-675.

phic Epidermolysis Bullosa. Human Gene Therapy 2004;15(10) 921-933.

Implications for Gene Therapy. Gene Ther 2000;7(19) 1631-1639.

Epidermolysis Bullosa. Nat Med 2004;10(7) 693-695.

Line. Hum Gene Ther 2002;13(13) 1655-1662.

2002;8(10) 1166-1170.

586 Gene Therapy - Tools and Potential Applications

2006;126(4) 766-772.

2006;126(12) 2614-2620.

Zone. J Invest Dermatol 2003;121(5) 1021-1028.


[73] Chao H, Mansfield SG, Bartel RC, Hiriyanna S, Mitchell LG, Garcia-Blanco MA, and Walsh CE. Phenotype Correction of Hemophilia A Mice by Spliceosome-Mediated RNA Trans-Splicing. Nat Med 2003;9(8) 1015-1019.

[84] Kierlin-Duncan MN and Sullenger BA. Using 5'-PTMs to Repair Mutant Beta-Globin

Gene Therapy for the *COL7A1* Gene http://dx.doi.org/10.5772/51926 589

[85] Wally V, Brunner M, Lettner T, Wagner M, Koller U, Trost A, Murauer EM, Hainzl S, Hintner H, and Bauer JW. K14 MRNA Reprogramming for Dominant Epidermolysis

[86] Rindt H, Yen PF, Thebeau CN, Peterson TS, Weisman GA, and Lorson CL. Replace‐

[87] Lorain S, Peccate C, Le Hir M, and Garcia L. Exon Exchange Approach to Repair

[88] Koller U, Wally V, Mitchell LG, Klausegger A, Murauer EM, Mayr E, Gruber C, Hainzl S, Hintner H, and Bauer JW. A Novel Screening System Improves Genetic

Correction by Internal Exon Replacement. Nucleic Acids Res 2011;39(16) e108-

[89] Wally V, Koller U, Bauer JW. High-Throughput Screening for Highly Functional RNA-Trans-Splicing Molecules: Correction of Plectin in Epidermolysis Bullosa Sim‐ plex. In: Plaseska-Karanfilska D (ed.) Human Genetic Diseases. Rijeka: InTech; 2011. p223-240 Available from: http://www.intechopen.com/books/human-genetic-diseases

ment of Huntingtin Exon 1 by Trans-Splicing. Cell Mol Life Sci 2012

Duchenne Dystrophin Transcripts. PLoS One 2010;5(5) e10894-

Bullosa Simplex. Hum Mol Genet 2010;19(23) 4715-4725.

Transcripts. RNA 2007

(Accessed 3 October 2011)


[84] Kierlin-Duncan MN and Sullenger BA. Using 5'-PTMs to Repair Mutant Beta-Globin Transcripts. RNA 2007

[73] Chao H, Mansfield SG, Bartel RC, Hiriyanna S, Mitchell LG, Garcia-Blanco MA, and Walsh CE. Phenotype Correction of Hemophilia A Mice by Spliceosome-Mediated

[74] Puttaraju M, DiPasquale J, Baker CC, Mitchell LG, and Garcia-Blanco MA. Messen‐ ger RNA Repair and Restoration of Protein Function by Spliceosome-Mediated RNA

[75] Liu X, Luo M, Zhang LN, Yan Z, Zak R, Ding W, Mansfield SG, Mitchell LG, and En‐ gelhardt JF. Spliceosome-Mediated RNA Trans-Splicing With Recombinant Adeno-Associated Virus Partially Restores Cystic Fibrosis Transmembrane Conductance Regulator Function to Polarized Human Cystic Fibrosis Airway Epithelial Cells.

[76] Dallinger G, Puttaraju M, Mitchell LG, Yancey KB, Yee C, Klausegger A, Hintner H, and Bauer JW. Development of Spliceosome-Mediated RNA Trans-Splicing (SMaRT (TM)) for the Correction of Inherited Skin Diseases. Experimental Dermatology

[77] Rodriguez-Martin T, Garcia-Blanco MA, Mansfield SG, Grover AC, Hutton M, Yu Q, Zhou J, Anderton BH, and Gallo JM. Reprogramming of Tau Alternative Splicing by Spliceosome-Mediated RNA Trans-Splicing: Implications for Tauopathies. Proc Natl

[78] Zayed H, Xia L, Yerich A, Yant SR, Kay MA, Puttaraju M, McGarrity GJ, Wiest DL, McIvor RS, Tolar J, and Blazar BR. Correction of DNA Protein Kinase Deficiency by Spliceosome-Mediated RNA Trans-Splicing and Sleeping Beauty Transposon Deliv‐

[79] Chen HY, Kathirvel P, Yee WC, and Lai PS. Correction of Dystrophia Myotonica Type 1 Pre-MRNA Transcripts by Artificial Trans-Splicing. Gene Ther 2009;16(2)

[80] Coady TH and Lorson CL. Trans-Splicing-Mediated Improvement in a Severe Mouse

[81] Wang J, Mansfield SG, Cote CA, Du Jiang P, Weng K, Amar MJA, Brewer BH, Rema‐ ley AT, McGarrity GJ, Garcia-Blanco MA, and Puttaraju M. Trans-Splicing Into High‐ ly Abundant Albumin Transcripts for Production of Therapeutic Proteins In Vivo.

[82] Gruber C, Gratz IK, Murauer EM, Mayr E, Koller U, Bruckner-Tuderman L, Mene‐ guzzi G, Hintner H, and Bauer JW. Spliceosome-Mediated RNA Trans-Splicing Facil‐ itates Targeted Delivery of Suicide Genes to Cancer Cells. Mol Cancer Ther 2011

[83] Mansfield SG, Clark RH, Puttaraju M, Kole J, Cohn JA, Mitchell LG, and Garcia-Blan‐ co MA. 5' Exon Replacement and Repair by Spliceosome-Mediated RNA Trans-Splic‐

Model of Spinal Muscular Atrophy. J Neurosci 2010;30(1) 126-130.

RNA Trans-Splicing. Nat Med 2003;9(8) 1015-1019.

Trans-Splicing. Molecular Therapy 2001;4(2) 105-114.

Hum Gene Ther 2005;16(9) 1116-1123.

Acad Sci U S A 2005;102(43) 15659-15664.

ery. Mol Ther 2007;15(7) 1273-1279.

Molecular Therapy 2009;17(2) 343-351.

ing. RNA 2003;9(10) 1290-1297.

2003;12(1) 37-46.

588 Gene Therapy - Tools and Potential Applications

211-217.


**Chapter 24**

**Molecular Therapy for Lysosomal Storage Diseases**

Lysosomes are organella involving the catabolism of biomolecules extracellularly and intra‐ cellularly incorporated, which contain more than 60 distinct acidic hydrolases (lysosomal enzymes) and their co-factors. Lysosomal storage diseases (LSDs) are caused by germ-line gene mutations encoding lysosomal enzymes, their activator proteins, integral membrane proteins, cholesterol transporters and proteins concerning intracellular trafficking of lysoso‐ mal enzymes [1,2]. The LSDs associate with excessive accumulation of natural substrates, in‐ cluding glycoconjugates (glycosphingolipids, oligosaccharides derived from glycoproteins, and glycosaminoglycans from proteoglycans) as well as heterogeneous manifestations in both visceral and nervous systems [1,2]. LSDs comprise greater than 40 diseases, of which incidence is about 1 per 100 thousand births, and recognized as so-called 'Orphan diseases'. In the biosynthesis of lysosomal matrix enzymes, newly synthesized enzymes are N-glyco‐ sylated in the endoplasmic reticulum (ER) and then phosphorylated in the Golgi apparatus on the 6 position of the terminal mannose residues (M6P) via two step reactions catalyzed by Golgi-localized phosphotransferase and uncovering enzyme necessary to expose the ter‐ minal M6P residues [3,4]. The M6P-carrying enzymes then bind the cation-dependent man‐ nose 6-phosphate receptor (CD-M6PR) at physiological pH in the Golgi. The enzyme– receptor complex is then transported to late-endosomes where the M6P-carrying enzymes dissociate from the receptor at acidic pH, while the CD-M6PR then traffics back to the Golgi as a shuttle. M6P-carrying enzymes are delivered to lysosomes via fusion with late-endo‐ somes. A small percentage of lysosomal enzymes is known secreted from the cell. The se‐ creted M6P-carrying enzymes or the dephosphorylated enzyme with terminal mannose residues can then bind either cation-independent M6P/IGFII receptor (CI-M6PR) or man‐ nose receptor (MR) on the plasma membrane [4,5]. Thus, the extracellular lysosomal en‐ zymes can be endocytosed via both glycan receptors to be delivered to the lysosomes where

Daisuke Tsuji and Kohji Itoh

http://dx.doi.org/10.5772/54074

**1. Introduction**

Additional information is available at the end of the chapter

the captured enzymes can exhibit their normal catabolic functions.

© 2013 Tsuji and Itoh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

### **Molecular Therapy for Lysosomal Storage Diseases**

Daisuke Tsuji and Kohji Itoh

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54074

#### **1. Introduction**

Lysosomes are organella involving the catabolism of biomolecules extracellularly and intra‐ cellularly incorporated, which contain more than 60 distinct acidic hydrolases (lysosomal enzymes) and their co-factors. Lysosomal storage diseases (LSDs) are caused by germ-line gene mutations encoding lysosomal enzymes, their activator proteins, integral membrane proteins, cholesterol transporters and proteins concerning intracellular trafficking of lysoso‐ mal enzymes [1,2]. The LSDs associate with excessive accumulation of natural substrates, in‐ cluding glycoconjugates (glycosphingolipids, oligosaccharides derived from glycoproteins, and glycosaminoglycans from proteoglycans) as well as heterogeneous manifestations in both visceral and nervous systems [1,2]. LSDs comprise greater than 40 diseases, of which incidence is about 1 per 100 thousand births, and recognized as so-called 'Orphan diseases'.

In the biosynthesis of lysosomal matrix enzymes, newly synthesized enzymes are N-glyco‐ sylated in the endoplasmic reticulum (ER) and then phosphorylated in the Golgi apparatus on the 6 position of the terminal mannose residues (M6P) via two step reactions catalyzed by Golgi-localized phosphotransferase and uncovering enzyme necessary to expose the ter‐ minal M6P residues [3,4]. The M6P-carrying enzymes then bind the cation-dependent man‐ nose 6-phosphate receptor (CD-M6PR) at physiological pH in the Golgi. The enzyme– receptor complex is then transported to late-endosomes where the M6P-carrying enzymes dissociate from the receptor at acidic pH, while the CD-M6PR then traffics back to the Golgi as a shuttle. M6P-carrying enzymes are delivered to lysosomes via fusion with late-endo‐ somes. A small percentage of lysosomal enzymes is known secreted from the cell. The se‐ creted M6P-carrying enzymes or the dephosphorylated enzyme with terminal mannose residues can then bind either cation-independent M6P/IGFII receptor (CI-M6PR) or man‐ nose receptor (MR) on the plasma membrane [4,5]. Thus, the extracellular lysosomal en‐ zymes can be endocytosed via both glycan receptors to be delivered to the lysosomes where the captured enzymes can exhibit their normal catabolic functions.

© 2013 Tsuji and Itoh; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Many therapeutic approaches developed for LSDs, including bone marrow transplantation (BMT), stem cell-based therapy (SCT), enzyme replacement therapy (ERT) and *ex vivo* gene therapy, are based on this physiologic secretion/uptake system (cross-correction). In success‐ ful intravenous ERT for LSDs involving mainly visceral symptoms, including type 1 Gauch‐ er disease [6,7] and mucopolysaccharidosis I (MPS I) [8], MPS VI [9], Fabry [10,11], and Pompe diseases [12,13], either MR or CI-M6PR have been utilized as delivery targets of the recombinant lysosomal enzyme drugs produced by mammalian cell lines including CHO cells and human fibrosarcoma cells. However, intravenous ERT has several disadvantages: i) long-life therapy, ii) requirement of large amounts of recombinant human enzymes, iii) high cost, iv) immune response to the exogenous enzymes [14], and v) ineffectiveness to‐ wards LSDs involving neurological signs because of the blood–brain barrier (BBB), although clinical trials are under-going of intrathecal ERT for MPS type I [15], II and IIIB patients. SCT using hematopoietic stem cell (HSC), hematopoietic precursor cell (HPC) and mesenchymal stem cell (MSC) derived from bone marrows has also been utilized as a treatment for LSDs animal models and patients [16-20]. BMT and SCT are based on that stem cells distribute widely *in vivo* as sources continuously producing the deficient enzymes. However, applica‐ tion of BMT is generally limited to LSDs that show a clear beneficial response and for which ERT is not available.

encoding the Hex α-subunit, and *HEXB* (locus 5q13) encoding the Hex β-subunit, respec‐ tively [34,35]. The genes exhibit sequence homology, and the gene products exhibit 57% sim‐ ilarity in amino acid sequence. In TSD, the genetic defect of *HEXA* causes a deficiency of HexA (αβ with excessive accumulation of GM2 in the central nervous system (CNS), result‐ ing in neurological disorders, including weakness, startle reaction, early blindness, progres‐ sive cerebellar ataxia, psychomotor retardation, and cherry red spots, and macrocephaly. In SD, the inherited defect of *HEXB* leads to simultaneous deficiencies of HexA and HexB with accumulation of GM2 in the CNS and of oligosaccharides carrying the terminal N-acetylhex‐ osaminyl residues at their non-reducing ends, resulting in involvement of the visceral or‐ gans including cardiomegaly and minimal hepatosplenomegaly as well as the neurological symptoms. GM2 gangliosidosis AB variant (MIM 272750) is very rare autosomal recessive LSD caused by the gene mutation of GM2 activator protein (*GM2A* locus 5q31.3-q33.1) [34,36]. The gene product GM2A specifically binds GM2 to pull up from membranes in lyso‐ somes, and present it to HexA for degradation of GM2. The deficiency of GM2A also cause the GM2 accumulation and neurological symptoms similar to those of TSD and SD [34,36]. The pathogenic mechanisms of these GM2 ganliosidoses has not been fully elucidated, al‐ though neurodegeneration and neuroinflammation have been reported to contribute to the

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GM2 gangliosidoses including TSD and SD exhibit a spectrum of clinical phenotypes, which vary from the severe infantile form (classical type), which is of early onset and fatal culmi‐ nating in death before the age of 4 years, to the late-onset and less severe form (atypical type), which allows survival into childhood (subacute form) or adulthood (chronic form) [34,35,37,38]. Many mutations have been identified for each gene, including missense, dele‐ tion and insertion mutations [34,39-41]. Structural information on the basis of the crystal structures of human Hex B [42,43] and HexA [44] allowed us to predict the effects of mis‐ sense mutaitons identified in TSD [34,39,40] and SD [34,39,41] on the protein structures of mutated gene products. According to these reports, the β-subunit of Hex comprises two do‐ mains (domain I and II). Domain I has an α/β topology, and domain II is folded into a (β/α)8 barrel with the active site pocket at the *C*-termini of the β-strands. An extrahelix that follows the eighth helix of the (β/α)8-barrel is located between domain I and the barrel structure. On‐ ly the α-subunit active site can hydrolyze GM2 due to a flexible loop structure that is re‐ moved post-translationally from β, and to the presence of αN423 and αR424. The loop structure is involved in binding the GM2A, while αR424 is critical for binding the carboxy‐ late group of the N-acetylneuraminic acid residue of GM2. The β-subunit lacks these key residues and has βD452 and βL453 in their place. The β-subunit therefore cleaves only neu‐ tral substrates efficiently. The representative amino acid substitutions have been reported in the α-subunit, including R170W, R178H, W420C, C458Y, L484P, R499C/H, and R504C/H, as well as in the β-subunit, R505Q and C534Y. The dysfunctional and destabilizing defects in Hex α- and β-subunits well reflect biochemical and phenotypic abnormalities in TSD and SD, respectively. Such structural information should be useful to develop novel therapeutic

pathogenesis [34,35,37,38].

approaches for these disorders [34,45].

On the other hand, the gene replacement therapy (GT) [21-24] has advantages, including i) long-lasting therapy by a single transduction utilizing recombinant viral gene transfer vec‐ tors [25-29], ii) cross-correction effects, and iii) possible CNS-directed application to LSDs in‐ volving neurological symptoms [23,24,30-33], whereas GT has disadvantages, including i) low levels and persistence of expression in all tissues of patients, ii) incomplete response to therapy dependent on clinical phenotypes, and iii) insertional mutagenesis resulting in neo‐ plasia. GT is one of the promising therapeutic approaches, especially toward LSDs involving CNS symptoms. In this review, we focus on the challenges to develop the CNS-directed GT for LSDs including GM2 gangliosidoses.

#### **2. GM2 gangliosidoses**

Lysosomal β-hexosaminidase (Hex, EC 3.2.1.52) is a glycosidase that catalyzes the hydroly‐ sis of terminal N-acetylhexosamine residues at the non-reducing ends of oligosaccharides of glycoconjugates [34,35]. There are two major Hex isozymes in mammals including man, HexA (αβ, a heterodimer of α- and β-subunits) and HexB (ββ, a homodimer of β-subunit), and a minor unstable isozyme, HexS (αα, a homodimer of α-subunit). All these Hex iso‐ zymes can degrade terminal β-1,4 linked N-acetylglucosamine (GlcNAc) and N-acetylgalac‐ tosamine (GalNAc) residues, while only HexA and HexS prefer negatively charged substrates and cleave off the terminal N-acetylglucosamine 6-sulfate residues in keratan sul‐ fate. Hex A is essential for cleavage of the GalNAc residue from GM2 ganglioside (GM2) in co-operation with GM2 activator protein (GM2A) [34,35].

Tay-Sachs disease (TSD) (MIM 272800) and Sandhoff disease (SD) (MIM 268800) are autoso‐ mal recessive GM2 gangliosidoses caused by germ-line mutations of *HEXA* (locus 15q23-24)

encoding the Hex α-subunit, and *HEXB* (locus 5q13) encoding the Hex β-subunit, respec‐ tively [34,35]. The genes exhibit sequence homology, and the gene products exhibit 57% sim‐ ilarity in amino acid sequence. In TSD, the genetic defect of *HEXA* causes a deficiency of HexA (αβ with excessive accumulation of GM2 in the central nervous system (CNS), result‐ ing in neurological disorders, including weakness, startle reaction, early blindness, progres‐ sive cerebellar ataxia, psychomotor retardation, and cherry red spots, and macrocephaly. In SD, the inherited defect of *HEXB* leads to simultaneous deficiencies of HexA and HexB with accumulation of GM2 in the CNS and of oligosaccharides carrying the terminal N-acetylhex‐ osaminyl residues at their non-reducing ends, resulting in involvement of the visceral or‐ gans including cardiomegaly and minimal hepatosplenomegaly as well as the neurological symptoms. GM2 gangliosidosis AB variant (MIM 272750) is very rare autosomal recessive LSD caused by the gene mutation of GM2 activator protein (*GM2A* locus 5q31.3-q33.1) [34,36]. The gene product GM2A specifically binds GM2 to pull up from membranes in lyso‐ somes, and present it to HexA for degradation of GM2. The deficiency of GM2A also cause the GM2 accumulation and neurological symptoms similar to those of TSD and SD [34,36]. The pathogenic mechanisms of these GM2 ganliosidoses has not been fully elucidated, al‐ though neurodegeneration and neuroinflammation have been reported to contribute to the pathogenesis [34,35,37,38].

Many therapeutic approaches developed for LSDs, including bone marrow transplantation (BMT), stem cell-based therapy (SCT), enzyme replacement therapy (ERT) and *ex vivo* gene therapy, are based on this physiologic secretion/uptake system (cross-correction). In success‐ ful intravenous ERT for LSDs involving mainly visceral symptoms, including type 1 Gauch‐ er disease [6,7] and mucopolysaccharidosis I (MPS I) [8], MPS VI [9], Fabry [10,11], and Pompe diseases [12,13], either MR or CI-M6PR have been utilized as delivery targets of the recombinant lysosomal enzyme drugs produced by mammalian cell lines including CHO cells and human fibrosarcoma cells. However, intravenous ERT has several disadvantages: i) long-life therapy, ii) requirement of large amounts of recombinant human enzymes, iii) high cost, iv) immune response to the exogenous enzymes [14], and v) ineffectiveness to‐ wards LSDs involving neurological signs because of the blood–brain barrier (BBB), although clinical trials are under-going of intrathecal ERT for MPS type I [15], II and IIIB patients. SCT using hematopoietic stem cell (HSC), hematopoietic precursor cell (HPC) and mesenchymal stem cell (MSC) derived from bone marrows has also been utilized as a treatment for LSDs animal models and patients [16-20]. BMT and SCT are based on that stem cells distribute widely *in vivo* as sources continuously producing the deficient enzymes. However, applica‐ tion of BMT is generally limited to LSDs that show a clear beneficial response and for which

On the other hand, the gene replacement therapy (GT) [21-24] has advantages, including i) long-lasting therapy by a single transduction utilizing recombinant viral gene transfer vec‐ tors [25-29], ii) cross-correction effects, and iii) possible CNS-directed application to LSDs in‐ volving neurological symptoms [23,24,30-33], whereas GT has disadvantages, including i) low levels and persistence of expression in all tissues of patients, ii) incomplete response to therapy dependent on clinical phenotypes, and iii) insertional mutagenesis resulting in neo‐ plasia. GT is one of the promising therapeutic approaches, especially toward LSDs involving CNS symptoms. In this review, we focus on the challenges to develop the CNS-directed GT

Lysosomal β-hexosaminidase (Hex, EC 3.2.1.52) is a glycosidase that catalyzes the hydroly‐ sis of terminal N-acetylhexosamine residues at the non-reducing ends of oligosaccharides of glycoconjugates [34,35]. There are two major Hex isozymes in mammals including man, HexA (αβ, a heterodimer of α- and β-subunits) and HexB (ββ, a homodimer of β-subunit), and a minor unstable isozyme, HexS (αα, a homodimer of α-subunit). All these Hex iso‐ zymes can degrade terminal β-1,4 linked N-acetylglucosamine (GlcNAc) and N-acetylgalac‐ tosamine (GalNAc) residues, while only HexA and HexS prefer negatively charged substrates and cleave off the terminal N-acetylglucosamine 6-sulfate residues in keratan sul‐ fate. Hex A is essential for cleavage of the GalNAc residue from GM2 ganglioside (GM2) in

Tay-Sachs disease (TSD) (MIM 272800) and Sandhoff disease (SD) (MIM 268800) are autoso‐ mal recessive GM2 gangliosidoses caused by germ-line mutations of *HEXA* (locus 15q23-24)

ERT is not available.

592 Gene Therapy - Tools and Potential Applications

for LSDs including GM2 gangliosidoses.

co-operation with GM2 activator protein (GM2A) [34,35].

**2. GM2 gangliosidoses**

GM2 gangliosidoses including TSD and SD exhibit a spectrum of clinical phenotypes, which vary from the severe infantile form (classical type), which is of early onset and fatal culmi‐ nating in death before the age of 4 years, to the late-onset and less severe form (atypical type), which allows survival into childhood (subacute form) or adulthood (chronic form) [34,35,37,38]. Many mutations have been identified for each gene, including missense, dele‐ tion and insertion mutations [34,39-41]. Structural information on the basis of the crystal structures of human Hex B [42,43] and HexA [44] allowed us to predict the effects of mis‐ sense mutaitons identified in TSD [34,39,40] and SD [34,39,41] on the protein structures of mutated gene products. According to these reports, the β-subunit of Hex comprises two do‐ mains (domain I and II). Domain I has an α/β topology, and domain II is folded into a (β/α)8 barrel with the active site pocket at the *C*-termini of the β-strands. An extrahelix that follows the eighth helix of the (β/α)8-barrel is located between domain I and the barrel structure. On‐ ly the α-subunit active site can hydrolyze GM2 due to a flexible loop structure that is re‐ moved post-translationally from β, and to the presence of αN423 and αR424. The loop structure is involved in binding the GM2A, while αR424 is critical for binding the carboxy‐ late group of the N-acetylneuraminic acid residue of GM2. The β-subunit lacks these key residues and has βD452 and βL453 in their place. The β-subunit therefore cleaves only neu‐ tral substrates efficiently. The representative amino acid substitutions have been reported in the α-subunit, including R170W, R178H, W420C, C458Y, L484P, R499C/H, and R504C/H, as well as in the β-subunit, R505Q and C534Y. The dysfunctional and destabilizing defects in Hex α- and β-subunits well reflect biochemical and phenotypic abnormalities in TSD and SD, respectively. Such structural information should be useful to develop novel therapeutic approaches for these disorders [34,45].

#### **3. General aspects of gene therapy for LSDs**

Gene therapy (GT) utilizing various vectors for gene transfer has been preclinically and clinically applied for LSDs in recent years [21-33]. Recombinant viral vectors including retroviruses [25,32], adenovirus [26-28], herpes simplex virus (HSV) [33], adeno-associat‐ ed virus (AAV) [29,46-48] and lentiviruses [24,49-51] are utilized currently as the effec‐ tive means of gene transfer and enzyme expression. The retroviruses have been used primarily in *ex vivo* applications to transduce the dividing cells, such as HPC, HSC and other stem cells in culture, which are then transplanted into a recipient. However, the retroviral vectors are not suitable for *in vivo* GT due to lack of ability to transduce nondividing cells. On the other hand, the adenoviruses can infect very efficiently non-divid‐ ing cells. However, the use of the early generation adenoviral vectors has been limited due to their strong antigenicity. In contrast, lentiviruses can infect both dividing and non-dividing cells, and are applicable to both *ex vivo* and *in vivo* GT. AAV vectors are able to transduce many cell types *in vivo* effectively, and it is often used as a safer tool for gene transfer because of the lower immunogenicity.

lating enzymes and reduction of storage materials in the affected tissues [27,55, 56]. However, these therapeutic effects were transient because of the severe immune reac‐ tions directed against the adenoviral vector. Single intravenous administration of a modi‐ fied adenoviral vector to Pompe disease mice was demonstrated to reduce glycogen

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The AAV vector has been also developed as an alternative gene transfer tool for direct *in vivo* GT for LSDs. Intramuscular injection of AAV2 serotype vector [58,] in the murine mod‐ els of Pompe disease, Fabry disease and MPS VII caused high level expression in the muscle tissues but lower levels of circulating enzyme activity [59-61], although the efficacy varied depending on the diseases. Intravenous injection of AAV2 vectors in young adult mice with MPS VII and Fabry disease reduced the lysosomal storage in many tissues [61,62] Significant improvement was observed in MPS VII and MPS I mice following intravenous delivery of AAV2 vector during the neonatal period [28,63]. These findings suggested the effectiveness of AAV vector delivery at early presymptomatic stage to prevent onset rather than delayed

As mentioned above, GT has therapeutic potency for LSDs involving neurological symp‐ toms superior to that of clinically applied intravenous ERT, in which the enzyme cannot cross the BBB. Several CNS-directed *ex vivo* and *in vivo* GT have been performed for animal models of LSDs with brain involvement. Genetically modified bone marrow stromal cells using retroviral vector improved CNS pathology and cognitive function in MPS VII and GM1-gangliosidosis mice following intraventricular transplantation [64, 65]. Genetically modified human neuronal precursor cells (NPCs) differentiated into neurons and astrocytes and expressed β-glucuronidase for at least 6 months after injection into striata of adult MPS VII model mice. However, the cells did not migrate and correction was limited to regions adjacent to the transplantation site [66] *In vivo* GT of metachromatic leukodystrophy (MLD) by lentiviral vector corrected neuropathology and protected against learning impairments in the model mice [49]. CNS-directed in vivo GT using AAV vectors have been demonstrated to have therapeutic effects on the mouse model of LSDs involving neurological signs, in‐ cluding MPS IIIB [67], MPS VII [68], Globoid cell leukodystrophy (GLD) [69], Nieman-Pick A (NPA)[70] and α-mannosidosis [71] by intracranial administration of recombinant AAV vectors. Thus, AAV vectors exhibit a number of properties that have made this vector sys‐ tem an excellent choice for both CNS gene therapy and basic neurobiological investigations. In vivo, the preponderance of AAV vector transduction occurs in neurons where it is possi‐ ble to obtain long-term, stable gene expression with very little accompanying toxicity. Pro‐ moter selection, however, significantly influences the pattern and longevity of neuronal transduction distinct from the tropism inherent to AAV vectors. AAV vectors have success‐ fully manipulated CNS function using a wide variety of approaches including expression of foreign genes, expression of endogenous genes, expression of antisense RNA and expression of RNAi. With the discovery and characterization of different AAV serotypes, as well as the creation of novel chimeric serotypes, the potential patterns of in vivo vector transduction have been expanded substantially, offering alternatives to the more studied AAV 2 serotype. Furthermore, the development of specific AAV chimeras offers the potential to further re‐

storage with minimal immune response [57].

intervention for progressive LSDs.

The application of recombinant viral vectors varies dependently on several factors, includ‐ ing ease of vector delivery, expression level in cell types and target tissues and organs main‐ ly affected with LSD. At initial stage of development of GT for LSDs, the *ex vivo* transduction of HPC derived from type 1 Gaucher disease [25] and fibroblasts obtained from MPS VII model mice [32] using retroviral vectors was successful to secrete high levels of the enzymes and corrected the deficiencies. The *ex vivo* GT using retroviral vector and au‐ tologous HSC or HPC (human CD34+ cells) derived from bone marrow of the patient as do‐ nor cells for transplantation was clinically applied to type 1 Gaucher disease patients, and demonstrated the production of therapeutically effective levels of enzyme activity, resulting in persistent circulating enzyme available to tissues and organs [52]. The transduced cells al‐ so migrated into many tissues, expressed high levels of enzyme and reduced lysosomal stor‐ age in several critical tissues. However, several problems had been emerged, including less efficiency in transduction of human HSC or HPC using murine-based retroviral vector and difficulty in continuous production of sufficient amounts of recombinant enzymes to main‐ tain the effectiveness.

The lentiviral vector based on human immunodeficiency virus had been expected to over‐ come the limitation of early generation of murine-based retroviral vectors in *ex vivo* GT [53]. Transduction efficiency of human HPC derived from Gaucher disease patient [54] was im‐ proved by using HIV-based lentiviral vector. β-Glucuronidase (GUSB)-deficient mobilized peripheral blood CD34(+) cells from a patient with MPSVII were transduced with a thirdgeneration lentiviral vector encoding human GUSB, and then xenotransplantation to murine model with MPSVII. The corrected cells distributed widely throughout recipient tissues, re‐ sulting in significant therapeutic effects including improvements in biochemical parameters and reduction of the lysosomal distension of several host tissues [24].

Direct *in vivo* GT using adenoviral vector have been preclinically applied to murine mod‐ els with Pompe, Fabry, and Wolman diseases, resulted in sufficient expression of circu‐ lating enzymes and reduction of storage materials in the affected tissues [27,55, 56]. However, these therapeutic effects were transient because of the severe immune reac‐ tions directed against the adenoviral vector. Single intravenous administration of a modi‐ fied adenoviral vector to Pompe disease mice was demonstrated to reduce glycogen storage with minimal immune response [57].

**3. General aspects of gene therapy for LSDs**

594 Gene Therapy - Tools and Potential Applications

for gene transfer because of the lower immunogenicity.

tain the effectiveness.

Gene therapy (GT) utilizing various vectors for gene transfer has been preclinically and clinically applied for LSDs in recent years [21-33]. Recombinant viral vectors including retroviruses [25,32], adenovirus [26-28], herpes simplex virus (HSV) [33], adeno-associat‐ ed virus (AAV) [29,46-48] and lentiviruses [24,49-51] are utilized currently as the effec‐ tive means of gene transfer and enzyme expression. The retroviruses have been used primarily in *ex vivo* applications to transduce the dividing cells, such as HPC, HSC and other stem cells in culture, which are then transplanted into a recipient. However, the retroviral vectors are not suitable for *in vivo* GT due to lack of ability to transduce nondividing cells. On the other hand, the adenoviruses can infect very efficiently non-divid‐ ing cells. However, the use of the early generation adenoviral vectors has been limited due to their strong antigenicity. In contrast, lentiviruses can infect both dividing and non-dividing cells, and are applicable to both *ex vivo* and *in vivo* GT. AAV vectors are able to transduce many cell types *in vivo* effectively, and it is often used as a safer tool

The application of recombinant viral vectors varies dependently on several factors, includ‐ ing ease of vector delivery, expression level in cell types and target tissues and organs main‐ ly affected with LSD. At initial stage of development of GT for LSDs, the *ex vivo* transduction of HPC derived from type 1 Gaucher disease [25] and fibroblasts obtained from MPS VII model mice [32] using retroviral vectors was successful to secrete high levels of the enzymes and corrected the deficiencies. The *ex vivo* GT using retroviral vector and au‐ tologous HSC or HPC (human CD34+ cells) derived from bone marrow of the patient as do‐ nor cells for transplantation was clinically applied to type 1 Gaucher disease patients, and demonstrated the production of therapeutically effective levels of enzyme activity, resulting in persistent circulating enzyme available to tissues and organs [52]. The transduced cells al‐ so migrated into many tissues, expressed high levels of enzyme and reduced lysosomal stor‐ age in several critical tissues. However, several problems had been emerged, including less efficiency in transduction of human HSC or HPC using murine-based retroviral vector and difficulty in continuous production of sufficient amounts of recombinant enzymes to main‐

The lentiviral vector based on human immunodeficiency virus had been expected to over‐ come the limitation of early generation of murine-based retroviral vectors in *ex vivo* GT [53]. Transduction efficiency of human HPC derived from Gaucher disease patient [54] was im‐ proved by using HIV-based lentiviral vector. β-Glucuronidase (GUSB)-deficient mobilized peripheral blood CD34(+) cells from a patient with MPSVII were transduced with a thirdgeneration lentiviral vector encoding human GUSB, and then xenotransplantation to murine model with MPSVII. The corrected cells distributed widely throughout recipient tissues, re‐ sulting in significant therapeutic effects including improvements in biochemical parameters

Direct *in vivo* GT using adenoviral vector have been preclinically applied to murine mod‐ els with Pompe, Fabry, and Wolman diseases, resulted in sufficient expression of circu‐

and reduction of the lysosomal distension of several host tissues [24].

The AAV vector has been also developed as an alternative gene transfer tool for direct *in vivo* GT for LSDs. Intramuscular injection of AAV2 serotype vector [58,] in the murine mod‐ els of Pompe disease, Fabry disease and MPS VII caused high level expression in the muscle tissues but lower levels of circulating enzyme activity [59-61], although the efficacy varied depending on the diseases. Intravenous injection of AAV2 vectors in young adult mice with MPS VII and Fabry disease reduced the lysosomal storage in many tissues [61,62] Significant improvement was observed in MPS VII and MPS I mice following intravenous delivery of AAV2 vector during the neonatal period [28,63]. These findings suggested the effectiveness of AAV vector delivery at early presymptomatic stage to prevent onset rather than delayed intervention for progressive LSDs.

As mentioned above, GT has therapeutic potency for LSDs involving neurological symp‐ toms superior to that of clinically applied intravenous ERT, in which the enzyme cannot cross the BBB. Several CNS-directed *ex vivo* and *in vivo* GT have been performed for animal models of LSDs with brain involvement. Genetically modified bone marrow stromal cells using retroviral vector improved CNS pathology and cognitive function in MPS VII and GM1-gangliosidosis mice following intraventricular transplantation [64, 65]. Genetically modified human neuronal precursor cells (NPCs) differentiated into neurons and astrocytes and expressed β-glucuronidase for at least 6 months after injection into striata of adult MPS VII model mice. However, the cells did not migrate and correction was limited to regions adjacent to the transplantation site [66] *In vivo* GT of metachromatic leukodystrophy (MLD) by lentiviral vector corrected neuropathology and protected against learning impairments in the model mice [49]. CNS-directed in vivo GT using AAV vectors have been demonstrated to have therapeutic effects on the mouse model of LSDs involving neurological signs, in‐ cluding MPS IIIB [67], MPS VII [68], Globoid cell leukodystrophy (GLD) [69], Nieman-Pick A (NPA)[70] and α-mannosidosis [71] by intracranial administration of recombinant AAV vectors. Thus, AAV vectors exhibit a number of properties that have made this vector sys‐ tem an excellent choice for both CNS gene therapy and basic neurobiological investigations. In vivo, the preponderance of AAV vector transduction occurs in neurons where it is possi‐ ble to obtain long-term, stable gene expression with very little accompanying toxicity. Pro‐ moter selection, however, significantly influences the pattern and longevity of neuronal transduction distinct from the tropism inherent to AAV vectors. AAV vectors have success‐ fully manipulated CNS function using a wide variety of approaches including expression of foreign genes, expression of endogenous genes, expression of antisense RNA and expression of RNAi. With the discovery and characterization of different AAV serotypes, as well as the creation of novel chimeric serotypes, the potential patterns of in vivo vector transduction have been expanded substantially, offering alternatives to the more studied AAV 2 serotype. Furthermore, the development of specific AAV chimeras offers the potential to further re‐ fine targeting strategies. These different AAV serotypes also provide a solution to the im‐ mune silencing that proves to be a realistic likelihood given broad exposure of the human population to the AAV 2 serotype. These advantageous CNS properties of AAV vectors have fostered a wide range of clinically relevant applications including Parkinson's disease, lysosomal storage diseases, Canavan's disease, epilepsy, Huntington's disease and ALS. In many cases the proposed therapies have progressed to phase I/II clinical trials. Each individ‐ ual application, however, presents a unique set of challenges that must be solved in order to attain clinically effective gene therapies [72].

We transfected an expression vector plasmid coding the human *HEXB* cDNA to fibroblasts derived from Sandhoff disease mice (*Hexb*-/- mice) and established a transformed murine cell line stably producing the human Hex β-subunit [80]. However, the GM2 accumulated in the transformed murine cell line was not reduced, while co-transfection of the human *HEXA*

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Yamaguchi *et al*. evaluated the systemic *in vivo* GT for *Hexb*-/- mice using cationic liposomemediated plasmid using the *Hexb*-/- mice [81]. The mice received a single intravenous injec‐ tion of two plasmids, encoding the human α and β subunits of hexosaminidase cDNAs. As a result, 10–35% of normal levels of Hex expression, theoretically therapeutic levels, were ach‐ ieved in most visceral organs, but not in the brain, 3 days after injection with decreased lev‐ els by day 7. Histochemical staining confirmed widespread enzyme activity in visceral organs. Both GA2 and GM2 were reduced by almost 10% and 50%, respectively, on day 3,

These findings suggested that brain-directed *in vivo* GT based on direct transduction of the affected tissues by single gene transfer or *ex vivo* GT utilizing double genes (i.e. *HEXA* and *HEXB* cDNAs) for producing the homo-specific HexA should be required to achieve the therapeutic effects on TSD and SD. Since then, studies on the CNS-directed *in vivo* GT and *ex vivo* GT have been performed as two streams of development of molecular therapy for GM2

Bourgoin *et al.* constructed the recombinant adenovirus coding the human *HEXB* cDNA, and transduced the fibroblasts derived from patient with SD resulting in high expression of HexA and HexB activities. They also administered the adenoviral vector intracerebrally to SD mice (*Hexb-/-* mice), and succeeded in expression of near-normal level of enzymatic ac‐ tivity in the entire brain. Co-injection of hyperosmotic concentrations of mannitol with low doses of the adenoviral vector enhanced the vector diffusion in the injected hemisphere without viral cytotoxicity. It was suggested that such combination will allow a high and dif‐

fuse transduction efficiency of adeno-viral vector in the brain with higher safety [82].

effects without adverse effects due to the viral vector [83].

Martino *et al*. also constructed a non-replicating herpes simplex viral (HSV) vector encoding *Hexa* cDNA. They transplanted the encapsuled recombinant HSV into the brain of *Hexa*-/ mice. The diffusion of recombinant HSV and the secreted HexA derived from transduced neural cells corrected the GM2 storage in the brain during one month due to cross correction

Caillaud and co-workers reported that mono and bicistronic lentiviral vectors based on a simian immunodeficiency virus (SIV) containing the human *HEXA* or/and *HEXB* cDNAs were constructed and tested on the fibroblasts derived from the SD patient [84]. The bicis‐ tronic SIV.ASB vector encoding both *HEXA* and *HEXB* cDNAs enabled a massive restora‐ tion of Hex activity. A large reduction of GM2 accumulation in SIV.ASB transduced cells. Moreover, the Hex isozymes secreted by transduced SD fibroblasts were endocytosed in de‐ ficient cells via CI-M6PR, allowing GM2 metabolism restoration in cross-corrected cells.

cDNA resulted in restoration of HexA activity and reduction of GM2 storage.

and by 60% and 70% on day 7 compared with untreated age-matched *Hexb*-/- mice.

gangliosidoses.

**4.2. CNS-directed** *in vivo* **gene therapy**

#### **4. Gene therapy for GM2 gangliosidoses**

#### **4.1. Experimental and preclinical gene therapy using animal models**

GM2 gangliosidoses, including Tay-Sachs disease (TSD), Sandhoff disease (SD) and the AB variant disorder, are characterized by excessive accumulation of GM2 and neurological symptoms due to progressive neurodegeneration and gliosis, as described above. However, there is no effective therapy for GM2 gangliosidoses at present, although we have reported and proposed the clinical potential of the intrathecal ERT using recombinant modified hu‐ man HexA [73] and HexB [74,75] in recent years. It is crucial for treatment of GM2 ganglio‐ sidoses to develop the CNS-directed molecular therapy including such intrathecal ERT, *ex vivo* and *in vivo* GT or the combined methods including SRT [76]. In this chapter, we would focus on the CNS-directed GT and summarize the preclinical approaches using small and large animal models with GM2 gangliosidoses.

At early stage of development of GT for GM2 gangliosidoses, gene transduction of cultured cells was performed by utilizing recombinant vectors (virus or plasmids), and examined the effect of cross correction due to the secreted Hex isozymes. Guidotti, JE. *et al*. constructed a retroviral vector encoding for the α-subunit of human HexA (*HEXA* cDNA) and transduced the HexA-deficient fibroblasts derived from Tay-Sach disease model mice (*Hexa*-/- mice) [77]. Transduced cells overexpressed the human Hex α-subunit to produce the chimeric HexA composed of human α-subunit and murine β-subunit, which were taken up via CI-M6PR by non-transduced cells and exhibited the cross-correction effect.

On the other hand, Martino *et al*. also constructed a retroviral vector encoding for the α-sub‐ unit of human HexA (*HEXA* cDNA) and transduced NIH3T3 murine fibroblasts, resulting in production of large amount of human Hex activity. The secreted Hex was incorporated into the fibroblasts derived from TSD patient, but failed to correct intracellular GM2 storage, probably because of the absence of HexA isozyme sufficient for degrading the accumulated GM2 [78]. Akli S et al. produced a replication-deficient recombinant adenovirus (AdRSV) coding the human *HEXA* cDNA, and transduced the fibroblasts derived from TSD patients. Transdused cells restored the Hex activity ranging from 40 to 84% of the normal, and secret‐ ed the Hex a-subunit, which were delivered to lysosomes and degraded the GM2 accumu‐ lated in TSD fibroblasts [79].

We transfected an expression vector plasmid coding the human *HEXB* cDNA to fibroblasts derived from Sandhoff disease mice (*Hexb*-/- mice) and established a transformed murine cell line stably producing the human Hex β-subunit [80]. However, the GM2 accumulated in the transformed murine cell line was not reduced, while co-transfection of the human *HEXA* cDNA resulted in restoration of HexA activity and reduction of GM2 storage.

Yamaguchi *et al*. evaluated the systemic *in vivo* GT for *Hexb*-/- mice using cationic liposomemediated plasmid using the *Hexb*-/- mice [81]. The mice received a single intravenous injec‐ tion of two plasmids, encoding the human α and β subunits of hexosaminidase cDNAs. As a result, 10–35% of normal levels of Hex expression, theoretically therapeutic levels, were ach‐ ieved in most visceral organs, but not in the brain, 3 days after injection with decreased lev‐ els by day 7. Histochemical staining confirmed widespread enzyme activity in visceral organs. Both GA2 and GM2 were reduced by almost 10% and 50%, respectively, on day 3, and by 60% and 70% on day 7 compared with untreated age-matched *Hexb*-/- mice.

These findings suggested that brain-directed *in vivo* GT based on direct transduction of the affected tissues by single gene transfer or *ex vivo* GT utilizing double genes (i.e. *HEXA* and *HEXB* cDNAs) for producing the homo-specific HexA should be required to achieve the therapeutic effects on TSD and SD. Since then, studies on the CNS-directed *in vivo* GT and *ex vivo* GT have been performed as two streams of development of molecular therapy for GM2 gangliosidoses.

#### **4.2. CNS-directed** *in vivo* **gene therapy**

fine targeting strategies. These different AAV serotypes also provide a solution to the im‐ mune silencing that proves to be a realistic likelihood given broad exposure of the human population to the AAV 2 serotype. These advantageous CNS properties of AAV vectors have fostered a wide range of clinically relevant applications including Parkinson's disease, lysosomal storage diseases, Canavan's disease, epilepsy, Huntington's disease and ALS. In many cases the proposed therapies have progressed to phase I/II clinical trials. Each individ‐ ual application, however, presents a unique set of challenges that must be solved in order to

GM2 gangliosidoses, including Tay-Sachs disease (TSD), Sandhoff disease (SD) and the AB variant disorder, are characterized by excessive accumulation of GM2 and neurological symptoms due to progressive neurodegeneration and gliosis, as described above. However, there is no effective therapy for GM2 gangliosidoses at present, although we have reported and proposed the clinical potential of the intrathecal ERT using recombinant modified hu‐ man HexA [73] and HexB [74,75] in recent years. It is crucial for treatment of GM2 ganglio‐ sidoses to develop the CNS-directed molecular therapy including such intrathecal ERT, *ex vivo* and *in vivo* GT or the combined methods including SRT [76]. In this chapter, we would focus on the CNS-directed GT and summarize the preclinical approaches using small and

At early stage of development of GT for GM2 gangliosidoses, gene transduction of cultured cells was performed by utilizing recombinant vectors (virus or plasmids), and examined the effect of cross correction due to the secreted Hex isozymes. Guidotti, JE. *et al*. constructed a retroviral vector encoding for the α-subunit of human HexA (*HEXA* cDNA) and transduced the HexA-deficient fibroblasts derived from Tay-Sach disease model mice (*Hexa*-/- mice) [77]. Transduced cells overexpressed the human Hex α-subunit to produce the chimeric HexA composed of human α-subunit and murine β-subunit, which were taken up via CI-M6PR by

On the other hand, Martino *et al*. also constructed a retroviral vector encoding for the α-sub‐ unit of human HexA (*HEXA* cDNA) and transduced NIH3T3 murine fibroblasts, resulting in production of large amount of human Hex activity. The secreted Hex was incorporated into the fibroblasts derived from TSD patient, but failed to correct intracellular GM2 storage, probably because of the absence of HexA isozyme sufficient for degrading the accumulated GM2 [78]. Akli S et al. produced a replication-deficient recombinant adenovirus (AdRSV) coding the human *HEXA* cDNA, and transduced the fibroblasts derived from TSD patients. Transdused cells restored the Hex activity ranging from 40 to 84% of the normal, and secret‐ ed the Hex a-subunit, which were delivered to lysosomes and degraded the GM2 accumu‐

attain clinically effective gene therapies [72].

596 Gene Therapy - Tools and Potential Applications

**4. Gene therapy for GM2 gangliosidoses**

large animal models with GM2 gangliosidoses.

lated in TSD fibroblasts [79].

non-transduced cells and exhibited the cross-correction effect.

**4.1. Experimental and preclinical gene therapy using animal models**

Bourgoin *et al.* constructed the recombinant adenovirus coding the human *HEXB* cDNA, and transduced the fibroblasts derived from patient with SD resulting in high expression of HexA and HexB activities. They also administered the adenoviral vector intracerebrally to SD mice (*Hexb-/-* mice), and succeeded in expression of near-normal level of enzymatic ac‐ tivity in the entire brain. Co-injection of hyperosmotic concentrations of mannitol with low doses of the adenoviral vector enhanced the vector diffusion in the injected hemisphere without viral cytotoxicity. It was suggested that such combination will allow a high and dif‐ fuse transduction efficiency of adeno-viral vector in the brain with higher safety [82].

Martino *et al*. also constructed a non-replicating herpes simplex viral (HSV) vector encoding *Hexa* cDNA. They transplanted the encapsuled recombinant HSV into the brain of *Hexa*-/ mice. The diffusion of recombinant HSV and the secreted HexA derived from transduced neural cells corrected the GM2 storage in the brain during one month due to cross correction effects without adverse effects due to the viral vector [83].

Caillaud and co-workers reported that mono and bicistronic lentiviral vectors based on a simian immunodeficiency virus (SIV) containing the human *HEXA* or/and *HEXB* cDNAs were constructed and tested on the fibroblasts derived from the SD patient [84]. The bicis‐ tronic SIV.ASB vector encoding both *HEXA* and *HEXB* cDNAs enabled a massive restora‐ tion of Hex activity. A large reduction of GM2 accumulation in SIV.ASB transduced cells. Moreover, the Hex isozymes secreted by transduced SD fibroblasts were endocytosed in de‐ ficient cells via CI-M6PR, allowing GM2 metabolism restoration in cross-corrected cells. Therefore, the bicistronic lentivector supplying both HexA and HexB isozymes may provide a potential therapeutic tool for the treatment of TSD and SD. A mechanistic link was demon‐ strated among GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplas‐ mic reticulum Ca2+-ATPase (SERCA) activity, and axonal outgrowth. Arfi *et al*. examined the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neu‐ rons derived from embryonic *Hexb*-/- mice, which were transduced with the lentival vector [85]. Normal axonal growth rates, the rate of Ca2+ uptake via the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity and the sensitivity of the neurons to thapsigargininduced cell death were restored concomitantly with a decrease in GM2 and GA2 levels. Thus, the bicistronic SIV.ASB vector was revealed to reverse the biochemical defects and down-stream consequences in SD neurons, suggesting its potential of systemic and CNS-di‐ rected *in vivo* GT. Kyrkanides S. *et al.* performed the system in vivo GT utilizing the re‐ combinant lentiviral vector FIV coding human *HEXB* cDNA to the neonatal *Hexb*-/- mice via intrapenitoneal administration. They also demonstrated the distribution of Hex isozymes in‐ to the CNS, including periventricular areas of the cerebrum as well as in the cerebellar cor‐ tex, and reduction of GM2 accumulated in these areas [86].

reduction therapy (SRT) utilizing deoxynojirimycin derivatives [91]. Transduction of neural cells derived from *Hexa*-/- and *Hexb*-/- mice by recombinant viral vectors was performed.

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599

Lacorazza HD *et al.* constructed the ecotropic retrovirus encoding the human *HEXA* cDNA and transduced multipotent neural progenitor cell lines, which stably expressed and secret‐ ed high levels of active HexA and cross-corrected the metabolic defect including GM2 stor‐ age in TSD fibroblastic cell line. The genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and neonatal mice, in which substantial amounts of human Hex α-subunit and activity were observed throughout the brain enough

Tsuji D. *et al.* constructed a recombinant lentiviral vector encoding the murine *Hexb* cDNA, and transduced microglial cells established from the brains of *Hexb*-/- mice [50]. Transduced microglial cells produced and secreted Hex activity, in which the intracellularly accumulat‐ ed GM2 and oligosaccharides with terminal N-acetylglucosamine residues were reduced.

Mesenchymal stem cells (MSCs) derived from bone marrow stromal cells are one of the can‐ didates for autologous donor cells for ex vivo GT, and have the multipotency to differentiate under specific culture conditions into other cell types such as osteoblasts, adipocytes, and chondrocytes [93,] as well as into neural lineages [94]. Recently, we established MSCs de‐ rived from bone marrow of adult *Hexb*-/- mice. The MSCs expressed cell-type specific mark‐ ers, including CD29, CD90 and CD54, but not CD45, and exhibited the ability to differentiate into various cell types, including neuron-restricted precursor cells (NRPs) expressing N-CAM carrying polysialic acid (PSA-NCAM). We produced a bicistronic retroviral vector (MSV-*modB*) encoding for the modified human *HEXB* cDNAs (*modB*) causing six α-subunit type amino acid substitutions as well as *EGFP* gene [75]. The gene products, modified HexB (modB, a homodimer of the modified β-subunits) different from the wild-type HexB, can recognize negatively charged artificial substrates and bind to GM2A to exhibit GM2-degrad‐ ing activity. We transduced the MSCs derived from *Hexb*-/- mice (SD MSCs) with the MSV*modB*, resulting in restoration of HexA-like activity and reduction of the accumulated GM2 and GlcNAc-oligosaccharides. The modB was also secreted from the transduced SD MSCs. In addition, we performed intraventricular engraftment of the transduced SD MSCs express‐ ing *modB* into the brain of *Hexb*-/- mice. As a result, the injected transduced SD MSCs ex‐ pressing HexA-like activity and EGFP were observed in periventricular region of the brain (Figure 1). Reduction of the immunoreactivity towards natural substrates including GM2 and GlcNAc-oligosaccharides were also observed around the periventricular region of *Hexb*-/- mice brain (Figure 2). These results suggest that genetically modified MSCs can be utilized as a brain-directed donor cells for *ex vivo* GT for LSDs involving neurological mani‐

Lee J-P. *et al.* demonstrated intracranial transplantation of neural stem cells (NSCs) delayed onset, improved motor function, reduced GM2 storage and prolonged life span in the *Hexb*-/ mice partly due to the cross correction effect of the Hex isozymes secreted from NSCs. Hu‐ man NSCs derived directly from the CNS and secondarily induced from embryonic stem

Transduced microglial cell line was expected as a donor for brain-directed *ex vivo* GT.

for therapeutic effect in TSD [92].

festations, including Tay-Sachs and Sandhoff diseases.

Cachon-Gonzalez *et al*. has reported that the *Hexb*-/- mice treated by stereotaxic intracranial inoculation of recombinant adeno-associated viral (rAAV) vectors encoding the human *HEXA* and *HEXB* cDNAs, including an HIV tat sequence as a protein transduction domain (PTD), to enhance protein expression and distribution [87]. *Hexb*-/- mice survived for >1 year with sustained, widespread and abundant enzyme delivery in the CNS. Onset of the disease was delayed with preservation of motor function; inflammation and GM2 storage in the brain and spinal cord was reduced. Gene delivery of the human HexA (αβ) by using AAV vectors has realistic potential for treating the TS and SD patients. Sargeant TJ. *et al.* demon‐ strated that intracranial co -injection of rAAV serotype 2/1 (rAAV2/1) vectors encoding the human *HEXA* and *HEXB* cDNAs prevents neuronal loss in the *Hexb*-/- mice brain tissues, in‐ cluding thalamus, brainstem and spinal cord, and correlated with increased lifespan [88]. Moreover, they performed intracranial co-injection of rAAV2/1 vectors into 1-month-old *Hexb*-/- mice [89]. As a result, the treated mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of Hex isozymes and their global distribution via axo‐ nal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue—long-term protein expression by transduced brain parenchy‐ ma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective en‐ zyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by intracranial vector delivery.

#### **4.3. CNS-directed** *ex vivo* **gene therapy**

*Ex vivo* GT for GM2 gangliosidoses is based on the results of BMT previously reported [90,91]. BMT was demonstrated to prolong life span and ameliorate neurological symptoms in *Hexb*-/- mice [90], and the synergistic effects was also shown in combination with substrate reduction therapy (SRT) utilizing deoxynojirimycin derivatives [91]. Transduction of neural cells derived from *Hexa*-/- and *Hexb*-/- mice by recombinant viral vectors was performed.

Therefore, the bicistronic lentivector supplying both HexA and HexB isozymes may provide a potential therapeutic tool for the treatment of TSD and SD. A mechanistic link was demon‐ strated among GM2 accumulation, neuronal cell death, reduction of sarcoplasmic/endoplas‐ mic reticulum Ca2+-ATPase (SERCA) activity, and axonal outgrowth. Arfi *et al*. examined the ability of the SIV.ASB vector to reverse these pathophysiological events, hippocampal neu‐ rons derived from embryonic *Hexb*-/- mice, which were transduced with the lentival vector [85]. Normal axonal growth rates, the rate of Ca2+ uptake via the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity and the sensitivity of the neurons to thapsigargininduced cell death were restored concomitantly with a decrease in GM2 and GA2 levels. Thus, the bicistronic SIV.ASB vector was revealed to reverse the biochemical defects and down-stream consequences in SD neurons, suggesting its potential of systemic and CNS-di‐ rected *in vivo* GT. Kyrkanides S. *et al.* performed the system in vivo GT utilizing the re‐ combinant lentiviral vector FIV coding human *HEXB* cDNA to the neonatal *Hexb*-/- mice via intrapenitoneal administration. They also demonstrated the distribution of Hex isozymes in‐ to the CNS, including periventricular areas of the cerebrum as well as in the cerebellar cor‐

Cachon-Gonzalez *et al*. has reported that the *Hexb*-/- mice treated by stereotaxic intracranial inoculation of recombinant adeno-associated viral (rAAV) vectors encoding the human *HEXA* and *HEXB* cDNAs, including an HIV tat sequence as a protein transduction domain (PTD), to enhance protein expression and distribution [87]. *Hexb*-/- mice survived for >1 year with sustained, widespread and abundant enzyme delivery in the CNS. Onset of the disease was delayed with preservation of motor function; inflammation and GM2 storage in the brain and spinal cord was reduced. Gene delivery of the human HexA (αβ) by using AAV vectors has realistic potential for treating the TS and SD patients. Sargeant TJ. *et al.* demon‐ strated that intracranial co -injection of rAAV serotype 2/1 (rAAV2/1) vectors encoding the human *HEXA* and *HEXB* cDNAs prevents neuronal loss in the *Hexb*-/- mice brain tissues, in‐ cluding thalamus, brainstem and spinal cord, and correlated with increased lifespan [88]. Moreover, they performed intracranial co-injection of rAAV2/1 vectors into 1-month-old *Hexb*-/- mice [89]. As a result, the treated mice gave unprecedented survival to 2 years and prevented disease throughout the brain and spinal cord. Classical manifestations of disease, including spasticity were resolved by localized gene transfer to the striatum or cerebellum, respectively. Abundant biosynthesis of Hex isozymes and their global distribution via axo‐ nal, perivascular, and cerebrospinal fluid (CSF) spaces, as well as diffusion, account for the sustained phenotypic rescue—long-term protein expression by transduced brain parenchy‐ ma, choroid plexus epithelium, and dorsal root ganglia neurons supplies the corrective en‐ zyme. Prolonged survival permitted expression of cryptic disease in organs not accessed by

*Ex vivo* GT for GM2 gangliosidoses is based on the results of BMT previously reported [90,91]. BMT was demonstrated to prolong life span and ameliorate neurological symptoms in *Hexb*-/- mice [90], and the synergistic effects was also shown in combination with substrate

tex, and reduction of GM2 accumulated in these areas [86].

598 Gene Therapy - Tools and Potential Applications

intracranial vector delivery.

**4.3. CNS-directed** *ex vivo* **gene therapy**

Lacorazza HD *et al.* constructed the ecotropic retrovirus encoding the human *HEXA* cDNA and transduced multipotent neural progenitor cell lines, which stably expressed and secret‐ ed high levels of active HexA and cross-corrected the metabolic defect including GM2 stor‐ age in TSD fibroblastic cell line. The genetically engineered CNS progenitors were transplanted into the brains of both normal fetal and neonatal mice, in which substantial amounts of human Hex α-subunit and activity were observed throughout the brain enough for therapeutic effect in TSD [92].

Tsuji D. *et al.* constructed a recombinant lentiviral vector encoding the murine *Hexb* cDNA, and transduced microglial cells established from the brains of *Hexb*-/- mice [50]. Transduced microglial cells produced and secreted Hex activity, in which the intracellularly accumulat‐ ed GM2 and oligosaccharides with terminal N-acetylglucosamine residues were reduced. Transduced microglial cell line was expected as a donor for brain-directed *ex vivo* GT.

Mesenchymal stem cells (MSCs) derived from bone marrow stromal cells are one of the can‐ didates for autologous donor cells for ex vivo GT, and have the multipotency to differentiate under specific culture conditions into other cell types such as osteoblasts, adipocytes, and chondrocytes [93,] as well as into neural lineages [94]. Recently, we established MSCs de‐ rived from bone marrow of adult *Hexb*-/- mice. The MSCs expressed cell-type specific mark‐ ers, including CD29, CD90 and CD54, but not CD45, and exhibited the ability to differentiate into various cell types, including neuron-restricted precursor cells (NRPs) expressing N-CAM carrying polysialic acid (PSA-NCAM). We produced a bicistronic retroviral vector (MSV-*modB*) encoding for the modified human *HEXB* cDNAs (*modB*) causing six α-subunit type amino acid substitutions as well as *EGFP* gene [75]. The gene products, modified HexB (modB, a homodimer of the modified β-subunits) different from the wild-type HexB, can recognize negatively charged artificial substrates and bind to GM2A to exhibit GM2-degrad‐ ing activity. We transduced the MSCs derived from *Hexb*-/- mice (SD MSCs) with the MSV*modB*, resulting in restoration of HexA-like activity and reduction of the accumulated GM2 and GlcNAc-oligosaccharides. The modB was also secreted from the transduced SD MSCs. In addition, we performed intraventricular engraftment of the transduced SD MSCs express‐ ing *modB* into the brain of *Hexb*-/- mice. As a result, the injected transduced SD MSCs ex‐ pressing HexA-like activity and EGFP were observed in periventricular region of the brain (Figure 1). Reduction of the immunoreactivity towards natural substrates including GM2 and GlcNAc-oligosaccharides were also observed around the periventricular region of *Hexb*-/- mice brain (Figure 2). These results suggest that genetically modified MSCs can be utilized as a brain-directed donor cells for *ex vivo* GT for LSDs involving neurological mani‐ festations, including Tay-Sachs and Sandhoff diseases.

Lee J-P. *et al.* demonstrated intracranial transplantation of neural stem cells (NSCs) delayed onset, improved motor function, reduced GM2 storage and prolonged life span in the *Hexb*-/ mice partly due to the cross correction effect of the Hex isozymes secreted from NSCs. Hu‐ man NSCs derived directly from the CNS and secondarily induced from embryonic stem (ES) cells also demonstrated a broad repertoire of potentially therapeutic actions, which are expected to be applied for the treatment of neurodegenerative diseases [95]

[2] Figura KV. Andrej H. Lysosomal Enzymes and Their Receptors. Annual Review of

Molecular Therapy for Lysosomal Storage Diseases

http://dx.doi.org/10.5772/54074

601

[3] Kornfeld S. Structure and Function of the Mannose 6-Phosphate/Insulin-like Growth

[4] Neufeld EF. Fratantoni, JC. Inborn Errors of Mucopolysaccharide Metabolism. Sci‐

[5] Achord DT. Human Beta-Glucuronidase: in Vivo Clearance and in Vitro Uptake by a Glycoprotein Recognition System on Reticuloendothelial Cells. Cell 1978;15: 269-278.

[6] Barton NW. Replacement Therapy for Inherited Enzyme Deficiency-Macrophage-Targeted Glucocerebrosidase for Gaucher's Disease. The New England Journal of

[7] Weinreb NJ. Effectiveness of Enzyme Replacement Therapy in 1028 Patients with Type 1 Gaucher Disease After 2 to 5 Years of Treatment: a Report from the Gaucher

[8] Kakkis ED. Enzyme-Replacement Therapy in Mucopolysaccharidosis I. The New

[9] Harmatz P. Enzyme Replacement Therapy in Mucopolysaccharidosis VI (Maroteaux-

[10] Schiffmann R. Enzyme Replacement Therapy in Fabry Disease A Randomized Con‐ trolled Trial. The Journal of American Medical Association 2001;285(21): 2743-2749.

[11] Eng CM. Safety and Efficacy of Recombinant Human α-Galactosidase a Replacement Therapy in Fabry's Disease. The New England Journal of Medicine 2001;345(1): 9-16.

[12] Amalfitano A. Recombinant Human Acid Alpha-Glucosidase Enzyme Therapy for Infantile Glycogen Storage Disease Type II: Results of a Phase I/II Clinical Trial. Ge‐

[13] Winkel LP. Enzyme Replacement Therapy in Late-Onset Pompe's Disease: a Three-

[14] Wang J. Neutralizing Antibodies to Therapeutic Enzymes: Considerations for Test‐

[15] Patricia ID. Intrathecal Enzyme Replacement Therapy for Mucopolysaccharidosis I: Translating Success in Animal Models to Patients. Current Pharmaceutical Biotech‐

[16] Shapiro EG. Neuropsychological Outcomes of Several Storage Diseases with and without Bone Marrow Transplantation. Journal of Inherited Metabolic Disease

ing, Prevention and Treatment. Nature Biotechnology 2008;26: 901-908.

Registry. The American Journal of Medicine 2002;113(2): 112-119.

Lamy Syndrome). The Journal of Pediatrics 2004;144(5): 574-580.

Year Follow-Up. Annals of Neurology 2004;55(4): 495-502.

England Journal of Medicine 2001;344(3): 182-188.

Factor II Receptors. Annual Review of Biochemistry 1992;61: 307-330.

Biochemistry 1986;55: 167-193.

Medicine 1991;324(21): 1464-1470.

netics in Medicine 2001;3(2): 132-138.

nology 2011;12(6): 946-955.

1995;18(4): 413-429.

ence 1970;169: 141-146.

#### **5. Conclusion**

A number of preclinical and therapeutic approaches for GM2 gangliosidoses, including stem cell therapy, substrate deprivation therapy, gene therapy, and enzyme replacement therapy, are being examined and evaluated with disease model mice, although there is no effective therapy for treatment of the patients with GM2 gangliosidoses at present. However, accord‐ ing to the preclinical results obtained by using animal disease models, CNS-directed *in vivo* gene therapy utilizing recombinant viral vectors and *ex vivo* gene therapy based on the cross-correction by transduced autologous and heterologous stem cells are promising for de‐ velopment of novel therapies for LSDs associated with neurological abnormalities, including GM2 gangliosidoses. Improvement of these GTs and their combination with other clinical approaches will facilitate the development of efficient therapies for neurodegenerative dis‐ orders caused by neuroinflammation and gliosis.

#### **Acknowledgements**

Recent our research was supported by NIBIO (Osaka, Japan). We would appreciate Ms. Mayuko Oe for assisting us to prepare this review.

#### **Author details**

Daisuke Tsuji1,2 and Kohji Itoh1,2

\*Address all correspondence to: dtsuji@tokushima-u.ac.jp

1 Department of Medicinal Biotechnology, Graduate School of Pharmaceutical Sciences, In‐ stitute for Medicinal Research, The University of Tokushima, Tokushima, Japan

2 NIBIO, Ibaraki, Osaka, Japan

#### **References**

[1] Scriver CR. In The Metabolic and Molecular Bases of Inherited Disease http:// www.ommbid.com ;part16: 134-154.

[2] Figura KV. Andrej H. Lysosomal Enzymes and Their Receptors. Annual Review of Biochemistry 1986;55: 167-193.

(ES) cells also demonstrated a broad repertoire of potentially therapeutic actions, which are

A number of preclinical and therapeutic approaches for GM2 gangliosidoses, including stem cell therapy, substrate deprivation therapy, gene therapy, and enzyme replacement therapy, are being examined and evaluated with disease model mice, although there is no effective therapy for treatment of the patients with GM2 gangliosidoses at present. However, accord‐ ing to the preclinical results obtained by using animal disease models, CNS-directed *in vivo* gene therapy utilizing recombinant viral vectors and *ex vivo* gene therapy based on the cross-correction by transduced autologous and heterologous stem cells are promising for de‐ velopment of novel therapies for LSDs associated with neurological abnormalities, including GM2 gangliosidoses. Improvement of these GTs and their combination with other clinical approaches will facilitate the development of efficient therapies for neurodegenerative dis‐

Recent our research was supported by NIBIO (Osaka, Japan). We would appreciate Ms.

1 Department of Medicinal Biotechnology, Graduate School of Pharmaceutical Sciences, In‐

[1] Scriver CR. In The Metabolic and Molecular Bases of Inherited Disease http://

stitute for Medicinal Research, The University of Tokushima, Tokushima, Japan

expected to be applied for the treatment of neurodegenerative diseases [95]

orders caused by neuroinflammation and gliosis.

Mayuko Oe for assisting us to prepare this review.

\*Address all correspondence to: dtsuji@tokushima-u.ac.jp

www.ommbid.com ;part16: 134-154.

**5. Conclusion**

600 Gene Therapy - Tools and Potential Applications

**Acknowledgements**

**Author details**

**References**

Daisuke Tsuji1,2 and Kohji Itoh1,2

2 NIBIO, Ibaraki, Osaka, Japan


[17] Miranda SR. Bone Marrow Transplantation in Acid Sphingomyelinase-Deficient Mice: Engraftment and Cell Migration into The Brain as a Function of Radiation, Age, and Phenotype. Blood 1997;90(1): 444-452.

[30] Biffi A. Gene Therapy of Metachromatic Leukodystrophy Reverses Neurological Damage and Deficits in Mice. The Journal of Clinical Investigation 2006;116(11):

Molecular Therapy for Lysosomal Storage Diseases

http://dx.doi.org/10.5772/54074

603

[31] Wang D. Reprogramming Erythroid Cells for Lysosomal Enzyme Production Leads to Visceral and CNS Cross-Correction in Mice with Hurler Syndrome. Proceedings of the National Academy of Sciences of the United States of America 2009; 106(47):

[32] Taylor RM. Decreased Lysosomal Storage in the Adult MPS VII Mouse Brain in the Vicinity of Grafts of Retroviral Vector-Corrected Fibroblasts Secreting High Levels of

[33] Berges BK. Widespread Correction of Lysosomal Storage in the Mucopolysaccharido‐ sis Type VII Mouse Brain with A Herpes Simplex Virus Type 1 Vector Expressing Be‐

[34] Gravel RA. GM2 Gangliosidoses in The Metabolic and Molecular Bases of Inherited

[35] Mahuran DJ. Biochemical Consequences of Mutations Causing the GM2 Ganglio‐

[36] Kytzia HJ. Evidence for Two Different Active Sites on Human β-Hexosaminidase A.

[37] Sakuraba H. Molecular Pathologies of and Enzyme Replacement Therapies for Lyso‐ somal Diseases. CNS & Neurological Disorders - Drug Targets 2006;5: 401-413.

[38] Itoh K. Neurochemical Aspects of Sandhoff Disease in Neurochemistry of Metabolic Diseases-Lysosomal Storage Diseases, Phenylketonuria and Canavan Disease 2007;

[39] Tanaka A. A New Point Mutation in the Beta-Hexosaminidase Alpha Subunit Gene Responsible for Infantile Tay-Sachs Disease in a Non-Jewish Caucasian Patient (a

[40] Ohno K. Mutation in GM2-Gangliosidosis B1 Variant. Journal of Neurochemistry

[41] Kytzia HJ. Variant of GM2-Gangliosidosis with Hexosaminidase a Having a Severely

[42] Mark BL. Crystal Structure of Human Beta-Hexosaminidase B: Understanding the Molecular Basis of Sandhoff and Tay-Sachs Disease. Journal of Molecular Biology

[43] Timm M. The X-ray Crystal Structure of Human β-Hexosaminidase B Provides New Insights into Sandhoff Disease. Journal of Molecular Biology 2003;328(3): 669-681.

Changed Substrate Specificity. The EMBO Journal 1983;2(7): 1201–1205.

Kpn Mutant). The American Journal of Human Genetics 1990;47(3): 568-574.

Bold Beta-Glucuronidase. Nature Medicine 1997;3(7): 771-774.

ta-Glucuronidase. Molecular Therapy 2006;13(5): 859-869.

sidoses. Biochimica et Biophysica Acta 1999;1455(2-3): 105-138.

The Journal of Biological Chemistry 1985;260: 7568-7572.

Disease. McGraw-Hill 2001; 3827-3876.

3070-3082.

19958-19963.

55-82.

1988;50(1): 316-318.

2003;327: 1093-1109.


[30] Biffi A. Gene Therapy of Metachromatic Leukodystrophy Reverses Neurological Damage and Deficits in Mice. The Journal of Clinical Investigation 2006;116(11): 3070-3082.

[17] Miranda SR. Bone Marrow Transplantation in Acid Sphingomyelinase-Deficient Mice: Engraftment and Cell Migration into The Brain as a Function of Radiation,

[18] Lönnqvist T. Hematopoietic Stem Cell Transplantation in Infantile Neuronal Ceroid

[19] Jin HK. Intracerebral Transplantation of Mesenchymal Stem Cells into Acid Sphingo‐ myelinase-Deficient Mice Delays the Onset of Neurological Abnormalities and Ex‐ tends Their Life Span. The Journal of Clinical Investigation 2002;109(9): 1183-1191.

[20] Hofling AA. Human CD34+ Hematopoietic Progenitor Cell-Directed Lentiviral-Mediated Gene Therapy in a Xenotransplantation Model of Lysosomal Storage Dis‐

[21] Mark SS. Gene Therapy for Lysosomal Storage Diseases. Molecular Therapy 2006;13:

[22] Haskins M. Gene Therapy for Lysosomal Storage Diseases (LSDs) in Large Animal

[23] Sands MS. CNS-Directed Gene Therapy for Lysosomal Storage Diseases. ACTA PAE‐

[25] Fink JK. Correction of Glucocerebrosidase Deficiency after Retroviral-Mediated Gene Transfer into Hematopoietic Progenitor Cells from Patients with Gaucher Disease. Proceedings of the National Academy of Sciences of the United States of America

[26] Nicolino MP. Adenovirus-Mediated Transfer of the Acid Alpha-Glucosidase Gene into Fibroblasts, Myoblasts and Myotubes From Patients with Glycogen Storage Dis‐ ease Type II Leads to High Level Expression of Enzyme and Corrects Glycogen Ac‐

[27] Ziegler RJ. Correction of Enzymatic and Lysosomal Storage Defects in Fabry Mice by Adenovirus-Mediated Gene Transfer. Human Gene Therapy 1999;10(10): 1667-1682.

[28] Daly TM. Neonatal Intramuscular Injection with Recombinant Adeno-Associated Vi‐ rus Results in Prolonged Beta-Glucuronidase Expression in Situ and Correction of Liver Pathology in Mucopolysaccharidosis Type VII Mice. Human Gene Therapy

[29] Griffey M. Adeno-Associated Virus 2-Mediated Gene Therapy Decreases Autofluor‐ escent Storage Material and Increases Brain Mass in a Murine Model of Infantile

Neuronal Ceroid Lipofuscinosis. Neurobiology of Disease 2004;16(2): 360-369.

cumulation. Human Molecular Genetics 1998;7(11): 1695-1702.

Donor-Derived Cells and Correction of Tissue Pathology in a Novel Murine Xeno‐ transplantation Model of Lysosomal Storage Disease. Blood 2003;101: 2054-2063.

Cells Leads to Widespread Distribution of

Age, and Phenotype. Blood 1997;90(1): 444-452.

Lipofuscinosis. Neurology 2001;57(8): 1411-1416.

ease. Molecular Therapy 2004;9(6): 856-865.

Models. The ILAR Journal 2009;50(2): 112-121.

DIATRICA SUPPLEMENT 2008;97(457): 22-27.

[24] Hofling A. Engrafment of Human CD34+

1990;87(6): 2334-2338.

1999;10: 85-94.

839–849.

602 Gene Therapy - Tools and Potential Applications


[44] Lemieux MJ. Crystallographic Structure of Human Beta-Hexosaminidase A: Interpre‐ tation of Tay-Sachs Mutations and Loss of GM2 ganglioside hydrolysis. Journal of Molecular Biology 2006; 359: 913-929.

[58] Hermonat PL. Use of Adeno-Associated Virus as a Mammalian DNA Cloning Vec‐ tor: Transduction of Neomycin Resistance into Macka-Kian Tissue Culture Cells. Pro‐

Molecular Therapy for Lysosomal Storage Diseases

http://dx.doi.org/10.5772/54074

605

[59] Fraites TJ. Correction of the Enzymatic and Functional Deficits in a Model of Pompe Disease using Adeno-Associated Virus Vectors. Molecular Therapy 2002;5: 571-578.

[60] Takahashi H. Long-Term Systemic Therapy of Fabry Disease in a Knockout Mouse by Adeno-Associated Virus-Mediated Muscle-Directed Gene Transfer. Proceedings

[61] Watson G. Intrathecal Administration of AAV Vectors for the Treatment of Lysoso‐

[62] Jung SC. Adeno-Associated Viral Vector-Mediated Gene Transfer Results in Long-Term Enzymatic and Functional Correction in Multiple Organs of Fabry Mice. Pro‐

[63] Hartung SD. Correction of Metabolic, Craniofacial, and Neurologic Abnormalities in MPS I Mice Treated at Birth with Adeno-Associated Virus Vector Transducing the

[64] Sakurai K. Brain Transplantation of Genetically Modified Bone Marrow Stromal Cells Corrects CNS Pathology and Cognitive Function in MPS VII Mice. Gene Thera‐

[65] Sano R. Chemokine-Induced Recruitment of Genetically Modified Bone Marrow Cells into the CNS of GM1-Gangliosidosis Mice Corrects Neuronal Pathology. Blood

[66] Buchet D. Long-Term Expression of Beta-Glucuronidase by Genetically Modified Human Neural Progenitor Cells Grafted into the Mouse Central Nervous System.

[67] Fu H. Neurological Correction of Lysosomal Storage in a Mucopolysaccharidossis IIIB Mouse Model by Adeno-Associated Virus-Mediated Gene Delivery. Molecular

[68] Frisella WA, Intracranial Injection of Recombinant Adeno-Associated Virus Im‐ proves Cognitive Function in a Murine Model of Mucopolysaccharidosis Type VII.

[69] Rafi MA. AAV-Mediated Expression of Galactocerebrosidase in Brain Results in At‐ tenuated Symptoms and Extended Life Span in Murine Models of Globoid Cell Leu‐

[70] Passini MA. Distribution of a Lysosomal Enzyme in the Adult Brain by Axonal Transport and by Cells of the Rostral Migratory Stream. The Journal of Neuroscience

mal Storage in the Brains of MPS I Mice. Gene Therapy 2006;13: 917-925

ceedings of the National Academy of Sciences 2001;98: 2676-2681.

Human α-L-Iduronidase Gene. Molecular Therapy 2004;9: 866-875.

Molecular and Cellular Neuroscience 2002;19: 389-401.

kodystrophy. Molecular Therapy 2005;11: 734-744.

py 2004;11: 1475-1481.

2005;106: 2259-2268.

Therapy 2002;5: 42-49.

2002;22: 6437-6446.

Molecular Therapy 2001;3: 351-358.

ceedings of the National Academy of Sciences 1984;81: 6466-6470.

of the National Academy of Sciences 2002;99: 13777-13782.


[58] Hermonat PL. Use of Adeno-Associated Virus as a Mammalian DNA Cloning Vec‐ tor: Transduction of Neomycin Resistance into Macka-Kian Tissue Culture Cells. Pro‐ ceedings of the National Academy of Sciences 1984;81: 6466-6470.

[44] Lemieux MJ. Crystallographic Structure of Human Beta-Hexosaminidase A: Interpre‐ tation of Tay-Sachs Mutations and Loss of GM2 ganglioside hydrolysis. Journal of

[45] Sakuraba H. Molecular and Structural Studies of the GM2 Gangliosidosis 0 Variant.

[46] Liu G. Functional Correction of CNS Phenotypes in a Lysosomal Storage Disease Model using Adeno-Associated Virus Type 4 Vectors. The Journal of Neuroscience

[47] Sevin C. Intracerebral Adeno-Associated Virus-Mediated Gene Transfer in Rapidly Progressive Forms of Metachromatic Leukodystrophy. Human Molecular Genetics

[48] Spampanato C. Efficacy of a Combined Intaracerebral and Systemic Gene Delivery Approach for the Treatment of a Severe Lysosomal Storage Disorder. Molecular

[49] Consiglio A, In Vivo Gene Therapy of Metachromatic Leukodystrophy by Lentiviral Vectors: Correction of Neuropathology and Protection Against Learning Impair‐

[50] Tsuji D. Metabolic Correction in Microglia Derived from Sandhoff Disease Model

[51] McIntyre C. Lentiviral-Mediated Gene Therapy for Murine Mucopolysaccharidosis

[52] Bahnson AB. Transduction of CD34+ Enriched Cord Blood and Gaucher Bone Mar‐ row Cells by a Retroviral Vector Carrying the Glucocerebrosidase Gene. Gene Thera‐

[53] Fabrega S. Gene Therapy of Gaucher's and Fabry's Diseases: Current Status and Pros‐

[54] Dunbar CE. Retroviral Transfer of the Glucocerebrosidase Gene into CD34+ Cells from Patients with Gaucher Disease: in Vivo Detection of Transduced Cells Without

[55] Amalfitano A. Systemic Correction of the Muscle Disorder Glycogen Storage Disease Type 2 after Hepatic Targeting of a Modified Adenovirus Vector Encording Human Acid-Alpha-Gulucosidase. Proceedings of the National Academy of Sciences 1999;96:

[56] Du H. Lysosomal Acid Lipase Deficiency: Correction of Lipid Storage by Adenovi‐ rus-Mediated Gene Transfer in Mice. Human Gene Therapy 2002;13: 1361-1372. [57] Xu F. Glycogen Storage in Multiple Muscles of Old GSD-II Mice can be Rapidly Cleared after a Single Intravenous Injection with a Modified Adenoviral Vector Ex‐

ments in Affected Mice. Nature Medicine 2001;7(3): 310-316.

Type IIIA. Molecular Genetics of Metabolism 2008;93(4): 411-418.

Mice. Journal of Neurochemistry 2005; 94(6): 1631-1638.

pects. Journal of Social Biology 2002;196(2): 175-181.

Myeloablation. Human Gene Therapy 1998;9(17): 2629-2640.

pressing hGAA. The Journal of Gene Medicine 2005;7: 171-178.

Molecular Biology 2006; 359: 913-929.

2005;25(41): 9321-9327.

604 Gene Therapy - Tools and Potential Applications

Therapy 2011;19(5): 860-869.

py 1994;1(3): 176-184.

8861-8866.

2006;15(1): 53-64.

Journal of Human Genetics 2002;47(4): 176-183.


[71] Vite CH. Effective Gene Therapy for an Inherited CNS Disease in a Large Animal Model. Annals of Neurology 2005;57: 355-364.

[85] Arfi A. Reversion of the Biochemical Defects in Murine Embryonic Sandhoff Neu‐ rons Using a Bicistronic Lentiviral Vector Encoding Hexosaminidase Alpha and Beta.

Molecular Therapy for Lysosomal Storage Diseases

http://dx.doi.org/10.5772/54074

607

[86] Kykanides S. Beta-Hexosaminidase Lentiviral Vectors: Transfer into the CNS via Sys‐

[87] Cachón-González MB. Effective Gene Therapy in an Authentic Model of Tay-Sachsrelated Diseases. Proceedings of the National Academy of Sciences of the United

[88] Sargeant TJ. Adeno-Associated Virus-Mediated Expression of β-Hexosaminidase Prevents Neuronal Loss in the Sandhoff Mouse Brain. Human Molecular Genetics

[89] Cachón-González M. Gene Transfer Correct Acute GM2 Gangliosidosis – Potential Therapeutic Contribution of Perivascular Enzyme Flow. Molecular Therapy

[90] Norflus F. Bone Marrow Transplantation Prolongs Life Span and Ameliorates Neu‐ rologic Manifestations in Sandhoff Disease Mice. Journal of Clinical Investigation

[91] Jeyakumar M. Enhanced Survival in Sandhoff Disease Mice Receiving a Combination of Substrate Deprivation Therapy and Bone Marrow Transplantation. Blood 2001;97:

[92] Lacorazza HD. Expression of Human Beta-Hexosaminidase Alpha-Subunit Gene (The Gene Defect of Tay-Sachs Disease) in Mouse Brains Upon Engraftment of Trans‐

[93] Pittenger MF. Multilineage Potential of Adult Human Mesenchymal Stem Cells. Sci‐

[94] Dezawa M. Specific Induction of Neuronal Cells from Bone Marrow Stromal Cells and Application for Autologous Transplantation. The Journal of Clinical Investiga‐

[95] Lee JP. Stem Cells Act through Multiple Mechanisms to Benefit Mice with Neurode‐

generative Metabolic Disease. Nature Medicine 2007;13(4): 439-447.

duced Progenitor Cells. Nature Medicine 1996;2(4): 424-429.

temic Administration. Molecular Brain Research 2005;133: 286-298.

Journal of Neurochemistry 2006;96(6): 1572-1579.

States of America 2006;103(27): 10373-10378.

2011;20(22): 4371-4380.

2012;20(8): 1489-1500.

1998;101: 1881-1888.

ence 1999;284(5411): 143-147.

tion 2004;113(12): 1701-1710.

327-329.


[85] Arfi A. Reversion of the Biochemical Defects in Murine Embryonic Sandhoff Neu‐ rons Using a Bicistronic Lentiviral Vector Encoding Hexosaminidase Alpha and Beta. Journal of Neurochemistry 2006;96(6): 1572-1579.

[71] Vite CH. Effective Gene Therapy for an Inherited CNS Disease in a Large Animal

[72] McCown TJ. Adeno-Asoociated Virus (AVV) Vectors in the CNS. Current Gene Ther‐

[73] Tsuji D. Highly Phosphomannosylated Enzyme Replacement Therapy for GM2 Gan‐

[74] Matsuoka K. Introduction of an N-Glycan Sequon into HEXA Enhances Human β-Hexosaminidase Cellular Uptake in a Model of Sandhoff Disease. Molecular Therapy

[75] Matsuoka K. Therapeutic Potential of Intracerebroventricular Replacement of Modi‐ fied Human β-Hexosaminidase B for GM2 Gangliosidosis. Molecular Therapy

[76] Pastores GM. Miglustat: Substrate Reduction Therapy for Lysosomal Storage Disor‐ ders Associated with Primary Central Nervous System Involvement. Recent Patents

[77] Guidotti J. Retrovirus-Mediated Enzymatic Correction of Tay-Sachs Defect in Trans‐ duced and Non-Transduced Cells. Human Molecular Genetics 1998; 7(5): 831-838.

[78] Martino S. A Direct Gene Transfer Strategy via Brain Internal Capsule Reverses the Biochemical Defect in Tay-Sachs Disease. Human Molecular Genetics 2005;14(15):

[79] Akli S. Restoration of Hexosaminidase A Activity in Human Tay-Sachs Fibroblasts via Adenoviral Vector-Mediated Gene Transfer. Gene Therapy 1996;3(9): 769-774.

[80] Itakura T. Inefficiency in GM2 Ganglioside Elimination by Human Lysosomal β-Hex‐ osaminidase β-Subunit Gene Transfer to Fibroblastic Cell Line Derived from Sandh‐ off Disease Model Mice. Biological & Pharmaceutical Bulletin 2006;29(8): 1564-1569.

[81] Yamaguchi A. Plasmid-Based Gene Transfer Ameliorates Visceral Storage in a Mouse Model of Sandhoff Disease. Journal of Molecular Medicine 2003;81(3):

[82] Bourgoin C. Widespread Distribution of Beta-Hexosaminidase Activity in the Brain of a Sandhoff Mouse Model After Co-injection Of Adenoviral Vector And Mannitol.

[83] Martino S. A Direct Gene Transfer Strategy via Brain Internal Capsule Reverses the Biochemical Defect in Tay-Sachs Disease. Human Molecular Genetics 2005;14(15)

[84] Arfi A. Bicistronic Lentiviral Vector Corrects Beta-Hexosaminidase Deficiency in Transduced and Cross-Corrected Human Sandhoff Fibroblasts. Neurobiology of Dis‐

Model. Annals of Neurology 2005;57: 355-364.

gliosidosis. Annals of Neurology 2011;69(4): 691-701.

apy 2005;5(3): 333-338.

606 Gene Therapy - Tools and Potential Applications

2010;18(8): 1519-1526.

2011;19(6): 1017-1024.

2113-2123.

185-193.

2113-2123.

ease 2005;20(2) 583-593.

on CNS Drug Discovery 2006;1(1): 77-82.

Gene Therapy 2003;10(21) 1841-1849.


**Section 5**

**Applications: Others**

**Section 5**

**Applications: Others**

**Chapter 25**

**Gene Therapy Perspectives Against Diseases of the**

Gene therapy uses a variety of techniques as the introduction of a normal allele of a gene in cases where the cell does not express the gene or in other cases where the gene is underexpressed. In order to achieve effective gene therapy for a specific gene in a certain type of cells a lot of work is needed. More specifically the following steps are essential: 1. Isolation of target gene, 2. Development of a specific gene vector, 3. Specification of the target cell, 4. Definition of route of administration, and 5. Identification of other potential uses of the

The value of gene of gene therapy is often discussed, especially in some diseases who have a known protein defect and the protein itself can be produced in a large scale and could then be administered to the patient. Genetic engineering could be beneficial in the production of the target protein. Nevertheless, the infusion of the protein is not curative, because of the

In order to isolate a specific gene, it is essential to produce a cDNA library that contains the total number of unique genes expressed in a specific tissue. The DNA contained in a cDNA

The standard procedure of the construction of a cDNA library includes 1) isolation of the total amount of mRNA that is produced in the target cells, 2) Hybridization using a multi-T promoter, 3) Synthesis of complementary DNA (cDNA) to the mRNA prototype using the enzyme reverse transcriptase, 4) Degradation of the mRNA by the means of an alkali, 5) Synthesis of the second DNA strand using nucleotides and the enzyme DNA polymerase.

> © 2013 Lykouras et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

library is not genomic, therefore it contains only the encoding sequences of the DNA.

half-life of the protein itself and the growth factors that are essential.

**Respiratory System**

http://dx.doi.org/10.5772/52775

**1. Introduction**

gene.

Dimosthenis Lykouras, Kiriakos Karkoulias, Christos Tourmousoglou, Efstratios Koletsis, Kostas Spiropoulos and Dimitrios Dougenis

Additional information is available at the end of the chapter

### **Gene Therapy Perspectives Against Diseases of the Respiratory System**

Dimosthenis Lykouras, Kiriakos Karkoulias, Christos Tourmousoglou, Efstratios Koletsis, Kostas Spiropoulos and Dimitrios Dougenis

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52775

#### **1. Introduction**

Gene therapy uses a variety of techniques as the introduction of a normal allele of a gene in cases where the cell does not express the gene or in other cases where the gene is underexpressed. In order to achieve effective gene therapy for a specific gene in a certain type of cells a lot of work is needed. More specifically the following steps are essential: 1. Isolation of target gene, 2. Development of a specific gene vector, 3. Specification of the target cell, 4. Definition of route of administration, and 5. Identification of other potential uses of the gene.

The value of gene of gene therapy is often discussed, especially in some diseases who have a known protein defect and the protein itself can be produced in a large scale and could then be administered to the patient. Genetic engineering could be beneficial in the production of the target protein. Nevertheless, the infusion of the protein is not curative, because of the half-life of the protein itself and the growth factors that are essential.

In order to isolate a specific gene, it is essential to produce a cDNA library that contains the total number of unique genes expressed in a specific tissue. The DNA contained in a cDNA library is not genomic, therefore it contains only the encoding sequences of the DNA.

The standard procedure of the construction of a cDNA library includes 1) isolation of the total amount of mRNA that is produced in the target cells, 2) Hybridization using a multi-T promoter, 3) Synthesis of complementary DNA (cDNA) to the mRNA prototype using the enzyme reverse transcriptase, 4) Degradation of the mRNA by the means of an alkali, 5) Synthesis of the second DNA strand using nucleotides and the enzyme DNA polymerase.

© 2013 Lykouras et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The cDNA library contains only the exons of the genes that are expressed in the specific tis‐ sue; therefore the cDNA can show the activity of the studied tissue.

an important role as a phosphorylation-regulated Cl− channel and a regulator of channels and transporters [1, 2]. More specifically, the activation of CFTR results in a parallel inhibi‐ tion of the epithelial Na+ channel (ENaC), which is lost when CFTR is absent or not func‐ tioning. There is a so called "low volume" hypothesis, which suggests that a loss of Cl− secretion and an increase in Na+ absorption reduce the thickness of the airway surface liq‐ uid (ASL), thus impairing mucociliary clearance [3]. Moreover, a reduction in the secretion of bicarbonates (mediated by the CFTR) might affect the hydration of the secreted mucus, thus altering its physical properties [4]. CFTR is also expressed in submucosal glands in the airways, which mainly participate in host defence. A loss of CFTR function in duct-lining serous cells prevents the secretion of mucus and anti-microbial factors by submucosal

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

613

Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 1989, it was thought that scientists could prevent or delay the onset or even the progres‐ sion of lung disease by using gene transfer. Although loss of CFTR function may affect a great number of different cells and tissues, progressive lung disease is responsible for the rates of morbidity and mortality. Therefore, the efforts of gene therapy have focused so far on gene transfer to the airways. CFTR is expressed in various epithelial cells in the lumen

The fact that CF is an autosomal recessive disease lead to the idea that the delivery of a CFTR cDNA to the airway epithelium with a viral or non-viral vector could have beneficial effect. The delivery method could be either direct instillation or aerosol delivery. Further‐ more, early studies indicated that the transfection of 6–10% of CF epithelia generated wild-

The selection of targets cells for gene therapy in CF is still controversial. The available strat‐ egies suggest correcting cells of the surface epithelium, the submucosal glands, or both [9, 10, 11]. The CFTR is expressed in the airways, including ciliated cells within the surface epi‐ thelium and a subpopulation of cells in submucosal gland ducts and acini. There are several epithelial cell types in the lung that seem to have progenitor functions, thus allowing longterm correction if these cells are targeted with selected vectors [12]. Experiments from sever‐ al species and model systems have revealed potential progenitor populations, including: basal cells [13] and non-ciliated columnar cells of the airways [14, 15], submucosal gland epi‐

Many viral and nonviral vectors have been tested for their usefulness in CF gene therapy. Adenoviral (AV) vectors have as a great disadvantage their low transduction efficiency of human airway epithelia and by their induction of strong immune responses [18]. In contrast adeno-associated viral (AAV) vectors may lead to long-term gene transfer and expression in

In addition to the DNA viruses, AV and AAV, various RNA viruses have been investigated for uses in airway gene transfer. Murine parainfluenza virus type 1, human respiratory syn‐ cytial virus (RSV) and human parainfluenza virus type 3 (PIV3) can effectively transfect air‐ way epithelial cells by attaching to sialic acid and cholesterol [19], which are found on the

and in submucosal glands of the airways, where the mRNA is expressed [6, 7].

thelia [16], Clara cells [17] and alveolar type II cells in the distal lung.

bronchial epithelia of rabbits and nonhuman primates.

type levels of chloride transport in vitro [8].

glands [5].

As soon as the isolation of the gene that is to be administered to the patient is achieved, an appropriate vector is needed in order to deliver the gene to the target cells. The most impor‐ tant vectors that are generally used in gene therapy applications in order to perform trans‐ fection of the targeted cellular population are:


The target cell has to be defined carefully in order to achieve the best curative result. In the case of gene therapy in the lung, the airway epithelia or even the lung vasculature may be efficient cellular targets.

The route of administration has to be defined so as the target gene is transported to the tar‐ get cells in order to perform the transfection of the target tissue cells.

The use of a target gene in the therapy of a certain condition of the lung does not exclude a possible use of the gene in another therapeutic strategy, where there is a similar pathophysi‐ ology (e.g. inflammation). Therefore, the identification of other potential uses of the target gene is always important.

#### **2. Gene therapy in cystic fibrosis**

Gene therapy is still far from becoming a curative treatment for cystic fibrosis (CF). Despite the outstanding technological and medical progress there is still number of interesting ge‐ netic, biological, pharmaceutical and ethical problems. Only when these issues are to be solved will gene therapy become an option for the treatment of CF.

As for the biology of CF, the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in airway epithelia, on the luminal side of the plasma membrane, where it plays an important role as a phosphorylation-regulated Cl− channel and a regulator of channels and transporters [1, 2]. More specifically, the activation of CFTR results in a parallel inhibi‐ tion of the epithelial Na+ channel (ENaC), which is lost when CFTR is absent or not func‐ tioning. There is a so called "low volume" hypothesis, which suggests that a loss of Cl− secretion and an increase in Na+ absorption reduce the thickness of the airway surface liq‐ uid (ASL), thus impairing mucociliary clearance [3]. Moreover, a reduction in the secretion of bicarbonates (mediated by the CFTR) might affect the hydration of the secreted mucus, thus altering its physical properties [4]. CFTR is also expressed in submucosal glands in the airways, which mainly participate in host defence. A loss of CFTR function in duct-lining serous cells prevents the secretion of mucus and anti-microbial factors by submucosal glands [5].

The cDNA library contains only the exons of the genes that are expressed in the specific tis‐

As soon as the isolation of the gene that is to be administered to the patient is achieved, an appropriate vector is needed in order to deliver the gene to the target cells. The most impor‐ tant vectors that are generally used in gene therapy applications in order to perform trans‐

**1.** Plasmids which are well-tolerated and safe, but transfer towards the nucleus is not so

**2.** Adeno-virus which may transfect differentiating as well as stale cells and have a very good percentage of transfection, but is not inserted in the nucleus and there is a possi‐

**3.** Retro-virus which are inserted in the genome and are stable during transport, but they

**4.** Lenti-virus which is a subtype of retro-virus that may be inserted in stable cells and it is

**5.** AAV (adeno-associated-based vector) which is inserted in the genome, is quite stable during the procedure and stable cells can be transfected as well, but only 4,7 kb can be

**6.** Liposomes – Oligonucleotides (ODN-based) which are very easy to use, selective for the endothelium, special alterations can improve the availability and reduce toxicity

The target cell has to be defined carefully in order to achieve the best curative result. In the case of gene therapy in the lung, the airway epithelia or even the lung vasculature may be

The route of administration has to be defined so as the target gene is transported to the tar‐

The use of a target gene in the therapy of a certain condition of the lung does not exclude a possible use of the gene in another therapeutic strategy, where there is a similar pathophysi‐ ology (e.g. inflammation). Therefore, the identification of other potential uses of the target

Gene therapy is still far from becoming a curative treatment for cystic fibrosis (CF). Despite the outstanding technological and medical progress there is still number of interesting ge‐ netic, biological, pharmaceutical and ethical problems. Only when these issues are to be

As for the biology of CF, the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in airway epithelia, on the luminal side of the plasma membrane, where it plays

sue; therefore the cDNA can show the activity of the studied tissue.

fection of the targeted cellular population are:

612 Gene Therapy - Tools and Potential Applications

bility of reaction against the adeno-virus

quite stable during the procedure

efficient cellular targets.

gene is always important.

**2. Gene therapy in cystic fibrosis**

can only used in transfection of multiplying cells

inserted and there is a possibility of mutations

get cells in order to perform the transfection of the target tissue cells.

solved will gene therapy become an option for the treatment of CF.

easy

Since the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 1989, it was thought that scientists could prevent or delay the onset or even the progres‐ sion of lung disease by using gene transfer. Although loss of CFTR function may affect a great number of different cells and tissues, progressive lung disease is responsible for the rates of morbidity and mortality. Therefore, the efforts of gene therapy have focused so far on gene transfer to the airways. CFTR is expressed in various epithelial cells in the lumen and in submucosal glands of the airways, where the mRNA is expressed [6, 7].

The fact that CF is an autosomal recessive disease lead to the idea that the delivery of a CFTR cDNA to the airway epithelium with a viral or non-viral vector could have beneficial effect. The delivery method could be either direct instillation or aerosol delivery. Further‐ more, early studies indicated that the transfection of 6–10% of CF epithelia generated wildtype levels of chloride transport in vitro [8].

The selection of targets cells for gene therapy in CF is still controversial. The available strat‐ egies suggest correcting cells of the surface epithelium, the submucosal glands, or both [9, 10, 11]. The CFTR is expressed in the airways, including ciliated cells within the surface epi‐ thelium and a subpopulation of cells in submucosal gland ducts and acini. There are several epithelial cell types in the lung that seem to have progenitor functions, thus allowing longterm correction if these cells are targeted with selected vectors [12]. Experiments from sever‐ al species and model systems have revealed potential progenitor populations, including: basal cells [13] and non-ciliated columnar cells of the airways [14, 15], submucosal gland epi‐ thelia [16], Clara cells [17] and alveolar type II cells in the distal lung.

Many viral and nonviral vectors have been tested for their usefulness in CF gene therapy. Adenoviral (AV) vectors have as a great disadvantage their low transduction efficiency of human airway epithelia and by their induction of strong immune responses [18]. In contrast adeno-associated viral (AAV) vectors may lead to long-term gene transfer and expression in bronchial epithelia of rabbits and nonhuman primates.

In addition to the DNA viruses, AV and AAV, various RNA viruses have been investigated for uses in airway gene transfer. Murine parainfluenza virus type 1, human respiratory syn‐ cytial virus (RSV) and human parainfluenza virus type 3 (PIV3) can effectively transfect air‐ way epithelial cells by attaching to sialic acid and cholesterol [19], which are found on the apical surface of these cells. These viruses replicate in the cytoplasm and do not seem to cause mutagenesis during the insertion in DNA. Although RSV and PIV3 are human patho‐ gens, SeV, the only RNA virus for which efficiency has been assessed in vivo, is not. Howev‐ er, gene expression mediated by recombinant SeV-based vectors needs repeated administration, which does not seem feasible because of the development of neutralizing an‐ tibodies against the vector itself [20].

to overcome is the identification of methods to efficiently deliver RNAi to differentiated air‐

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

615

Lung transplantation is currently the only definitive treatment for end-stage CF lung dis‐ ease. However, the availability of donors is limited and the survival of transplantation is hardly 10–20% at 10 years [29]. Recently, two groups independently used similar tissue-en‐ gineering strategies to develop an autologous bioartificial lung that may begin to help over‐ come the limited availability of donor tissues [30, 31]. Evidence for gas exchange within the resulting grafts was demonstrated. Following the development of this technology, the ex vivo correction of patient-derived cells and the transplantation of these cells could lead to the cure of the disease. Although these initial results are very exciting, several steps need to be further optimized before long-term tissue-engineered lung function can be used in pa‐

**3. Gene therapy in Chronic Obstructive Pulmonary Disease (COPD)**

Chronic obstructive pulmonary disease is a disease characterised by the presence of airflow obstruction generally progressive due to chronic bronchitis or emphysema and may be par‐ tially reversible. COPD is the 4th leading cause of death in the United States. In 2000, the WHO estimated 2.74 million deaths worldwide from COPD. In-patient hospitalization and emergency department care accounts for >73% of this cost COPD costs \$1,522 per person per

Tobacco smoke is by far the most important risk factor for COPD worldwide. Other impor‐ tant risk factors are occupational exposure, socioeconomic status and genetic predisposition [33]. Thus, investigating into copd and into management possibilities is of high importance in order to provide the essential help to the patients. The currently used drugs can manage effectively the main symptoms of copd and may control the symptoms of this condition.

To date, there are no effective treatments for emphysema, nor are there efficient clinical management strategies. Novel approaches using gene therapy and stem cell technologies may offer new opportunities. However, this will remain almost entirely dependent on a more thorough understanding of the pathogenesis of COPD [34]. Currently, the most ac‐ cepted theory for the development of COPD is protease/ antiprotease imbalance similar to emphysema due to hereditary 1-antitrypsin deficiency [35]. Newer studies [36] have shown that the pathogenesis of COPD involves not only elastases, but also collagenases and gelati‐ nases. Experimental models [37] have suggested a role for 1-antitrypsin and secretory leuko‐ protease inhibitor in the treatment of this disorder. However, there is still need for a convincing study proving the concept of antiprotease treatment for COPD and emphysema [38] Neutrophils are a major source of proteases and reactive oxygen, so gene therapy could also target adhesion molecules for neutrophils to reduce their accumulation into the lung

way epithelia.

**2.2. Lung tissue engineering**

tient applications [32].

parenchyma.

year (3 times asthma costs) [GOLD 2008].

Lentiviral (LV) vectors derived from human immunodeficiency virus type 1 (HIV-1) and fe‐ line immunodeficiency virus (FIV) are integrating retroviruses which can be adequately uti‐ lized to achieve efficient transfection of airway epithelia [21].

Among the many nonviral gene therapy vectors investigated so far, GL67 ([Cholest-5-en-3 ol(3b)- 3-[(3-aminopropyl)[4-[(3-aminopropyl)amino]butyl] carbamate]) has emerged as a promising lipid for efficient lung transfection [22].

Finally, mRNA-based nonviral gene transfer is a new strategy in order to express the CFTR in target cells [23]. By the use of mRNA instead of plasmid DNA as the transgene, transfec‐ tion efficacy depends on the cytoplasmic expression machinery. However, when compared to DNA, much less is known about immune responses to RNA, although responses to both seem to be mediated by Toll-like receptors (TLRs).

As a chronic, lifelong disease, CF will be best treated with a continuous level of CFTR ex‐ pression. This could be achieved either by repeated application or with a long-duration ex‐ pression system. Viral vectors, which are mainly used in gene therapy appear difficult to administer repeatedly [24], in contrast to synthetic approaches [25].

The use of genomic DNA that contains all the control elements that allow gene expression at physiological levels has been utilized [26]. Extensive knowledge of the critical regulatory el‐ ements in the CFTR locus is required.

The CFTR gene maps at 7q31.2 and the expression is regulated during development and in different tissues. The CFTR locus is in connection with genes with different tissue-specific expression profiles, suggesting the presence of specific control promoters and insulators. Nuclear localization studies of CFTR and its adjacent gene loci in humans and mice demon‐ strate that different chromatin regions behave independently, depending on their expression profiles [27].

#### **2.1. Applications of RNA interference to treat CF**

The recent knowledge in the field of small interfering RNAs has led to the development of applications in relevance to CF. The RNAi technology has been used in order to identify gene products that contribute to steps in wild-type and mutant CFTR production and action [28]. Therefore, there is a possibility that RNAi-based strategies could be developed to in‐ crease the expression of △F508 CFTR, to rescue △F508 CFTR from proteosomal degradation or prolong its action on cell membrane. Similarly, targeting other cellular pathways, such as the inflammatory process, might lead to the reduction of symptoms. A significant obstacle to overcome is the identification of methods to efficiently deliver RNAi to differentiated air‐ way epithelia.

#### **2.2. Lung tissue engineering**

apical surface of these cells. These viruses replicate in the cytoplasm and do not seem to cause mutagenesis during the insertion in DNA. Although RSV and PIV3 are human patho‐ gens, SeV, the only RNA virus for which efficiency has been assessed in vivo, is not. Howev‐ er, gene expression mediated by recombinant SeV-based vectors needs repeated administration, which does not seem feasible because of the development of neutralizing an‐

Lentiviral (LV) vectors derived from human immunodeficiency virus type 1 (HIV-1) and fe‐ line immunodeficiency virus (FIV) are integrating retroviruses which can be adequately uti‐

Among the many nonviral gene therapy vectors investigated so far, GL67 ([Cholest-5-en-3 ol(3b)- 3-[(3-aminopropyl)[4-[(3-aminopropyl)amino]butyl] carbamate]) has emerged as a

Finally, mRNA-based nonviral gene transfer is a new strategy in order to express the CFTR in target cells [23]. By the use of mRNA instead of plasmid DNA as the transgene, transfec‐ tion efficacy depends on the cytoplasmic expression machinery. However, when compared to DNA, much less is known about immune responses to RNA, although responses to both

As a chronic, lifelong disease, CF will be best treated with a continuous level of CFTR ex‐ pression. This could be achieved either by repeated application or with a long-duration ex‐ pression system. Viral vectors, which are mainly used in gene therapy appear difficult to

The use of genomic DNA that contains all the control elements that allow gene expression at physiological levels has been utilized [26]. Extensive knowledge of the critical regulatory el‐

The CFTR gene maps at 7q31.2 and the expression is regulated during development and in different tissues. The CFTR locus is in connection with genes with different tissue-specific expression profiles, suggesting the presence of specific control promoters and insulators. Nuclear localization studies of CFTR and its adjacent gene loci in humans and mice demon‐ strate that different chromatin regions behave independently, depending on their expression

The recent knowledge in the field of small interfering RNAs has led to the development of applications in relevance to CF. The RNAi technology has been used in order to identify gene products that contribute to steps in wild-type and mutant CFTR production and action [28]. Therefore, there is a possibility that RNAi-based strategies could be developed to in‐ crease the expression of △F508 CFTR, to rescue △F508 CFTR from proteosomal degradation or prolong its action on cell membrane. Similarly, targeting other cellular pathways, such as the inflammatory process, might lead to the reduction of symptoms. A significant obstacle

tibodies against the vector itself [20].

614 Gene Therapy - Tools and Potential Applications

lized to achieve efficient transfection of airway epithelia [21].

promising lipid for efficient lung transfection [22].

seem to be mediated by Toll-like receptors (TLRs).

**2.1. Applications of RNA interference to treat CF**

ements in the CFTR locus is required.

profiles [27].

administer repeatedly [24], in contrast to synthetic approaches [25].

Lung transplantation is currently the only definitive treatment for end-stage CF lung dis‐ ease. However, the availability of donors is limited and the survival of transplantation is hardly 10–20% at 10 years [29]. Recently, two groups independently used similar tissue-en‐ gineering strategies to develop an autologous bioartificial lung that may begin to help over‐ come the limited availability of donor tissues [30, 31]. Evidence for gas exchange within the resulting grafts was demonstrated. Following the development of this technology, the ex vivo correction of patient-derived cells and the transplantation of these cells could lead to the cure of the disease. Although these initial results are very exciting, several steps need to be further optimized before long-term tissue-engineered lung function can be used in pa‐ tient applications [32].

#### **3. Gene therapy in Chronic Obstructive Pulmonary Disease (COPD)**

Chronic obstructive pulmonary disease is a disease characterised by the presence of airflow obstruction generally progressive due to chronic bronchitis or emphysema and may be par‐ tially reversible. COPD is the 4th leading cause of death in the United States. In 2000, the WHO estimated 2.74 million deaths worldwide from COPD. In-patient hospitalization and emergency department care accounts for >73% of this cost COPD costs \$1,522 per person per year (3 times asthma costs) [GOLD 2008].

Tobacco smoke is by far the most important risk factor for COPD worldwide. Other impor‐ tant risk factors are occupational exposure, socioeconomic status and genetic predisposition [33]. Thus, investigating into copd and into management possibilities is of high importance in order to provide the essential help to the patients. The currently used drugs can manage effectively the main symptoms of copd and may control the symptoms of this condition.

To date, there are no effective treatments for emphysema, nor are there efficient clinical management strategies. Novel approaches using gene therapy and stem cell technologies may offer new opportunities. However, this will remain almost entirely dependent on a more thorough understanding of the pathogenesis of COPD [34]. Currently, the most ac‐ cepted theory for the development of COPD is protease/ antiprotease imbalance similar to emphysema due to hereditary 1-antitrypsin deficiency [35]. Newer studies [36] have shown that the pathogenesis of COPD involves not only elastases, but also collagenases and gelati‐ nases. Experimental models [37] have suggested a role for 1-antitrypsin and secretory leuko‐ protease inhibitor in the treatment of this disorder. However, there is still need for a convincing study proving the concept of antiprotease treatment for COPD and emphysema [38] Neutrophils are a major source of proteases and reactive oxygen, so gene therapy could also target adhesion molecules for neutrophils to reduce their accumulation into the lung parenchyma.

#### **3.1. A1-antitrypsin deficiency**

A1-antitrypsin (AAT), is a major anti-protease serum protein, counteracting the effects of neutrophil elastase and other pro-inflammatory molecules released at sites of lung inflam‐ mation [39]. There are not effective treatments using protein therapy so gene therapy is be‐ ing evaluated as an alternative approach.

tightness, and coughing, usually associated with widespread, but variable, airflow obstruc‐ tion that is often reversible either spontaneously or with treatment [GINA 2000]. Since its pathogenesis is not clear, this definition is descriptive and inclusive of different phenotypes that are being increasingly recognized. Worldwide, 300 million people are supposed to be affected by asthma [53]. It appears that the global prevalence of asthma ranges 1–18% of the population in different countries. The WHO has estimated that 15 million disability-adjust‐ ed life-yrs are lost annually due to asthma, representing 1% of the total global disease bur‐ den [54]. Annual worldwide deaths from asthma have been estimated at 250,000 and

Gene Therapy Perspectives Against Diseases of the Respiratory System

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617

The best treatment of asthma is inhaled corticosteroids and bronchodilators, for the majority of asthmatic patients [55]. Gene therapy could bring some benefit for asthmatic patients with uncontrolled asthma who require high doses of corticosteroids and for patients with corticosteroid- resistant asthma. The target of gene therapy in bronchial asthma could be the overexpression of T-helper (Th) type 1 cytokines that influence the Th2 cytokine reactions [56]. Moreover the overexpression of IL-12 also restored local antiviral immunity, which is impaired in a Th2-dominated environment particulary during exacerbations of bronchial asthma due to viral infections [57]. Another study [58],examined the gene transfer of IFN that is a very interesting mediator in the airway hyper-responsiveness. Furthermore, anoth‐ er newer study [59] has shown that the transfer of the glucocorticoid receptor gene in vitro mediated the inhibition of nuclear factor-B activities even in absence of exogenous cortico‐ steroids, and the authors suggested that this approach could restore corticosteroid sensitivi‐

Lung cancer is the most common cancer worldwide, it is responsible for 12.4% of new cases of cancer in 2002. The overall mortality is 87% and 5-year survival is estimated to range from 15% in USA to 8.9% in developing countries [60]. It ranks first as the cause of death and it is responsible for 1.18 million deaths in 2002 and it is accounted by the World Health Organi‐ zation for 18.4% of all cancer deaths by 2015. Non-small cell cancer (NSCLC) accounts for

Even if there have been a lot of advances in surgery, radiation and chemotherapy, the 5-year survival for lung cancer remains poor. There is now great interest in gene therapy ap‐ proaches for thoracic malignancies. Lung cancer is usually metastatic at the time of diagno‐

Gene therapy for thoracic malignancies represents a therapeutic approach that has been evaluated in clinical trials for the last two decades. Using viral vectors or antisense RNA, strategies have included induction of apoptosis, suicide gene expression,cytokine based

therapy, various vaccinations and adoptive transfer of modified immune cells.

mortality does not appear to correlate well with prevalence.

ty in patients.

**5. Gene therapy in lung cancer**

approximately 85% of lung cancers [61].

sis and systemic therapy is needed rather than local therapy.

Early studies in cotton rats using first-generation Ad vectors resulted in detection of AAT in bronchoalveolar fluid for only 1 week post-administration [40]. Cationic liposomes have also been used to express human AAT in the rabbit lung following aerosolisation [41]. Recombi‐ nant AAV vectors are being evaluated for more persistent expression of therapeutic serum levels of human AAT in murine and non-human primate models following intramuscular injection [42]. A1-antitrypsin deficiency is a pulmonary disease with an underlying single gene defect and a target for gene therapy. One specific treatment for AAT deficiency availa‐ ble is the administration of AAT intravenously, but only 2–3% of the infused AAT actually reach the lungs. Another method of administration is the inhalation of nebulized AAT pow‐ der or aerosolized AAT solution [43]. However, the treatment by the means of an alternative therapy, namely gene therapy, provides long term solution [44]. Several vectors containing cDNA of AAT have been constructed for treating AAT deficiency diseases. These vectors are retroviral [45], adenoviral [46] and adeno-associated viral [47]. Besides this, AAT gene can also be transferred by liposomal vectors [48]. First clinical trial has demonstrated that AAT gene could be transferred in humans [49]. Patients with AAT deficiency received a single dose of non-viral cationic liposome. Protein Gene Therapy for Alpha-1-Antitrypsin Deficien‐ cy Diseases was detected in nasal lavage fluid, with maximum levels on fifth day, which is approximately one third of the normal levels. The retroviral vector containing cDNA of hu‐ man AAT with constitutive promoter have also been used as a delivery system. The disad‐ vantage of retroviral vector system is that transgene expression is low. The adenoviral vectors containing human AAT cDNA have been delivered to different organs and cells [50]. Results in vitro demonstrated that human alpha-1-antitrypsin was synthesized as well as se‐ creted. The adenoviruses are pathogenic in nature as well as immunogenic, therefore they have limited applications in treating AAT deficiency diseases. Recombinant adeno-associat‐ ed viral vectors have been most successful delivery system so far, as they are capable of ach‐ ieving therapeutic levels of AAT [51], and are less likely to induce an inflammatory response than adenoviral vectors. These viral and non-viral vectors showed advantages as well as dis‐ advantages in curing AAT deficiency diseases. Among tested rAAV serotypes, the rAAV8 was found to be more powerful gene therapy vector for treating lungs and liver diseases [52]. Newly developed AAV vector looks promising for treating AAT deficiency diseases.

#### **4. Gene therapy in asthma**

Asthma is a disorder defined by certain clinical, physiological and pathological characteris‐ tics. Asthma is a chronic inflammatory disorder of the airways associated with airway hy‐ per-responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, usually associated with widespread, but variable, airflow obstruc‐ tion that is often reversible either spontaneously or with treatment [GINA 2000]. Since its pathogenesis is not clear, this definition is descriptive and inclusive of different phenotypes that are being increasingly recognized. Worldwide, 300 million people are supposed to be affected by asthma [53]. It appears that the global prevalence of asthma ranges 1–18% of the population in different countries. The WHO has estimated that 15 million disability-adjust‐ ed life-yrs are lost annually due to asthma, representing 1% of the total global disease bur‐ den [54]. Annual worldwide deaths from asthma have been estimated at 250,000 and mortality does not appear to correlate well with prevalence.

The best treatment of asthma is inhaled corticosteroids and bronchodilators, for the majority of asthmatic patients [55]. Gene therapy could bring some benefit for asthmatic patients with uncontrolled asthma who require high doses of corticosteroids and for patients with corticosteroid- resistant asthma. The target of gene therapy in bronchial asthma could be the overexpression of T-helper (Th) type 1 cytokines that influence the Th2 cytokine reactions [56]. Moreover the overexpression of IL-12 also restored local antiviral immunity, which is impaired in a Th2-dominated environment particulary during exacerbations of bronchial asthma due to viral infections [57]. Another study [58],examined the gene transfer of IFN that is a very interesting mediator in the airway hyper-responsiveness. Furthermore, anoth‐ er newer study [59] has shown that the transfer of the glucocorticoid receptor gene in vitro mediated the inhibition of nuclear factor-B activities even in absence of exogenous cortico‐ steroids, and the authors suggested that this approach could restore corticosteroid sensitivi‐ ty in patients.

#### **5. Gene therapy in lung cancer**

**3.1. A1-antitrypsin deficiency**

616 Gene Therapy - Tools and Potential Applications

ing evaluated as an alternative approach.

**4. Gene therapy in asthma**

A1-antitrypsin (AAT), is a major anti-protease serum protein, counteracting the effects of neutrophil elastase and other pro-inflammatory molecules released at sites of lung inflam‐ mation [39]. There are not effective treatments using protein therapy so gene therapy is be‐

Early studies in cotton rats using first-generation Ad vectors resulted in detection of AAT in bronchoalveolar fluid for only 1 week post-administration [40]. Cationic liposomes have also been used to express human AAT in the rabbit lung following aerosolisation [41]. Recombi‐ nant AAV vectors are being evaluated for more persistent expression of therapeutic serum levels of human AAT in murine and non-human primate models following intramuscular injection [42]. A1-antitrypsin deficiency is a pulmonary disease with an underlying single gene defect and a target for gene therapy. One specific treatment for AAT deficiency availa‐ ble is the administration of AAT intravenously, but only 2–3% of the infused AAT actually reach the lungs. Another method of administration is the inhalation of nebulized AAT pow‐ der or aerosolized AAT solution [43]. However, the treatment by the means of an alternative therapy, namely gene therapy, provides long term solution [44]. Several vectors containing cDNA of AAT have been constructed for treating AAT deficiency diseases. These vectors are retroviral [45], adenoviral [46] and adeno-associated viral [47]. Besides this, AAT gene can also be transferred by liposomal vectors [48]. First clinical trial has demonstrated that AAT gene could be transferred in humans [49]. Patients with AAT deficiency received a single dose of non-viral cationic liposome. Protein Gene Therapy for Alpha-1-Antitrypsin Deficien‐ cy Diseases was detected in nasal lavage fluid, with maximum levels on fifth day, which is approximately one third of the normal levels. The retroviral vector containing cDNA of hu‐ man AAT with constitutive promoter have also been used as a delivery system. The disad‐ vantage of retroviral vector system is that transgene expression is low. The adenoviral vectors containing human AAT cDNA have been delivered to different organs and cells [50]. Results in vitro demonstrated that human alpha-1-antitrypsin was synthesized as well as se‐ creted. The adenoviruses are pathogenic in nature as well as immunogenic, therefore they have limited applications in treating AAT deficiency diseases. Recombinant adeno-associat‐ ed viral vectors have been most successful delivery system so far, as they are capable of ach‐ ieving therapeutic levels of AAT [51], and are less likely to induce an inflammatory response than adenoviral vectors. These viral and non-viral vectors showed advantages as well as dis‐ advantages in curing AAT deficiency diseases. Among tested rAAV serotypes, the rAAV8 was found to be more powerful gene therapy vector for treating lungs and liver diseases [52]. Newly developed AAV vector looks promising for treating AAT deficiency diseases.

Asthma is a disorder defined by certain clinical, physiological and pathological characteris‐ tics. Asthma is a chronic inflammatory disorder of the airways associated with airway hy‐ per-responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest Lung cancer is the most common cancer worldwide, it is responsible for 12.4% of new cases of cancer in 2002. The overall mortality is 87% and 5-year survival is estimated to range from 15% in USA to 8.9% in developing countries [60]. It ranks first as the cause of death and it is responsible for 1.18 million deaths in 2002 and it is accounted by the World Health Organi‐ zation for 18.4% of all cancer deaths by 2015. Non-small cell cancer (NSCLC) accounts for approximately 85% of lung cancers [61].

Even if there have been a lot of advances in surgery, radiation and chemotherapy, the 5-year survival for lung cancer remains poor. There is now great interest in gene therapy ap‐ proaches for thoracic malignancies. Lung cancer is usually metastatic at the time of diagno‐ sis and systemic therapy is needed rather than local therapy.

Gene therapy for thoracic malignancies represents a therapeutic approach that has been evaluated in clinical trials for the last two decades. Using viral vectors or antisense RNA, strategies have included induction of apoptosis, suicide gene expression,cytokine based therapy, various vaccinations and adoptive transfer of modified immune cells.

#### **5.1. Clinical trials**

#### *5.1.1. Replacement of tumor suppressors*

The goal of this strategy is to use a gene vector in order to encode a tumor-suppressor pro‐ tein in tumor cells that is mutated or absent in the majority of lung cancers.

Gene-Modified Dentritic Cell-Based Vaccination: Dentritic cells (DC) are the most potent an‐ tigen presenting cells in the immune system and they have been used for vaccination as vac‐ cine vehicles. They have been used in two ways. The first one is to modify DC ex vivo with chemokines or cytokines and inject them directly into tumors and then they take antigen and induce immune response. The second one is to load immature, phagocytic DC with an‐ tigen with the aid of purified protein, cell extracts, mRNA and gene vectors and after that

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

619

Ad.p53: p53 protein: It is proposed as a tumor antigen for vaccines as mutant p53 exists in very high levels in tumor cells and has more prolonged half-time than normal cells. p53 based gene therapy (p53 transduced DC) with standard chemotherapy showed promising results [74]. In a phase I trial, 29 patients with small cell lung cancer were vaccinated with DC transduced with Ad.p53 and the result was 1 patient with partial response and 7 cases with stable disease. Besides, out of the 21 patients that received a second line of chemothera‐ py, there was 62% response rate much higher from the rate that it is known for the second line therapy in small cell lung cancer. There was also a better survival (12.1 months instead

CCL21: CCL21 is a CC chemokine which is expressed in high levels in high endothelial ven‐ ules and T cell zones of spleen and lymph nodes and also it attracts mature DC, naive T cells and induces T-cell activation [75]. Preclinical data showed that there was potent activity

Gene-Modified Tumor Cell-Based Vaccination: Killed tumor cells (usually irradiated) have been injected into patients as vaccines against recurrent cancers for many years with partial

Transforming growth factor β2 antisense vector modified cells: It is known that increased levels of transforming growth factor (TGF-β2) are associated with greater immunosuppres‐ sion and poorer prognosis in patients with NSCLC. Preclinical studies showed that the de‐ livery of an antisense gene to TGF-β2 to ex vivo tumor cells inhibited cellular TGF-β2 expression and resulted in increased immunogenicity when these tumor cells were adminis‐ tered as a vaccine. In a phase II trial this method of vaccination with irradiated tumor cells modified with a TGF-β2 antisense vector (belagenpumatucel-L) was evaluated. There was better survival (dose-related) with minimal toxicity. Besides there were different immuno‐ logic end points such as increased levels of cytokines (INF-γ, interleukin-6,interleukin-4) and increased levels of antibody production to vaccine HLAs. In a trial, 21 patients received belagenpumatucel-L at a single dose [76]. It was shown that 70% of cases were stable, but there was no complete or partial response. There is an ongoing phase III trial in which this

Tumor cells modified to secrete granulocyte-monocyte colony stimulating factor(GVAX): Granulocyte-monocyte colony stimulating factor (GM-CSF) is a cytokine that is involved in the maturation and proliferation of myeloid progenitor cells and stimulates proliferation, maturation and migration of DC and that leads to induction of T-cell immune responses against cancer. There are preclinical studies in which the transfection of tumor cells with the

of 9.6 months) in patients that showed an immune response to vaccination.

against lung cancers when DC transduced with CCL1 were injected into tumors.

they inject these DC subcutaneously.

successful results.

vaccination is evaluated.

Tumor-based p53 therapy: It has been shown that the replacement of the normal p53 tumor suppressor gene in tumor cells induces rapid cell death by studies in cellular and animal models. In several early-phase clinical studies the strategy of the restoring the wild-type p53 expression in lung tumor cells was studied. The first study to demonstrate the feasibility of tumor suppressor gene replacement mediating tumor regression was a phase I study in which a retrovirus vector carrying wild-type p53 was administered to 7 patients with lung cancer with direct intra-tumoral injection. There was evidence of increased apoptosis in 6 patients and tumor regression in 3 patients [62]. Other phase I studies of p53 replacement with adenoviral vectors resulted in few partial responses and several patients with stabiliza‐ tion of disease[63, 64, 65]. Weill et al. delivered Ad.p53 to obstructive lesions endobronchial‐ ly and they had several partial responses [66]. In another large phase I study of Ad.p53 gene transfer that was delivered intra-tumoraly in combination with chemotherapy, it was shown that there was increased apoptosis in transduced tumors when examined histologically [67]. A single-arm phase II study of intratumoral Ad.p53 in combination with radiation showed tumor regression in 63% (12 of 19) and was well tolerated [68]. But in another phase II study there was no difference in response rates for Ad.p53/chemotherapy-treated lesions for pri‐ mary tumor lesions versus lesions treated with chemotherapy alone and this showed that Ad.p53 provided little local benefit over chemotherapy [69]. Keedy et al. delivered repeated‐ ly Ad.p53 by bronchoalveolar lavage (BAL) to patients with bronchoalveolar carcinoma. It was shown that this delivery resulted in transient expression of p53 in 19% (3 of 16) of pa‐ tients,2 of the 3 patients achieved stable disease. It was suggested that BAL could be used for adenoviral delivery, but toxicity was a serious issue with this approach [70]. Guan et al. delivered Ad.p53 alone or in combination with bronchial artery instillation (BIA) of chemo‐ therapy (fluorouracil, navelbine or cisplatin). The delivery of Ad.p53 was performed via di‐ rect percutaneous delivery or via BIA. There was 47% response rate in the combination group and an improvement in time to progression when compared with BAI alone [71]. The Adp53 has been approved for usage in neck and head cancers in China, but there are not any trials using Ad.p53 in lung cancer in USA. Vanchani et al. believed that there is a strong issue with the application of this method in lung cancer (especially treating endobronchial lesions) as there is no bystander effect in combination with low transfection efficiency of ad‐ enoviral vectors [72].

FUS1 Replacement: FUS1 is a novel tumor suppressor gene that was identified in human chromosome 3p21.3 region where allele losses and genetic alterations occur for some human cancers. In most premalignant lung lesions and lung cancers the expression of FUS1 protein is absent. It was shown that wt-FUS1 function was restored in 3p21.3-deficient non-small cell lung carcinoma cells and this function inhibited tumor cell growth by induction of apop‐ tosis and alteration of cell cycle kinetics [73].

Gene-Modified Dentritic Cell-Based Vaccination: Dentritic cells (DC) are the most potent an‐ tigen presenting cells in the immune system and they have been used for vaccination as vac‐ cine vehicles. They have been used in two ways. The first one is to modify DC ex vivo with chemokines or cytokines and inject them directly into tumors and then they take antigen and induce immune response. The second one is to load immature, phagocytic DC with an‐ tigen with the aid of purified protein, cell extracts, mRNA and gene vectors and after that they inject these DC subcutaneously.

**5.1. Clinical trials**

enoviral vectors [72].

tosis and alteration of cell cycle kinetics [73].

*5.1.1. Replacement of tumor suppressors*

618 Gene Therapy - Tools and Potential Applications

The goal of this strategy is to use a gene vector in order to encode a tumor-suppressor pro‐

Tumor-based p53 therapy: It has been shown that the replacement of the normal p53 tumor suppressor gene in tumor cells induces rapid cell death by studies in cellular and animal models. In several early-phase clinical studies the strategy of the restoring the wild-type p53 expression in lung tumor cells was studied. The first study to demonstrate the feasibility of tumor suppressor gene replacement mediating tumor regression was a phase I study in which a retrovirus vector carrying wild-type p53 was administered to 7 patients with lung cancer with direct intra-tumoral injection. There was evidence of increased apoptosis in 6 patients and tumor regression in 3 patients [62]. Other phase I studies of p53 replacement with adenoviral vectors resulted in few partial responses and several patients with stabiliza‐ tion of disease[63, 64, 65]. Weill et al. delivered Ad.p53 to obstructive lesions endobronchial‐ ly and they had several partial responses [66]. In another large phase I study of Ad.p53 gene transfer that was delivered intra-tumoraly in combination with chemotherapy, it was shown that there was increased apoptosis in transduced tumors when examined histologically [67]. A single-arm phase II study of intratumoral Ad.p53 in combination with radiation showed tumor regression in 63% (12 of 19) and was well tolerated [68]. But in another phase II study there was no difference in response rates for Ad.p53/chemotherapy-treated lesions for pri‐ mary tumor lesions versus lesions treated with chemotherapy alone and this showed that Ad.p53 provided little local benefit over chemotherapy [69]. Keedy et al. delivered repeated‐ ly Ad.p53 by bronchoalveolar lavage (BAL) to patients with bronchoalveolar carcinoma. It was shown that this delivery resulted in transient expression of p53 in 19% (3 of 16) of pa‐ tients,2 of the 3 patients achieved stable disease. It was suggested that BAL could be used for adenoviral delivery, but toxicity was a serious issue with this approach [70]. Guan et al. delivered Ad.p53 alone or in combination with bronchial artery instillation (BIA) of chemo‐ therapy (fluorouracil, navelbine or cisplatin). The delivery of Ad.p53 was performed via di‐ rect percutaneous delivery or via BIA. There was 47% response rate in the combination group and an improvement in time to progression when compared with BAI alone [71]. The Adp53 has been approved for usage in neck and head cancers in China, but there are not any trials using Ad.p53 in lung cancer in USA. Vanchani et al. believed that there is a strong issue with the application of this method in lung cancer (especially treating endobronchial lesions) as there is no bystander effect in combination with low transfection efficiency of ad‐

FUS1 Replacement: FUS1 is a novel tumor suppressor gene that was identified in human chromosome 3p21.3 region where allele losses and genetic alterations occur for some human cancers. In most premalignant lung lesions and lung cancers the expression of FUS1 protein is absent. It was shown that wt-FUS1 function was restored in 3p21.3-deficient non-small cell lung carcinoma cells and this function inhibited tumor cell growth by induction of apop‐

tein in tumor cells that is mutated or absent in the majority of lung cancers.

Ad.p53: p53 protein: It is proposed as a tumor antigen for vaccines as mutant p53 exists in very high levels in tumor cells and has more prolonged half-time than normal cells. p53 based gene therapy (p53 transduced DC) with standard chemotherapy showed promising results [74]. In a phase I trial, 29 patients with small cell lung cancer were vaccinated with DC transduced with Ad.p53 and the result was 1 patient with partial response and 7 cases with stable disease. Besides, out of the 21 patients that received a second line of chemothera‐ py, there was 62% response rate much higher from the rate that it is known for the second line therapy in small cell lung cancer. There was also a better survival (12.1 months instead of 9.6 months) in patients that showed an immune response to vaccination.

CCL21: CCL21 is a CC chemokine which is expressed in high levels in high endothelial ven‐ ules and T cell zones of spleen and lymph nodes and also it attracts mature DC, naive T cells and induces T-cell activation [75]. Preclinical data showed that there was potent activity against lung cancers when DC transduced with CCL1 were injected into tumors.

Gene-Modified Tumor Cell-Based Vaccination: Killed tumor cells (usually irradiated) have been injected into patients as vaccines against recurrent cancers for many years with partial successful results.

Transforming growth factor β2 antisense vector modified cells: It is known that increased levels of transforming growth factor (TGF-β2) are associated with greater immunosuppres‐ sion and poorer prognosis in patients with NSCLC. Preclinical studies showed that the de‐ livery of an antisense gene to TGF-β2 to ex vivo tumor cells inhibited cellular TGF-β2 expression and resulted in increased immunogenicity when these tumor cells were adminis‐ tered as a vaccine. In a phase II trial this method of vaccination with irradiated tumor cells modified with a TGF-β2 antisense vector (belagenpumatucel-L) was evaluated. There was better survival (dose-related) with minimal toxicity. Besides there were different immuno‐ logic end points such as increased levels of cytokines (INF-γ, interleukin-6,interleukin-4) and increased levels of antibody production to vaccine HLAs. In a trial, 21 patients received belagenpumatucel-L at a single dose [76]. It was shown that 70% of cases were stable, but there was no complete or partial response. There is an ongoing phase III trial in which this vaccination is evaluated.

Tumor cells modified to secrete granulocyte-monocyte colony stimulating factor(GVAX): Granulocyte-monocyte colony stimulating factor (GM-CSF) is a cytokine that is involved in the maturation and proliferation of myeloid progenitor cells and stimulates proliferation, maturation and migration of DC and that leads to induction of T-cell immune responses against cancer. There are preclinical studies in which the transfection of tumor cells with the GM-CSF gene has led these cells to induce antitumor immune responses. The clinical trials in lung cancer started using a vaccine platform with intradermal vaccination of irradiated autologous tumor cells that were virally enginnered to secrete GM-CSF [77, 78]. In the first trial of cases of metastatic NSCLC, GM-CSF was transduced into autologous tumor cells with the aid of adenoviral vector before irradiation and vaccination. There were a few clini‐ cal responses with a strong immune response. A delayed hypersensitivity reaction to irradi‐ ated, autologous nontransfected tumor cells was observed in patients. Nemunaitis J,et al. used a similar strategy in early-stage and late-stage patients and they showed that there were several clinical responses with similar immunologic outcomes [79]. In another trial, Nemunaitis J,et al. used a vaccine of unmodified, irradiated autologous tumor cells mixed with a GM-CSF-secreting bystander cell line. The vaccine GM-CSF secretion was higher than with the autologous vaccine, but the frequency of vaccine site reactions, tumor responses and survival were less favorable with the bystander vaccine [80]. Finally, the GVAX ap‐ proach was not used more in lung cancer because the results were not satisfied and now on‐ ly studies in pancreatic cancer are going on.

Disease control was observed for 4 of 5 patients. The existence of MUC1 specific responses

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621

L523S vaccination: L523S is an immunogetic lung antigen that is expressed in 80% of lung cancer cells. Nemunaitis et al. in a phase I study, they gave two doses of intramuscular re‐ combinant DNA followed by two doses of Ad.L523S (given 4 weeks apart) to 13 patients with early stage NSCLC(stage 1B,IIA and IIB). The authors found that only 1 patient showed

This technology is able to downregulate a lot of molecules that promote lung cancer tumor growth. There are 3 trials with antisense therapy. In the first trial aprinocarsen was used. Aprinocarsen is an oligonucleotide that binds to mRNA for protein kinase C-a and inhibits its expression. It was demonstrated that this molecule was safe in patients with lung cancer and it was characterized by modest activity in combination with chemotherapy [85]. In an‐ other trial with chemotherapy with or without aprinocarsen as first line therapy, it was shown that there was no better survival but with some toxicity as well [86]. In phase I stud‐ ies it was shown that few patients had prolonged stable disease and 1 patient had response with the administration of Raf antisense molecules [87]. In patients with lung cancer in two phase II studies, these molecules did not show any antitumor activity [88]. Next, in other tri‐ als, the authors used Bcl-2, an apoptotic inhibitor which is overexpressed by many tumors and especially by 80-90% of SCLCs; the existence of this inhibitor means increased resistance to chemotherapy. In two trials there were encouraging results [89, 90], but in another trial of standard chemotherapy with or without a bcl-2 antisense oligonucleotide (oblimersen) more hematologic toxicity and worse overall survival was observed in the experimental arm [91].

The trials that have been made about gene therapy in lung cancer, preclinically and clinical‐ ly, have demonstrated intermittent efficacy. The technology of gene transfer is promising but it is not easy to transduce more than a small number of tumor cells. This is a very impor‐ tant issue especially with approaches that they do not have bystander effects. It is very im‐ portant to create vectors that they are able to induce long term in vivo expression as

Another interesting strategy is the immune-gene therapy, which requires gene transduction for stimulating an endogenous immune response and in this way a bystander effect is gener‐ ated. There are some engouraging approaches with gene therapy to stimulate anti-tumor re‐

There is another important field of creating adoptive transfer of gene-modified autologous lymphocytes that are modified ex vivo by using lentiviruses or retroviruses. This approach

sponses by delivering immunostimulatory cytokines or by administering a vaccine.

is directed against mesothelioma and lung cancer cells.

was translated to longer time to progression and better overall survival [83].

a L523S-specific antibody response [84].

**5.2. Antisense therapy**

**5.3. New directions**

lentiviruses and AAVs.

a(1,3)Galactosyltransferase: The gene that encodes a(1,3) Galactosyltransferase is not active in humans and it is functional in other mammalian cells. The major mechanism of hyper‐ acute rejection of xenotransplants is the production of anti-a Gal antibodies in humans. Mor‐ ris J et al. used allogeneic NSCLC tumor cells that were retrovirally modified to express aGT. It was shown that 6 of 17 patients, that received intradermal treatments, had prolonged stable disease [81].

B7.1/HLA vaccination: B7.1 is the one that costimulates T cells during priming by an anti‐ gen-priming cell. In a phase I trial of 19 patients with advanced NSCLC, treatment with an allogeneic lung cancer cell line vaccine transfected with B7.1,HLA-A1 and HLA-A2 was done. There was one partial response and 5 cases with stable disease. In the 6 responders, the CD8 T cell titers to tumor cell stimulations were elevated steadily till 150 weeks after therapy [82].A phase II trial is ongoing in patients with stage IIIB/IV who fail after the first line chemotherapy.

#### *5.1.2. Vaccines*

MUC-1 vaccination: MUC-1 is a tumor-associated mucin-type surface antigen normally found on epithelial cells in many tissues. In cases of lung cancer the targeting of MUC-1 has been used in a lot of ways with gene and non-gene therapy approaches. Ramlau R et al. in their 2 arm phase II trial with 65 patients with IIIB/IV NSCLC used a vaccinia virus contain‐ ing the coding sequences for MUC1 and IL-2 (TG4010).The patients that participated in the trial had MUC-1 antigen expression on the primary tumor or metastases. In the 1ST arm (44 patients), combination therapy with TG4010 and cisplatin/vinorelbine was given, and in the 2nd arm TG4010 monotherapy was given followed by combination therapy at progression. In the 1st arm there was partial response in 29.5% and survival rate of 53% for the 1st year. In the 2nd arm, two of the 21 patients had stable disease for more than 6 months with mono‐ therapy of TG4010 and this arm was terminated early as the results were not satisfied. There were MUC1-specific responses for 12 of 21 patients with stable disease or partial response. Disease control was observed for 4 of 5 patients. The existence of MUC1 specific responses was translated to longer time to progression and better overall survival [83].

L523S vaccination: L523S is an immunogetic lung antigen that is expressed in 80% of lung cancer cells. Nemunaitis et al. in a phase I study, they gave two doses of intramuscular re‐ combinant DNA followed by two doses of Ad.L523S (given 4 weeks apart) to 13 patients with early stage NSCLC(stage 1B,IIA and IIB). The authors found that only 1 patient showed a L523S-specific antibody response [84].

#### **5.2. Antisense therapy**

GM-CSF gene has led these cells to induce antitumor immune responses. The clinical trials in lung cancer started using a vaccine platform with intradermal vaccination of irradiated autologous tumor cells that were virally enginnered to secrete GM-CSF [77, 78]. In the first trial of cases of metastatic NSCLC, GM-CSF was transduced into autologous tumor cells with the aid of adenoviral vector before irradiation and vaccination. There were a few clini‐ cal responses with a strong immune response. A delayed hypersensitivity reaction to irradi‐ ated, autologous nontransfected tumor cells was observed in patients. Nemunaitis J,et al. used a similar strategy in early-stage and late-stage patients and they showed that there were several clinical responses with similar immunologic outcomes [79]. In another trial, Nemunaitis J,et al. used a vaccine of unmodified, irradiated autologous tumor cells mixed with a GM-CSF-secreting bystander cell line. The vaccine GM-CSF secretion was higher than with the autologous vaccine, but the frequency of vaccine site reactions, tumor responses and survival were less favorable with the bystander vaccine [80]. Finally, the GVAX ap‐ proach was not used more in lung cancer because the results were not satisfied and now on‐

a(1,3)Galactosyltransferase: The gene that encodes a(1,3) Galactosyltransferase is not active in humans and it is functional in other mammalian cells. The major mechanism of hyper‐ acute rejection of xenotransplants is the production of anti-a Gal antibodies in humans. Mor‐ ris J et al. used allogeneic NSCLC tumor cells that were retrovirally modified to express aGT. It was shown that 6 of 17 patients, that received intradermal treatments, had prolonged

B7.1/HLA vaccination: B7.1 is the one that costimulates T cells during priming by an anti‐ gen-priming cell. In a phase I trial of 19 patients with advanced NSCLC, treatment with an allogeneic lung cancer cell line vaccine transfected with B7.1,HLA-A1 and HLA-A2 was done. There was one partial response and 5 cases with stable disease. In the 6 responders, the CD8 T cell titers to tumor cell stimulations were elevated steadily till 150 weeks after therapy [82].A phase II trial is ongoing in patients with stage IIIB/IV who fail after the first

MUC-1 vaccination: MUC-1 is a tumor-associated mucin-type surface antigen normally found on epithelial cells in many tissues. In cases of lung cancer the targeting of MUC-1 has been used in a lot of ways with gene and non-gene therapy approaches. Ramlau R et al. in their 2 arm phase II trial with 65 patients with IIIB/IV NSCLC used a vaccinia virus contain‐ ing the coding sequences for MUC1 and IL-2 (TG4010).The patients that participated in the trial had MUC-1 antigen expression on the primary tumor or metastases. In the 1ST arm (44 patients), combination therapy with TG4010 and cisplatin/vinorelbine was given, and in the 2nd arm TG4010 monotherapy was given followed by combination therapy at progression. In the 1st arm there was partial response in 29.5% and survival rate of 53% for the 1st year. In the 2nd arm, two of the 21 patients had stable disease for more than 6 months with mono‐ therapy of TG4010 and this arm was terminated early as the results were not satisfied. There were MUC1-specific responses for 12 of 21 patients with stable disease or partial response.

ly studies in pancreatic cancer are going on.

620 Gene Therapy - Tools and Potential Applications

stable disease [81].

line chemotherapy.

*5.1.2. Vaccines*

This technology is able to downregulate a lot of molecules that promote lung cancer tumor growth. There are 3 trials with antisense therapy. In the first trial aprinocarsen was used. Aprinocarsen is an oligonucleotide that binds to mRNA for protein kinase C-a and inhibits its expression. It was demonstrated that this molecule was safe in patients with lung cancer and it was characterized by modest activity in combination with chemotherapy [85]. In an‐ other trial with chemotherapy with or without aprinocarsen as first line therapy, it was shown that there was no better survival but with some toxicity as well [86]. In phase I stud‐ ies it was shown that few patients had prolonged stable disease and 1 patient had response with the administration of Raf antisense molecules [87]. In patients with lung cancer in two phase II studies, these molecules did not show any antitumor activity [88]. Next, in other tri‐ als, the authors used Bcl-2, an apoptotic inhibitor which is overexpressed by many tumors and especially by 80-90% of SCLCs; the existence of this inhibitor means increased resistance to chemotherapy. In two trials there were encouraging results [89, 90], but in another trial of standard chemotherapy with or without a bcl-2 antisense oligonucleotide (oblimersen) more hematologic toxicity and worse overall survival was observed in the experimental arm [91].

#### **5.3. New directions**

The trials that have been made about gene therapy in lung cancer, preclinically and clinical‐ ly, have demonstrated intermittent efficacy. The technology of gene transfer is promising but it is not easy to transduce more than a small number of tumor cells. This is a very impor‐ tant issue especially with approaches that they do not have bystander effects. It is very im‐ portant to create vectors that they are able to induce long term in vivo expression as lentiviruses and AAVs.

Another interesting strategy is the immune-gene therapy, which requires gene transduction for stimulating an endogenous immune response and in this way a bystander effect is gener‐ ated. There are some engouraging approaches with gene therapy to stimulate anti-tumor re‐ sponses by delivering immunostimulatory cytokines or by administering a vaccine.

There is another important field of creating adoptive transfer of gene-modified autologous lymphocytes that are modified ex vivo by using lentiviruses or retroviruses. This approach is directed against mesothelioma and lung cancer cells.

#### **6. Conclusion**

Gene therapy is a very promising tool for the respiratory clinician and a few clinical trials have been performed. All these trials have shown safety but intermittent efficacy. Gene ther‐ apy for pulmonary diseases has not yet reached the point of clinical practice. But we can say that this tool will find a very interesting role in our efforts for treating respiratory diseases in the future.

[9] Engelhardt, J.F., Yankaskas, J.R., Ernst, S.A., Yang, Y., Marino, C.R., Boucher, R.C., Cohn, J.A. and Wilson, J.M. (1992) Submucosal glands are the predominant site of

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623

[10] Zhou, L., Dey, C.R., Wert, S.E., DuVall, M.D., Frizzell, R.A. and Whitsett, J.A. (1994) Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human

[11] Joo, N.S., Cho, H.J., Khansaheb, M. and Wine, J.J. (2010) Hyposecretion of fluid from tracheal submucosal glands of CFTR-deficient pigs. J. Clin. Invest., 120, 3161–3166

[12] Liu, X., Luo, M., Guo, C., Yan, Z., Wang, Y., Lei-Butters, D.C. and Engelhardt, J.F. (2009) Analysis of adeno-associated virus progenitor cell transduction in mouse lung.

[13] Rock, J.R., Onaitis, M.W., Rawlins, E.L., Lu, Y., Clark, C.P., Xue, Y., Randell, S.H. and Hogan, B.L. (2009) Basal cells as stem cells of the mouse trachea and human airway

[14] Randell, S.H. (1992) Progenitor-progeny relationships in airway epithelium. Chest,

[15] Ford, J.R. and Terzaghi-Howe, M. (1992) Basal cells are the progenitors of primary

[16] Borthwick, D.W., Shahbazian, M., Krantz, Q.T., Dorin, J.R. and Randell, S.H. (2001) Evidence for stem-cell niches in the tracheal epithelium. Am. J. Respir. Cell Mol. Bi‐

[17] Hong, K.U., Reynolds, S.D., Giangreco, A., Hurley, C.M. and Stripp, B.R. (2001) Clara cell secretory protein-expressing cells of the airway neuroepithelial body microenvir‐ onment include a label-retaining subset and are critical for epithelial renewal after

[18] Flotte TR, Ng P, Dylla DE, et al. Viral vector-mediated and cell-based therapies for

[19] Zhang L, Bukreyev A, Thompson CI, et al. Infection of ciliated cells by human para‐ influenza virus type 3 in an in vitro model of human airway epithelium. J Virol

[20] Griesenbach U, Boyton RJ, Somerton L, et al. Effect of tolerance induction to immu‐ nodominant T-cell epitopes of Sendai virus on gene expression following repeat ad‐

[21] Copreni E, Penzo M, Carrabino S, Conese M. Lentiviral-mediated gene transfer to the respiratory epithelium: a promising approach to gene therapy of Cystic Fibrosis.

progenitor cell depletion. Am. J. Respir. Cell Mol. Biol., 24, 671–681

CFTR expression in the human bronchus. Nat. Genet., 2, 240–248

epithelium. Proc. Natl Acad. Sci. USA, 106, 12771–12775

tracheal epithelial cell cultures. Exp. Cell Res., 198, 69–77

treatment of cystic fibrosis. Mol Ther 2007;15:229–41

ministration. Gene Ther 2006;13:449–56

Gene Ther 2004;11:S67–75

CFTR. Science, 266, 1705–1708

Mol. Ther., 17, 285–293

101, 11S–16S

ol., 24, 662–670

2005;79:1113–24

#### **Author details**

Dimosthenis Lykouras1 , Kiriakos Karkoulias1 , Christos Tourmousoglou2 , Efstratios Koletsis2 , Kostas Spiropoulos1 and Dimitrios Dougenis2

1 Department of Pulmonary Medicine, University Hospital of Patras, Greece

2 Department of Cardiothoracic Surgery, University Hospital of Patras, Greece

#### **References**


[9] Engelhardt, J.F., Yankaskas, J.R., Ernst, S.A., Yang, Y., Marino, C.R., Boucher, R.C., Cohn, J.A. and Wilson, J.M. (1992) Submucosal glands are the predominant site of CFTR expression in the human bronchus. Nat. Genet., 2, 240–248

**6. Conclusion**

622 Gene Therapy - Tools and Potential Applications

the future.

**Author details**

Dimosthenis Lykouras1

Kostas Spiropoulos1

**References**

Gene therapy is a very promising tool for the respiratory clinician and a few clinical trials have been performed. All these trials have shown safety but intermittent efficacy. Gene ther‐ apy for pulmonary diseases has not yet reached the point of clinical practice. But we can say that this tool will find a very interesting role in our efforts for treating respiratory diseases in

, Christos Tourmousoglou2

, Efstratios Koletsis2

,

, Kiriakos Karkoulias1

1 Department of Pulmonary Medicine, University Hospital of Patras, Greece

2 Department of Cardiothoracic Surgery, University Hospital of Patras, Greece

[1] Mall M, Bleich M, Greger R, Schreiber R, Kunzelmann K. The amiloride-inhibitable Na+ conductance is reduced by the cystic fibrosis transmembrane conductance regu‐

[2] Stutts MJ, Canessa CM, Olsen JC, et al. CFTR as a cAMP-dependent regulator of so‐

[3] Matsui H, Grubb BR, Tarran R, et al. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airway disease.

[4] Quinton PM. Cystic fibrosis: impaired bicarbonate secretion and mucoviscidosis.

[5] Wine JJ, Joo NS. Submucosal glands and airway defense. Proc Am Thorac Soc

[6] Devidas, S. and Guggino, W.B. (1997) CFTR: domains, structure, and function. J. Bio‐

[7] Riordan, J.R. (2008) CFTR function and prospects for therapy. Annu. Rev. Biochem.,

[8] Johnson, L.G., Olsen, J.C., Sarkadi, B., Moore, K.L., Swanstrom, R. and Boucher, R.C. (1992) Efficiency of gene transfer for restoration of normal airway epithelial function

lator in normal but not in cystic fibrosis airways. J Clin Invest 1998;102:15–21

and Dimitrios Dougenis2

dium channel. Science 1995;269:847–50. 1–3

Cell 1998;95:1005–15

Lancet 2008;372:415–7

energ. Biomembr., 29, 443–451

in cystic fibrosis. Nat. Genet., 2, 21–25

2004;1:47–53

77, 701–726


[22] Lee ER, Marshall J, Siegel CS, et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 1996;7:1701– 17

[35] Barnes, P. J. Mediators of chronic obstructive pulmonary disease. Pharm. Rev. 56,

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

625

[36] Segura-Valdez L, Pardo A, Gaxiola M, et al. Upregulation of gelatinases A and B, col‐ lagenases 1 and 2, and increased parenchymal cell death in COPD. Chest 2000;

[37] Tomee JF, Koeter GH, Hiemstra PS, et al. Secretory leukoprotease inhibitor: a native antimicrobial protein presenting a new therapeutic option? Thorax 1998; 53:114–116

[38] Rogers DF, Laurent GJ. New ideas on the pathophysiology and treatment of lung

[39] Carrell R. W. (1986) alpha 1-Antitrypsin: molecular pathology, leukocytes, and tissue

[40] Rosenfeld M. A., Siegfried W., Yoshimura K., Yoneyama K., Fukayama M., Stier L. E. et al. (1991) Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene

[41] Canonico A. E., Conary J. T., Meyrick B. O. and Brigham K. L. (1994) Aerosol and in‐ travenous transfection of human alpha- 1-antitrypsin gene to lungs of rabbits. Am J

[42] Song S., Scott-Jorgensen M., Wang J., Poirier A., Crawford J. and Campbell-Thomp‐ son M. et al. (2002) Intramuscular administration of recombinant adeno-associated virus 2 alpha- 1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate mod‐

[43] Crystal, R. G.( 1992). Gene therapy strategies for pulmonary diseases. American Jour‐ nal of Medicine, Vol 92, No. 6A, pp.44S-52S, ISSN 1175-6365]. [Hubbard, R. C. & Crystal, R. G. (1990). Strategies for aerosol therapy of alpha l-antitrypsin deficiency

by the aerosol route. Lung, Vol 168, Supplement, pp.565-578, ISSN 0341- 2040

[44] Flotte, T. (March 2011).Alpha-1-antitrypsin Deficiency, In:Project, 20.3.2011, Availa‐

[45] Kay, M. A.; Baley, P.; Rothenberg, S.; Leland, F.; Fleming, L.; Ponder, K. P.; Liu, T.; Finegold, M.; Darlington, G.; Pokorny, W, et al. (1992). Expression of human α- 1- an‐ titrypsin in dogs after autologous transplantation of retroviral transduced hepato‐ cytes. Proceeding of the National Academy of Science of the United State of America,

[46] Jaffe, H. A.; Danel, C.; Longenecker, G.; Metzger, M.; Setoguchi,Y.; Rosenfeld, M. A.; Gant, T.W.; Thorgeirsson, S. S.; Stratford-Perricaudet, L. D.; Perricaudet, M, et al. (1992). Adenovirus-mediated in vivo gene transfer and expression in normal rat liv‐

[47] Song, S.; Morgan, M.; Ellis, T.; Poirier, A.; Chesnut, K.; Wang, J.; Brantley, M.; Mu‐ zyczka, N.; Byrne, B. J.; Atkinson, M. & Flotte, T. R. (1998). Sustained secretion of hu‐

515–548 (2004)

117:684–694

disease. Thorax 1998; 53:200–203

damage. J Clin Invest 78: 1427–1431

Respir Cell Mol. Biol. 10: 24–29

to the lung epithelium in vivo. Science 252: 431–434

el: safety and immunologic aspects. Mol. Ther. 6: 329–335

ble from http://www.gtc.ufl.edu/research/gtc-rppulm.htm#aa

er. Nature Genetics, Vol 1, No.5, pp. 372–378, ISSN 1061-4036

Vol 89, No. 1, pp. 89–93, ISSN 0027-8424


[35] Barnes, P. J. Mediators of chronic obstructive pulmonary disease. Pharm. Rev. 56, 515–548 (2004)

[22] Lee ER, Marshall J, Siegel CS, et al. Detailed analysis of structures and formulations of cationic lipids for efficient gene transfer to the lung. Hum Gene Ther 1996;7:1701–

[23] Yamamoto A, Kormann M, Rosenecker J, Rudolph C. Current prospects for mRNA

[24] Moss, R. B.,Milla, C., Colombo, J., Accurso, F., Zeitlin, P. L., Clancy, J. P., et al. (2007) Repeated aerosolized AAV-CFTR for treatment of cystic fibrosis: a randomized pla‐

[25] Hyde, S. C., Southern, K. W., Gileadi, U., Fitzjohn, E. M., Mofford, K. A., Waddell, B. E., et al. (2000) Repeat administration of DNA/liposomes to the nasal epithelium of

[26] Conese M, Boyd AC, Di Gioia S, Auriche C, Ascenzioni F. Genomic context vectors and artificial chromosomes for cystic fibrosis gene therapy. Curr Gene Ther

[27] Zink D, Amaral MD, Englmann A, et al. Transcription-dependent spatial arrange‐ ments of CFTR and adjacent genes in human cell nuclei. J Cell Biol 2004;166:815–25 [28] Wang, X., Venable, J., LaPointe, P., Hutt, D.M., Koulov, A.V., Coppinger, J., Gurkan, C., Kellner, W., Matteson, J., Plutner, H. et al. (2006) Hsp90 cochaperone Aha1 down‐

[29] Orens, J.B. and Garrity, E.R. Jr (2009) General overview of lung transplantation and

[30] Petersen, T.H., Calle, E.A., Zhao, L., Lee, E.J., Gui, L., Raredon, M.B., Gavrilov, K., Yi, T., Zhuang, Z.W., Breuer, C. et al. (2010) Tissue-engineered lungs for in vivo implan‐

[31] Ott, H.C., Clippinger, B., Conrad, C., Schuetz, C., Pomerantseva, I., Ikonomou, L., Kotton, D. and Vacanti, J.P. (2010) Regeneration and orthotopic transplantation of a

[32] de Perrot, M., Fischer, S., Liu, M., Imai, Y., Martins, S., Sakiyama, S., Tabata, T., Bai, X.H., Waddell, T.K., Davidson, B.L. et al. (2003) Impact of human interleukin-10 on vector-induced inflammation and early graft function in rat lung transplantation.

[33] Menzies D, Nair A, Williamson PA, Schembri S, Al-Khairalla MZ, Barnes M, et al. Respiratory symptoms, pulmonary function, and markers of inflammation among bar workers before and after a legislative ban on smoking in public places. JAMA

[34] Eisner MD, Balmes J, Katz BP, Trupin L, Yelin E, Blanc P. Lifetime environmental to‐ bacco smoke exposure and the risk of chronic obstructive pulmonary disease. Envi‐

regulation rescues misfolding of CFTR in cystic fibrosis. Cell, 127, 803–815

review of organ allocation. Proc. Am. Thorac. Soc., 6, 13–19

gene delivery. Eur J Pharm Biopharm 2009;71:484–9

patients with cystic fibrosis. Gene Ther. 7, 1156–1165

cebocontrolled phase 2B trial. Hum. Gene Ther. 18, 726–732

17

624 Gene Therapy - Tools and Potential Applications

2007;7:175–87

tation. Science, 329, 538–541

2006;296(14):1742-8

ron Health Perspect 2005;4:7-15

bioartificial lung. Nat. Med., 16, 927–933

Am. J. Respir. Cell Mol. Biol., 28, 616–625


man α -1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proceeding of the National Academy of Science of the United State of Ameri‐ ca, Vol 95, No.24, pp.14384–14388, ISSN 0027-8424

[59] Mathieu M, Gougat C, Jaffuel D, et al. The glucocorticoid receptor gene as a candi‐

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

627

[60] Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics, 2002. CA Cancer J Clin

[62] Roth JA, Nguyen D, Lawrence DD, et al. Retrovirusmediated wild-type p53 gene transfer to tumors of patients with lung cancer. Nat Med 1996;2(9): 985–91

[63] Schuler M, Rochlitz C, Horowitz JA, et al. A phase I study of adenovirus-mediated wild-type p53 gene transfer in patients with advanced non-small cell lung cancer.

[64] Swisher SG, Roth JA, Nemunaitis J, et al. Adenovirus- mediated p53 gene transfer in

[65] Fujiwara T, Tanaka N, Kanazawa S, et al. Multicenter phase I study of repeated intra‐ tumoral delivery of adenoviral p53 in patients with advanced nonsmall- cell lung

[66] Weill D,Mack M,Roth J,et al. Adenoviral-mediated p53 gene transfer to non-small

[67] Nemunaitis J, Swisher SG, Timmons T, et al. Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J

[68] Swisher SG, Roth JA, Komaki R, et al. Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery of adenoviral p53

[69] Schuler M, Herrmann R, De Greve JL, et al. Adenovirus-mediated wild-type p53 gene transfer in patients receiving chemotherapy for advanced non-small-cell lung

[70] Keedy V, Wang W, Schiller J, et al. Phase I study of adenovirus p53 administered by bronchoalveolarlavage in patients with bronchioloalveolar cell lung carcinoma:

[71] Guan YS,Liu Y, Zou Q,et al.Adenovirus-mediated wild type p53 gene transfer in combination with bronchial arterial infusion for treatment of advanced non-small

[72] Vanchani A,Moon E,Wakeam E, et al.Gene therapy for mesothelioma and lung can‐

[73] Ji L, Roth JA. Tumor suppressor FUS1 signaling pathway. J Thorac Oncol 2008;3(4):

cell lung cancer,one year follow up.J Zhejiang Univ Sci B 2009; 10,5: 331-40

cancer: results of a multicenter phase II study. J Clin Oncol 2001;19(6):1750–8

advanced non-small-cell lung cancer. J Natl Cancer Inst1999;91(9):763–71

cell lung cancer through endobronchial injection.Chest 2000,118:966-970

(INGN 201) and radiation therapy. Clin Cancer Res 2003;9(1):93–101

[61] Sher T, Dy GK, Adjei AA. Small cell lung cancer. Mayo Clin Proc 2008; 83:355-67

date for gene therapy in asthma. Gene Ther 1999; 6:245–252.30

2005; 55:74-108

Hum Gene Ther 1998;9(14):2075–82

cancer. J Clin Oncol 2006;24(11): 1689–99

ECOG 6597. J Clin Oncol 2008;26(25):4166–71

cer.Am J Respir Cell Mol Biol, 2010,42,385-393

327–30

Clin Oncol 2000;18(3):609–22


[59] Mathieu M, Gougat C, Jaffuel D, et al. The glucocorticoid receptor gene as a candi‐ date for gene therapy in asthma. Gene Ther 1999; 6:245–252.30

man α -1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proceeding of the National Academy of Science of the United State of Ameri‐

[48] Canonico, A. E.; Conary, J. T.; Meyrick, B.O. & Brigham, K.L.(1994).Aerosol and in‐ travenous transfection of human -1 antitrypsin gene to lungs of rabbits. American Journal of Respiratory Cell and Molecular Biology, Vol 10, No.1, pp. 24–29, ISSN

[49] Brigham, K. L.; Lane, K. B.; Meyrick, B.; Stecenko, A. A.; Strack, S.; Cannon, D. R.; Caudill, M. & Canonico, A. E. (2000).Transfection of nasal mucosa with a normal al‐ pha-1 antitrypsin (AAT) gene in AAT deficient subjects: comparison with protein

therapy. Human Gene Therapy, Vol 11, No.7, pp. 1023–1032, ISSN 1043-0342

lung epithelium in vivo. Science, Vol 252, No.5004, pp. 431–434

ca, Vol 95, No.24, pp.14384–14388, ISSN 0027-8424

or unnecessary effort? Gene Ther 1999; 6:155–156

mice. Hum Gene Ther 1999; 10:1905–1914.29

date for gene therapy in asthma. Gene Ther 1999; 6:245–252

7330–7335, ISSN 0021-9258

[50] Rosenfeld, M. A.; Siegfried, W.; Yoshimura, K.; Yoneyama, K.; Fukayama, M.; Stier, L. E.; Paakko, P. K.; Gilardi, P.; Stratford-Perricaudet, L. D.; Perricaudet, M, et al. (1991).Adenovirus-mediated transfer of a recombinant α-1-antitrypsin gene to the

[51] Song, S.; Morgan, M.; Ellis, T.; Poirier, A.; Chesnut, K.; Wang, J.; Brantley, M.; Mu‐ zyczka, N.; Byrne, B. J.; Atkinson, M. & Flotte, T. R. (1998). Sustained secretion of hu‐ man α -1-antitrypsin from murine muscle transduced with adeno-associated virus vectors. Proceeding of the National Academy of Science of the United State of Ameri‐

[52] Sifers, R. N.; Brashears-Macatee, S.; Kidd, V. J.; Muensch, H. & Woo, S.L. (1988). A frameshift mutation results in a truncated α-1-antitrypsin that is retained within the rough endoplasmic reticulum. Journal of Biological Chemistry, Vol 263, No.15, pp.

[53] Global Strategy for Asthma Management and Prevention. Global Initiative for Asth‐ ma (GINA), 2006. Available from www.ginasthma.org Date last updated, 2006

[54] Masoli M, Fabian D, Holt S, Beasley R. The global burden of asthma: executive sum‐ mary of the GINA Dissemination Committee report. Allergy 2004; 59: 469 478

[55] Alton EW, Griesenbach U, Geddes DM. Gene therapy for asthma: inspired research

[56] Mathieu M, Gougat C, Jaffuel D, et al. The glucocorticoid receptor gene as a candi‐

[57] Hogan SP, Foster PS, Tan X, et al. Mucosal IL-12 gene delivery inhibits allergic air‐ ways disease and restores local antiviral immunity. Eur J Immunol 1998; 28:413–423

[58] Dow SW, Schwarze J, Heath TD, et al. Systemic and local interferon gamma gene de‐ livery to the lungs for treatment of allergen-induced airway hyperresponsiveness in

ca, Vol 95, No.24, pp.14384–14388, ISSN 0027-8424

1044-1549]

626 Gene Therapy - Tools and Potential Applications


[74] Antonia SJ, Mirza N, Fricke I, et al. Combination of p53 cancer vaccine with chemo‐ therapy in patients with extensive stage small cell lung cancer. Clin Cancer Res 2006;12(3 Pt 1):878–87

[86] Paz-Ares L, Douillard JY, Koralewski P, et al. Phase III study of gemcitabine and cis‐ platin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleo‐ tide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2006;

Gene Therapy Perspectives Against Diseases of the Respiratory System

http://dx.doi.org/10.5772/52775

629

[87] Rudin CM, Holmlund J, Fleming GF, et al. Phase I trial of ISIS 5132, an antisense oli‐ gonucleotide inhibitor of c-Raf-1, administered by 24-hour weekly infusion to pa‐

[88] Coudert B, Anthoney A, Fiedler W, et al. Phase II trial with ISIS 5132 in patients with small-cell (SCLC) and non-small cell (NSCLC) lung cancer. A European Organization for Research and Treatment of Cancer (EORTC) early clinical studies group report.

[89] Rudin CM, Otterson GA, Mauer AM, et al. A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory smallcell lung can‐

[90] Rudin CM, Kozloff M, Hoffman PC, et al. Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell

[91] Rudin CM, Salgia R, Wang X, et al. Randomized phase II study of carboplatin and etoposide with or without the bcl-2 antisense oligonucleotide oblimersen for exten‐

sive-stage small-cell lung cancer: CALGB 30103. J Clin Oncol 2008;26(6):870–6

tients with advanced cancer. Clin Cancer Res 2001;7(5):1214–20

24(9):1428–34

Eur J Cancer 2001;37 (17):2194–8

cer. Ann Oncol 2002;13(4):539–45

lung cancer. J Clin Oncol 2004;22(6): 1110–7


[86] Paz-Ares L, Douillard JY, Koralewski P, et al. Phase III study of gemcitabine and cis‐ platin with or without aprinocarsen, a protein kinase C-alpha antisense oligonucleo‐ tide, in patients with advanced-stage non-small-cell lung cancer. J Clin Oncol 2006; 24(9):1428–34

[74] Antonia SJ, Mirza N, Fricke I, et al. Combination of p53 cancer vaccine with chemo‐ therapy in patients with extensive stage small cell lung cancer. Clin Cancer Res

[75] Baratelli F, Takedatsu H, Hazra S, et al. Pre-clinical characterization of GMP grade CCL21-gene modified dendritic cells for application in a phase I trial in non-small

[76] Nemunaitis J, Dillman RO, Schwarzenberger PO, et al. Phase II study of belagenpu‐ matucel-L, a transforming growth factor beta-2 antisense gene modified allogeneic tumor cell vaccine in nonsmall-cell lung cancer. J Clin Oncol 2006;24(29): 4721–30

[77] Nemunaitis J, Nemunaitis M, Senzer N, et al. Phase II trial of belagenpumatucel-L, a TGF-beta2 antisense gene modified allogeneic tumor vaccine in advanced non small

[78] Salgia R, Lynch T, Skarin A, et al. Vaccination with irradiated autologous tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor augments antitumor immunity in some patients with metastatic non-small-cell lung carcinoma.

[79] Nemunaitis J, Sterman D, Jablons D, et al. Granulocyte- macrophage colony-stimulat‐ ing factor genemodified autologous tumor vaccines in non-small-cell lung cancer. J

[80] Nemunaitis J, Jahan T, Ross H, et al. Phase 1/2 trial of autologous tumor mixed with an allogeneic GVAX vaccine in advanced-stage non-small-cell lung cancer. Cancer

[81] Morris JC, Vahanian N, Janik JE, et al. Phase I study of an antitumor vaccination us‐ ing alpha(1,3)galactosyltransferase expressing allogeneic tumor cells in patients with refractory or recurrent non-small cell lung cancer (NSCLC). J Clin Oncol

[82] Raez LE, Cassileth PA, Schlesselman JJ, et al. Allogeneic vaccination with a B7.1 HLA-A gene-modified adenocarcinoma cell line in patients with advanced non-

[83] Ramlau R, Quoix E, Rolski J, et al. A phase II study of Tg4010 (mva-Muc1-Il2) in as‐ sociation with chemotherapy in patients with stage III/IV non-small cell lung cancer.

[84] Nemunaitis J, Meyers T, Senzer N, et al. Phase I trial of sequential administration of recombinant DNA and adenovirus expressing L523S protein in early stage non-

[85] Ritch P, Rudin CM, Bitran JD, et al. Phase II study ofnPKC-alpha antisense oligonu‐ cleotide aprinocarsen in combination with gemcitabine and carboplatin in patients

with advanced non-small cell lung cancer.Lung Cancer 2006;52(2):173–80

small-cell lung cancer. J Clin Oncol 2004;22(14):2800–7

small-cell lung cancer. Mol Ther 200613(6):1185–91

cell lung cancer (NSCLC) patients. Cancer Gene Ther 2009;16(8):620–4

2006;12(3 Pt 1):878–87

628 Gene Therapy - Tools and Potential Applications

cell lung cancer. J Transl Med 2008;6:38

J Clin Oncol 2003;21(4):624–30

Natl Cancer Inst 2004;96(4):326–31

Gene Ther 2006;13(6):555–62

J Thorac Oncol 2008;3(7):735–44

2005;23(16S): 2586


**Chapter 26**

**Gene Therapy in Critical Care Medicine**

Critical care medicine is directed toward patients with a wide spectrum of illnesses. These have the common denominators of marked exacerbation of an existing disease, severe acute new problems, or severe complications from disease or treatments. In recent years has been an explosion of evidence based medicine with improvement in outcome, however there are several conditions in critical care patients that maintains a high morbidity and high mortali‐ ty that is necessary to be addressed [1]. Of these, severe sepsis and the acute respiratory dis‐ tress syndrome (ARDS), including acute lung injury (ALI) (syndromes consisting of acute respiratory failure associated with pulmonary infiltrates due to intra- or extra-pulmonary diseases) are two important conditions that have increased mortality in critical care units

In 1991, a Consensus Conference of the American College of Chest Physicians an the Society for Critical Care (ACCP-SCCM) introduced the term systemic inflammatory response syn‐ drome (SIRS) as the presence of at least two of four clinical criteria: body temperature more than 38˚C or less than 36˚C, heart rate more than 90 beats per minute, respiratory rate more than 20 breaths per minute or hyperventilation with PaCO<sup>2</sup> less than 32 mmHg, white blood

trophils [4]. In 2001, a new consensus suggests that other signs and symptoms could reflect the clinical response to infection, including: fever/hypothermia, tachypnea/respiratory alka‐ losis, positive fluid balance/edema, general inflammatory reaction, altered white blood count, increased biomarkers (C-reactive protein, IL-6, pro-calcitonin), hemodynamic altera‐ tions, arterial hypotension, tachycardia, increased cardiac outflow/low systemic vascular re‐ sistance/high venous saturation O2, altered skin perfusion, decreased urine output,

> © 2013 Moreno-González and Zarain-Herzberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is

or with more than 10% immature neu‐

, less than 4000/mm<sup>3</sup>

Gabriel J. Moreno-González and

Additional information is available at the end of the chapter

Angel Zarain-Herzberg

http://dx.doi.org/10.5772/52701

**1. Introduction**

around the world [2, 3].

cell count more than 12000/mm<sup>3</sup>

properly cited.

### **Gene Therapy in Critical Care Medicine**

Gabriel J. Moreno-González and Angel Zarain-Herzberg

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52701

#### **1. Introduction**

Critical care medicine is directed toward patients with a wide spectrum of illnesses. These have the common denominators of marked exacerbation of an existing disease, severe acute new problems, or severe complications from disease or treatments. In recent years has been an explosion of evidence based medicine with improvement in outcome, however there are several conditions in critical care patients that maintains a high morbidity and high mortali‐ ty that is necessary to be addressed [1]. Of these, severe sepsis and the acute respiratory dis‐ tress syndrome (ARDS), including acute lung injury (ALI) (syndromes consisting of acute respiratory failure associated with pulmonary infiltrates due to intra- or extra-pulmonary diseases) are two important conditions that have increased mortality in critical care units around the world [2, 3].

In 1991, a Consensus Conference of the American College of Chest Physicians an the Society for Critical Care (ACCP-SCCM) introduced the term systemic inflammatory response syn‐ drome (SIRS) as the presence of at least two of four clinical criteria: body temperature more than 38˚C or less than 36˚C, heart rate more than 90 beats per minute, respiratory rate more than 20 breaths per minute or hyperventilation with PaCO<sup>2</sup> less than 32 mmHg, white blood cell count more than 12000/mm<sup>3</sup> , less than 4000/mm<sup>3</sup> or with more than 10% immature neu‐ trophils [4]. In 2001, a new consensus suggests that other signs and symptoms could reflect the clinical response to infection, including: fever/hypothermia, tachypnea/respiratory alka‐ losis, positive fluid balance/edema, general inflammatory reaction, altered white blood count, increased biomarkers (C-reactive protein, IL-6, pro-calcitonin), hemodynamic altera‐ tions, arterial hypotension, tachycardia, increased cardiac outflow/low systemic vascular re‐ sistance/high venous saturation O2, altered skin perfusion, decreased urine output,

<sup>© 2013</sup> Moreno-González and Zarain-Herzberg; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

hyperlactacemia, signs of organ dysfunction, hypoxemia, coagulation abnormalities, altered mental status, hyperglycemia, thrombocytopenia, disseminated intravascular coagulation, altered liver function, intolerance to feeding [5].

**2. Physiopathology of sepsis**

and c) RIG-I-like receptors (RLRs) [22].

initiating TLR-induced signal transduction [27].

TLR signaling cascades [28-33].

Microorganisms express macromolecular motifs, named pathogen-associated molecular pat‐ terns (PAMs) such as lipopolysaccharide (LPS), flagellin, double-stranded RNA and CpG DNA [22]. These molecules are recognized by the immune system through a family of transmembrane or intra-cytoplasmic receptors, the pattern recognition receptors (PRRs), classi‐ fied in three general families: a) Toll-like receptors (TLRs); b) NOD-like receptors (NLRs);

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 633

The TLRs are type I integral membrane glycoproteins characterized by the extracellular do‐ mains containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/ IL-1R homology (TIR) domain [23]. Based on their primary sequences, TLRs can be divides into several subfamilies, each of which recognized related PAMPs: the subfamily of TLR1, TLR2 and TLR6 recognize lipids, whereas the highly related TLR7, TLR8, TLR9 recognize nucleic acids. TLR4 recognize a very divergent collection of ligands [24]. The NLRs proteins are implicated in the recognition of bacterial components. Proteins in this family possess LRRs that mediate ligand sensing: a nucleotide binding oligomerization domain (NOD) and a domain for the initiation of signaling such as CARDs, PYRIN of baculovirus inhibitor of apoptosis repeat (BIR) domains [25]. The retinoic-acid inducible protein-I (RIG-I) is an INFinducible protein containing CARDs and a DExD/H box helicase domain and has been iden‐ tified as a cytoplasmic detector in viral infection in the TLR3 independent manner [26]. In addition to the numerous exogenous pathogen-derived ligands that activate different TLRs, endogenous TLR ligands have been identified, including hyaluronic acid, high mobility group box-1 (HMGB1) and heat shock proteins (HSPs), termed as damaged-associated mo‐ lecular patterns (DAMPs). During tissue injury or proteolysis, extracellular matrix compo‐ nents undergo cleavage, exposing moieties that can act as ligands for TLRs and therefore

The PAM/PPR interaction leads to immune cell activation with initiation of microbe-killing systems, production and secretion of pro-inflammatory cytokines and chemokines, en‐ hanced expression of co-stimulatory receptors essential for efficient T cell activation, pro‐ duction of arachinoid acid metabolites and initiation of extrinsic coagulation cascade [28-33]. The activation of the TLR signaling originated from the cytoplasmic Toll/IL-1 receptor (TIR) domain requires the association with the TIR domain-containing adaptor protein, MyD88. With ligands binding, MyD88 recruits IL-1 receptor-associates kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules. IRAK-1 activated by phosphor‐ ylation then associates with TRAF6, finally leading to activation of MAP kinases and NFκB. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP mediated downstream signaling by ST2L, TRIAD3A and SOCS1. MyD88-independent pathways induce activation of IRF3 and expression of interferon-β. TIR-domain containing adaptors such as TIRAP, TRIF and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual

Systemic inflammatory response syndrome can result from diverse etiologies, including, but not limited to infectious, trauma, pancreatitis, ischemia-reperfusion injury, and burns [6]. Sepsis is defined as the presence of infection and some of the listed signs and symptoms of SIRS, whereas severe sepsis is defined as sepsis associated with organ dysfunction and shock septic as severe sepsis with hypotension, despite adequate fluid resuscitation [7].

Over 18 million cases of severe sepsis occur each year. The number of severe sepsis cases is set to grow a rate of 1.5% per year from the annual incidence of 3 cases per 1000 of the popu‐ lation in 2001 [8, 9]. Sepsis is a major cause of mortality throughout the world, killing ap‐ proximately 1400 people every day, being as high as an additional fifty per cent as deaths are often attributed to complications from cancer or pneumonia, and not related to sepsis [10]. Death is common among sepsis patients, with around 28-50% of patients dying within the first month of diagnosis [11-13]. Sepsis impacts the lives of many people, including the patient and their families, in addition to doctors, nursing and care staff. The intense de‐ mands made on hospital staff, equipment and facilities to treat septic patients places a sig‐ nificant burden on healthcare resources, accounting for 40% of total ICU expenditure [10]. Each year the cost of treating septic patients increases and is as high as 7.6 billion euro in Europe [10] and 17.4 billion euro in the USA [8].

One common complication of SIRS and sepsis is acute lung injury/adult respiratory distress syndrome (ALI/ARDS). According to a Join North American European consensus commit‐ tee (NAECC), ARDS is defined as an inflammatory process in the lungs with acute onset of respiratory failure, new bilateral pulmonary infiltrates on frontal chest radiograph or com‐ puted tomography, absence of left ventricular failure (clinically diagnosed or a pulmonary artery occlusion pressure <18mmHg) and hypoxemia with a ratio between the partial pres‐ sure of arterial oxygen and the fraction of inspired oxygen (PaO2/FiO2 ratio) of ≤27 kPa inde‐ pendent of the level of positive end-expiratory pressure (PEEP) [14]. ALI is defined by the same criteria except that the PaO2/FiO2 ratio is between 27 kPa and 40 kPa[14-16]. Sepsis is the most common cause of ALI/ARDS and also the most common cause of death after pa‐ tients develop ALI/ARDS [17]. The incidence of ALI/ARDS is estimated to be 20 to 50 cases per 100000 person-year, with approximately 18% to 25% of cases meeting oxygenation crite‐ ria for ALI but not for ARDS [18, 19].

The reported rate of mortality from ARDS ranges from 31% to 74% depending on the char‐ acteristics of patients, with most deaths occurring as a consequence of multiple organ failure and sepsis [18, 19]. ALI has a significant lower crude hospital mortality (32%) compared with those with ARDS (57.9%) [20]. Crude estimates of the health care costs associated with ALI/ARDS may exceed 5 billion dollars per year in the United States alone [21].

#### **2. Physiopathology of sepsis**

hyperlactacemia, signs of organ dysfunction, hypoxemia, coagulation abnormalities, altered mental status, hyperglycemia, thrombocytopenia, disseminated intravascular coagulation,

Systemic inflammatory response syndrome can result from diverse etiologies, including, but not limited to infectious, trauma, pancreatitis, ischemia-reperfusion injury, and burns [6]. Sepsis is defined as the presence of infection and some of the listed signs and symptoms of SIRS, whereas severe sepsis is defined as sepsis associated with organ dysfunction and shock septic as severe sepsis with hypotension, despite adequate fluid resuscitation [7].

Over 18 million cases of severe sepsis occur each year. The number of severe sepsis cases is set to grow a rate of 1.5% per year from the annual incidence of 3 cases per 1000 of the popu‐ lation in 2001 [8, 9]. Sepsis is a major cause of mortality throughout the world, killing ap‐ proximately 1400 people every day, being as high as an additional fifty per cent as deaths are often attributed to complications from cancer or pneumonia, and not related to sepsis [10]. Death is common among sepsis patients, with around 28-50% of patients dying within the first month of diagnosis [11-13]. Sepsis impacts the lives of many people, including the patient and their families, in addition to doctors, nursing and care staff. The intense de‐ mands made on hospital staff, equipment and facilities to treat septic patients places a sig‐ nificant burden on healthcare resources, accounting for 40% of total ICU expenditure [10]. Each year the cost of treating septic patients increases and is as high as 7.6 billion euro in

One common complication of SIRS and sepsis is acute lung injury/adult respiratory distress syndrome (ALI/ARDS). According to a Join North American European consensus commit‐ tee (NAECC), ARDS is defined as an inflammatory process in the lungs with acute onset of respiratory failure, new bilateral pulmonary infiltrates on frontal chest radiograph or com‐ puted tomography, absence of left ventricular failure (clinically diagnosed or a pulmonary artery occlusion pressure <18mmHg) and hypoxemia with a ratio between the partial pres‐ sure of arterial oxygen and the fraction of inspired oxygen (PaO2/FiO2 ratio) of ≤27 kPa inde‐ pendent of the level of positive end-expiratory pressure (PEEP) [14]. ALI is defined by the same criteria except that the PaO2/FiO2 ratio is between 27 kPa and 40 kPa[14-16]. Sepsis is the most common cause of ALI/ARDS and also the most common cause of death after pa‐ tients develop ALI/ARDS [17]. The incidence of ALI/ARDS is estimated to be 20 to 50 cases per 100000 person-year, with approximately 18% to 25% of cases meeting oxygenation crite‐

The reported rate of mortality from ARDS ranges from 31% to 74% depending on the char‐ acteristics of patients, with most deaths occurring as a consequence of multiple organ failure and sepsis [18, 19]. ALI has a significant lower crude hospital mortality (32%) compared with those with ARDS (57.9%) [20]. Crude estimates of the health care costs associated with

ALI/ARDS may exceed 5 billion dollars per year in the United States alone [21].

altered liver function, intolerance to feeding [5].

632 Gene Therapy - Tools and Potential Applications

Europe [10] and 17.4 billion euro in the USA [8].

ria for ALI but not for ARDS [18, 19].

Microorganisms express macromolecular motifs, named pathogen-associated molecular pat‐ terns (PAMs) such as lipopolysaccharide (LPS), flagellin, double-stranded RNA and CpG DNA [22]. These molecules are recognized by the immune system through a family of transmembrane or intra-cytoplasmic receptors, the pattern recognition receptors (PRRs), classi‐ fied in three general families: a) Toll-like receptors (TLRs); b) NOD-like receptors (NLRs); and c) RIG-I-like receptors (RLRs) [22].

The TLRs are type I integral membrane glycoproteins characterized by the extracellular do‐ mains containing varying numbers of leucine-rich-repeat (LRR) motifs and a cytoplasmic signaling domain homologous to that of the interleukin 1 receptor (IL-1R), termed the Toll/ IL-1R homology (TIR) domain [23]. Based on their primary sequences, TLRs can be divides into several subfamilies, each of which recognized related PAMPs: the subfamily of TLR1, TLR2 and TLR6 recognize lipids, whereas the highly related TLR7, TLR8, TLR9 recognize nucleic acids. TLR4 recognize a very divergent collection of ligands [24]. The NLRs proteins are implicated in the recognition of bacterial components. Proteins in this family possess LRRs that mediate ligand sensing: a nucleotide binding oligomerization domain (NOD) and a domain for the initiation of signaling such as CARDs, PYRIN of baculovirus inhibitor of apoptosis repeat (BIR) domains [25]. The retinoic-acid inducible protein-I (RIG-I) is an INFinducible protein containing CARDs and a DExD/H box helicase domain and has been iden‐ tified as a cytoplasmic detector in viral infection in the TLR3 independent manner [26]. In addition to the numerous exogenous pathogen-derived ligands that activate different TLRs, endogenous TLR ligands have been identified, including hyaluronic acid, high mobility group box-1 (HMGB1) and heat shock proteins (HSPs), termed as damaged-associated mo‐ lecular patterns (DAMPs). During tissue injury or proteolysis, extracellular matrix compo‐ nents undergo cleavage, exposing moieties that can act as ligands for TLRs and therefore initiating TLR-induced signal transduction [27].

The PAM/PPR interaction leads to immune cell activation with initiation of microbe-killing systems, production and secretion of pro-inflammatory cytokines and chemokines, en‐ hanced expression of co-stimulatory receptors essential for efficient T cell activation, pro‐ duction of arachinoid acid metabolites and initiation of extrinsic coagulation cascade [28-33]. The activation of the TLR signaling originated from the cytoplasmic Toll/IL-1 receptor (TIR) domain requires the association with the TIR domain-containing adaptor protein, MyD88. With ligands binding, MyD88 recruits IL-1 receptor-associates kinase-4 (IRAK-4) to TLRs through interaction of the death domains of both molecules. IRAK-1 activated by phosphor‐ ylation then associates with TRAF6, finally leading to activation of MAP kinases and NFκB. Additional modes of regulation for these pathways include TRIF-dependent induction of TRAF6 signaling by RIP1 and negative regulation of TIRAP mediated downstream signaling by ST2L, TRIAD3A and SOCS1. MyD88-independent pathways induce activation of IRF3 and expression of interferon-β. TIR-domain containing adaptors such as TIRAP, TRIF and TRAM regulate TLR-mediated signaling pathways by providing specificity for individual TLR signaling cascades [28-33].

The interaction of PAMs with NRL recruits the receptor-interacting protein-2 (RIP2) kinase activating NFκB and MAPK kinases. A number of the NRL molecules have been shown to form a complex with caspase-1 and the adaptor molecule apoptosis associated speck-like pro‐ tein containing CARD (ASC) termed inflammasome. The central effector molecule of the in‐ flammasome is the cysteine protease caspase-1 that, upon activation cleaves cytosolic pro-IL-1β, pro-IL-18 and pro-IL-33 to their active forms enabling them to be secreted into the extracellular/systemic compartments [34]. The important fact is that NRLs and TLRs may syn‐ ergize. T-cell subgroups are modified in sepsis. Helper (CD4<sup>+</sup> ) T-cells can be categorized as type 1 helper (Th1) or 2 (Th2). Th1 cells generally secrete pro-inflammatory cytokines such as tumor necrosis factor-α(TNFα) and interleukin-1β (IL-1β); Th2 cells secrete anti-inflammato‐ ry cytokines such as IL-4 and IL-10, depending on the infecting organism, the burden of infec‐ tion and other factors during sepsis may also induce apoptosis of lung and intestinal cells [35]. Activated helper T cells evolve from a Th1 phenotype, producing pro-inflammatory cyto‐ kines, to a Th2 phenotype producing anti-inflammatory cytokines [35]. In addition, apoptosis of circulating and tissue lymphocytes (B cells and CD4<sup>+</sup> T cells) contributes to immunosup‐ pression [36]. The increased pro-inflammatory cytokines, activated B cells and T cells and cir‐ culating glucocorticoid levels causes apoptosis in septic patients [37]. Increased levels of TNFα and lipopolysaccharide during sepsis may also induce apoptosis [35].

Loss of epithelial cells and endothelial cell injury are involved in pathogenesis of ALI/ARDS. The former is due to the activation of Fas related apoptosis and the secretion of cytokines and chemokines by lung epithelial cells [44]. The latter is caused by the interaction of endo‐ thelial cells with neutrophils that stimulate release of vasoactive compounds, increased pul‐ monary vascular resistance with pulmonary hypertension [45], but also endothelial cells can be directly stimulated by endotoxin via TLR-1 with the release of vasoactive mediators and molecules altering lung permeability, such as TNFα, thromboxane-A2 and endothelin-1 [46].

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 635

Resolution from lung injury is an actively regulated program involving a removal of apop‐ totic neutrophils, remodeling of matrix, clearance of protein-rich alveolar fluid [47]. Recent‐ ly, has been demonstrated that CD4+ lymphocytes as well as plasmacytoid dendritic cells are

Gene therapy is defined as the introduction of nucleic acids into cells for the purpose of altering the course of a medical condition o disease [50]. In general, the advantages of gene therapy over the other treatments are the selective treatment of affected tissues, the possibility of using locally endogenous proteins in cases where its systemic application would incur in serious adverse secondary effects, and the possibility of therapeutic long term after a single application [51]. Currently, there are three categories of gene delivery methods: viral vector based, non-viral vector based and physical methods [52]. Viralbased gene delivery systems is accomplished by using replication-deficient viruses con‐ taining the gene of interest, but with the disease-causing sequences deleted from the viral genome [53] including RNA-based viral vectors [54, 55], DNA-based viral vectors such as adenoviral vectors [56], adeno-associated viruses (AAV) vectors orherpes simplex viral vector [57].The non-viral gene delivery methods use synthetic or natural compounds or physical forces to deliver a piece of DNA into a cell [58]. Two main groups of non-viral delivery methods have developed: chemical-based, including lipofection [59] and inorgan‐ ic nanoparticles that are usually prepared from metals, inorganic salts or ceramics [60]; and using physical forces such as local or rapid systemic injection [61], particle impact

Currently, there is evidence that applying therapeutic maneuvers such as early effective an‐ tibiotic administration, intensive fluid resuscitation, mechanical ventilation in selected pa‐ tients and use of C activated protein in sickest patients improve significantly the survive in these patients [66]. There are several clinical studies that are trying to validate another kind of therapies such as extra-renal depuration, levosimendan, the use of immunoglobulins, ni‐ tric oxide, statins, selenium, the use of enteral nutrition with eicosapentaenoic acid (EPA)/ψ-

active players in this process [48, 49].

**4. Vectors for gene therapy**

[62, 63], electric pulse [64] or laser irradiation [65].

**5. Gene therapy in sepsis**

#### **3. Physiopathology of acute respiratory distress syndrome**

There are two general types of ALI/ARDS, direct and indirect. Independent of the initial in‐ sult, the final result is that alveolar-capillary barrier becomes compromised. Direct ALI/ ARDS is often associated with direct mechanical, chemical or infectious stimuli, or other di‐ rect interactions capable to induce damage to lung structures [38]. Indirect pulmonary in‐ sults such as extra-pulmonary sepsis, trauma, shock, pancreatitis, brain injury or massive transfusion are the mainly causes of indirect ALI/ARDS. However, the highest incidence of indirect ALI/ARDS is seen during sepsis.

The emigration of activated PMNs and passage through the endothelium in the lungs, one of the characteristics of ALI, is regulated via adhesion molecules. Among them, L-selectin (CD62L) on PMNs appears to be involved in the initial rolling proceed on the endothelial surface, while CD11b/CD18 on PMNs mediate a tighter contact between them. CD31 of PE‐ CAM-1 is needed in the final step for the vascular diapedesis of leukocytes [38-40]. Neutro‐ phils are able to release a variety of harmful substances, such as proteolytic enzymes, reactive oxygen/nitrogen species, cytokines and chemokynes, which may be injurious to the adjacent endothelial cell and to the alveoli [39]. PMN apoptosis is a crucial injury-limiting mechanism of inflammatory resolution. Several inflammatory agents such as LPS, TNF, IL-8, IL-6, IL-1 and granulocyte colony stimulating factor (G-CSF) can delay apoptotic re‐ sponse, providing PMN with a longer life, allowing accumulating at local tissues [41]. NFκB has been reported as a modulator of apoptosis in inflammatory cells [42, 43] allowing a proinflammatory state.

Loss of epithelial cells and endothelial cell injury are involved in pathogenesis of ALI/ARDS. The former is due to the activation of Fas related apoptosis and the secretion of cytokines and chemokines by lung epithelial cells [44]. The latter is caused by the interaction of endo‐ thelial cells with neutrophils that stimulate release of vasoactive compounds, increased pul‐ monary vascular resistance with pulmonary hypertension [45], but also endothelial cells can be directly stimulated by endotoxin via TLR-1 with the release of vasoactive mediators and molecules altering lung permeability, such as TNFα, thromboxane-A2 and endothelin-1 [46].

Resolution from lung injury is an actively regulated program involving a removal of apop‐ totic neutrophils, remodeling of matrix, clearance of protein-rich alveolar fluid [47]. Recent‐ ly, has been demonstrated that CD4+ lymphocytes as well as plasmacytoid dendritic cells are active players in this process [48, 49].

#### **4. Vectors for gene therapy**

The interaction of PAMs with NRL recruits the receptor-interacting protein-2 (RIP2) kinase activating NFκB and MAPK kinases. A number of the NRL molecules have been shown to form a complex with caspase-1 and the adaptor molecule apoptosis associated speck-like pro‐ tein containing CARD (ASC) termed inflammasome. The central effector molecule of the in‐ flammasome is the cysteine protease caspase-1 that, upon activation cleaves cytosolic pro-IL-1β, pro-IL-18 and pro-IL-33 to their active forms enabling them to be secreted into the extracellular/systemic compartments [34]. The important fact is that NRLs and TLRs may syn‐

type 1 helper (Th1) or 2 (Th2). Th1 cells generally secrete pro-inflammatory cytokines such as tumor necrosis factor-α(TNFα) and interleukin-1β (IL-1β); Th2 cells secrete anti-inflammato‐ ry cytokines such as IL-4 and IL-10, depending on the infecting organism, the burden of infec‐ tion and other factors during sepsis may also induce apoptosis of lung and intestinal cells [35]. Activated helper T cells evolve from a Th1 phenotype, producing pro-inflammatory cyto‐ kines, to a Th2 phenotype producing anti-inflammatory cytokines [35]. In addition, apoptosis

pression [36]. The increased pro-inflammatory cytokines, activated B cells and T cells and cir‐ culating glucocorticoid levels causes apoptosis in septic patients [37]. Increased levels of TNF-

There are two general types of ALI/ARDS, direct and indirect. Independent of the initial in‐ sult, the final result is that alveolar-capillary barrier becomes compromised. Direct ALI/ ARDS is often associated with direct mechanical, chemical or infectious stimuli, or other di‐ rect interactions capable to induce damage to lung structures [38]. Indirect pulmonary in‐ sults such as extra-pulmonary sepsis, trauma, shock, pancreatitis, brain injury or massive transfusion are the mainly causes of indirect ALI/ARDS. However, the highest incidence of

The emigration of activated PMNs and passage through the endothelium in the lungs, one of the characteristics of ALI, is regulated via adhesion molecules. Among them, L-selectin (CD62L) on PMNs appears to be involved in the initial rolling proceed on the endothelial surface, while CD11b/CD18 on PMNs mediate a tighter contact between them. CD31 of PE‐ CAM-1 is needed in the final step for the vascular diapedesis of leukocytes [38-40]. Neutro‐ phils are able to release a variety of harmful substances, such as proteolytic enzymes, reactive oxygen/nitrogen species, cytokines and chemokynes, which may be injurious to the adjacent endothelial cell and to the alveoli [39]. PMN apoptosis is a crucial injury-limiting mechanism of inflammatory resolution. Several inflammatory agents such as LPS, TNF, IL-8, IL-6, IL-1 and granulocyte colony stimulating factor (G-CSF) can delay apoptotic re‐ sponse, providing PMN with a longer life, allowing accumulating at local tissues [41]. NFκB has been reported as a modulator of apoptosis in inflammatory cells [42, 43] allowing a pro-

) T-cells can be categorized as

T cells) contributes to immunosup‐

ergize. T-cell subgroups are modified in sepsis. Helper (CD4<sup>+</sup>

634 Gene Therapy - Tools and Potential Applications

of circulating and tissue lymphocytes (B cells and CD4<sup>+</sup>

indirect ALI/ARDS is seen during sepsis.

inflammatory state.

α and lipopolysaccharide during sepsis may also induce apoptosis [35].

**3. Physiopathology of acute respiratory distress syndrome**

Gene therapy is defined as the introduction of nucleic acids into cells for the purpose of altering the course of a medical condition o disease [50]. In general, the advantages of gene therapy over the other treatments are the selective treatment of affected tissues, the possibility of using locally endogenous proteins in cases where its systemic application would incur in serious adverse secondary effects, and the possibility of therapeutic long term after a single application [51]. Currently, there are three categories of gene delivery methods: viral vector based, non-viral vector based and physical methods [52]. Viralbased gene delivery systems is accomplished by using replication-deficient viruses con‐ taining the gene of interest, but with the disease-causing sequences deleted from the viral genome [53] including RNA-based viral vectors [54, 55], DNA-based viral vectors such as adenoviral vectors [56], adeno-associated viruses (AAV) vectors orherpes simplex viral vector [57].The non-viral gene delivery methods use synthetic or natural compounds or physical forces to deliver a piece of DNA into a cell [58]. Two main groups of non-viral delivery methods have developed: chemical-based, including lipofection [59] and inorgan‐ ic nanoparticles that are usually prepared from metals, inorganic salts or ceramics [60]; and using physical forces such as local or rapid systemic injection [61], particle impact [62, 63], electric pulse [64] or laser irradiation [65].

#### **5. Gene therapy in sepsis**

Currently, there is evidence that applying therapeutic maneuvers such as early effective an‐ tibiotic administration, intensive fluid resuscitation, mechanical ventilation in selected pa‐ tients and use of C activated protein in sickest patients improve significantly the survive in these patients [66]. There are several clinical studies that are trying to validate another kind of therapies such as extra-renal depuration, levosimendan, the use of immunoglobulins, ni‐ tric oxide, statins, selenium, the use of enteral nutrition with eicosapentaenoic acid (EPA)/ψlinolenic acid (GLA) that are in progress [67]. Basic research and clinical trials have focused on alternative therapeutic approaches [68].

siRNA-987, 5'-CCCACUCGGAGAAGUUUAATT-3' against mCD14. *In vitro* experiments with RAW264.7 cells (a transformed murine macrophage cell line) shown that siRNA-224 ef‐ fectively inhibited LPS-induced TNFα, MIP-2 and IL-6 release and NO production [78].

Severely burned patients are greatly susceptible to infection with various pathogens [79]. Macrophages (MΦs) have an important role in antibacterial innate immunity. In methicillin-

converted by the TLRs stimulation and has the ability to kill bacteria, to produce reactive nitrogen intermediates, and to release antimicrobial peptides [80], playing a pivotal role in host microbial resistance. M2MΦ have reduced ability to kill bacteria; IL-10 and CCL7 re‐ leased by M2MΦ are inhibitory molecules on the pathogen-stimulated MΦ conversion to M1MΦ. IL-10 is also a deactivator of antibacterial immunocompetent cells [81] and an inhib‐ itory molecule on various immunocompetent cell functions. Asai et al have demonstrated that IL-10 antisense oligonucleotides in a severely burned mice prevents the burn associated conversion of MΦ to M2MΦ and infectious complications stemming for MRSA local infec‐

CCL2 is a chemokine that attracts and activates mononuclear cells. The necessity of this che‐ mokine for Th2-cell generation has been demonstrated. In a study Shigematsu K et al [83] tried to protect thermally injured mice orally infected with a lethal dose of *E. faecalis* by gene therapy utilizing phosphorothioate-CCL2 antisense oligodeoxynucleotides. They demon‐ strate that sepsis stemming from *E. faecalis* translocation in severely burned mice is control‐ lable by the gene therapy using CCL2 antisense ODNs, through the elimination of mesenteric lymph node macrophages (MLN- MΦ)-M2aMΦs and M2cMΦs subtypes. [83].

IL-1β binds the type-1 IL-1 receptor, while LPS binds to TLR4, both activates intracellular pathways by phosphorylation of IRAK family members including IRAK-1, which involve the MyD88 adaptor protein [84]. The group of Johns RE et al developed a family of "smart" polymeric carriers, termed encrypted polymers that enhance the cytoplasmic delivery of therapeutic antisense oligonucleotides (ASONs). This group has demonstrated that these ASONs block LPS activation of the transcription factor NFκB reducing the LPS-induced ex‐ pression of cytokines and chemokines. IL-6 shows a 2-fold decrease whereas TNFα expres‐ sion trended to decrease. There was a 2-fold decrease in expression of several genes

Caspases are pro-enzymes of the aspartate-specific cysteine protease family and its activa‐ tion plays a central role in the execution of apoptosis [86]. Depending of the stimuli, two cas‐ pase-activation pathways have been described, the mitochondria-initiated caspase-8 dependent pathway and mitochondria-initiated caspase-9-mediated pathway. Activation of these pathways initiates a downstream cascade of effector caspases, such as caspase-3 that

) and M2MΦ (IL-12- IL-10<sup>+</sup>

) differentiate in two dif‐

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 637

). The former are

resistant *Staphylococcus aureus* infection (MRSA), MΦs (IL-12- IL-10-

ferent subpopulations, M1MΦ (IL-12<sup>+</sup> IL-10-

including MCP1, MCP3, eotaxin and IP10 [85].

**5.2. Intracellular signaling**

tion did not develop [82].

**5.3. Apoptosis**

#### **5.1. Pattern associated membrane receptors**

Different approaches have designed trying to block the interaction between PAMs and PPRs. One is the generation of antibodies that bind TLRs. Studies conducted with anti-lipo‐ polysaccharide binding protein or anti-CD14 in experimental models of endotoxic shock and Gram-negative bacterial sepsis, failed to show a protection when treatment was admin‐ istered after LPS o simultaneously with or shortly after bacterial inoculation [69-71]. By us‐ ing a recombinant chimeric fusion protein composed of the N-terminal and central domains (amino-acids 1-334) of the extracellular part of TLR4 and the Fc portion of the human IgG1, Roger et al [72] produced an anti-TLR4 antibodies that inhibited LPS-induced intracellular signaling and cytokine production and protected mice from lethal endotoxic shock and E. coli bacterial sepsis, not only in pre-treatment with the antibodies, but also even when treat‐ ment was delayed for several hours after endotoxemia of the onset of sepsis.

The RAGEs (receptor for advanced glycation end products) are part of DAMPs that may play a role in the perpetuation of inflammation that carries to severe sepsis or septic shock. RAGEs are up-regulated in acute and chronic inflammation and bind multiple endogenous mediators involved in sepsis and products of oxidative stress [73]. In a recent work, Christa‐ ki et al demonstrated that blocking RAGEs either before or after infection protected mice from lethality in sepsis due to *S. pneumoniae* pneumonia [74] probably by indirect inhibition of NFκB activation.

Exposure to Staphylococcal enterotoxin (SE) or SE plus lipopolysaccharide (LPS, endotoxin) in mice, triggers vigorous intracellular signaling that leads to hyper-inflammation and re‐ lease of pro-inflammatory cytokines such as TNFα, INFγ, IL-1β, IL-1α, IL-2 and IL-6 by acti‐ vation of innate immunity [75]. In order to evaluate the role of MyD88, the anchor adaptor protein that integrates and transduces intracellular signals from TLRs and IL-1 receptor su‐ perfamily, Kisssner et al evaluates a synthetic molecule, hydrocinnamoyl-L-valyl-pyrroli‐ dine (Compd1), which mimics the BB-loop in the TIR domain of MyD88. They observed an inhibited pro-inflammatory cytokine production in human primary cells. Also, administra‐ tion of Compd1 to mice inhibited pro-inflammatory cytokine response and increased surviv‐ al from toxic shock induced death-limiting hyper-inflammation [76].

Recently, the knockdown or TLR2 by three different small interfering RNAs (siRNA) (A: 5' aactatccactggtgaaacaa-3', B: 5'-aaacttgtcagtggccagaaa-3', C: 5'-aaagtcttgattgattggcca-3') re‐ duce de tumorigenesis generated by the injection of BEL-7402 cells in an athymic mouse. Also, the levels of cytokines IL-6 and IL-8 were found to be markedly depressed [77]. In this line, Lei Ming et al have designed four siRNA:

siRNA-180, 5'-GCCUGGAAUACCUUCUAAATT-3'; siRNA-224, 5'-GGGCAGUUCACUGAUAUUATT-3'; siRNA-341, 5'-CAGGAACUGACUCUUGAAATT-3';

siRNA-987, 5'-CCCACUCGGAGAAGUUUAATT-3' against mCD14. *In vitro* experiments with RAW264.7 cells (a transformed murine macrophage cell line) shown that siRNA-224 ef‐ fectively inhibited LPS-induced TNFα, MIP-2 and IL-6 release and NO production [78].

#### **5.2. Intracellular signaling**

linolenic acid (GLA) that are in progress [67]. Basic research and clinical trials have focused

Different approaches have designed trying to block the interaction between PAMs and PPRs. One is the generation of antibodies that bind TLRs. Studies conducted with anti-lipo‐ polysaccharide binding protein or anti-CD14 in experimental models of endotoxic shock and Gram-negative bacterial sepsis, failed to show a protection when treatment was admin‐ istered after LPS o simultaneously with or shortly after bacterial inoculation [69-71]. By us‐ ing a recombinant chimeric fusion protein composed of the N-terminal and central domains (amino-acids 1-334) of the extracellular part of TLR4 and the Fc portion of the human IgG1, Roger et al [72] produced an anti-TLR4 antibodies that inhibited LPS-induced intracellular signaling and cytokine production and protected mice from lethal endotoxic shock and E. coli bacterial sepsis, not only in pre-treatment with the antibodies, but also even when treat‐

The RAGEs (receptor for advanced glycation end products) are part of DAMPs that may play a role in the perpetuation of inflammation that carries to severe sepsis or septic shock. RAGEs are up-regulated in acute and chronic inflammation and bind multiple endogenous mediators involved in sepsis and products of oxidative stress [73]. In a recent work, Christa‐ ki et al demonstrated that blocking RAGEs either before or after infection protected mice from lethality in sepsis due to *S. pneumoniae* pneumonia [74] probably by indirect inhibition

Exposure to Staphylococcal enterotoxin (SE) or SE plus lipopolysaccharide (LPS, endotoxin) in mice, triggers vigorous intracellular signaling that leads to hyper-inflammation and re‐ lease of pro-inflammatory cytokines such as TNFα, INFγ, IL-1β, IL-1α, IL-2 and IL-6 by acti‐ vation of innate immunity [75]. In order to evaluate the role of MyD88, the anchor adaptor protein that integrates and transduces intracellular signals from TLRs and IL-1 receptor su‐ perfamily, Kisssner et al evaluates a synthetic molecule, hydrocinnamoyl-L-valyl-pyrroli‐ dine (Compd1), which mimics the BB-loop in the TIR domain of MyD88. They observed an inhibited pro-inflammatory cytokine production in human primary cells. Also, administra‐ tion of Compd1 to mice inhibited pro-inflammatory cytokine response and increased surviv‐

Recently, the knockdown or TLR2 by three different small interfering RNAs (siRNA) (A: 5' aactatccactggtgaaacaa-3', B: 5'-aaacttgtcagtggccagaaa-3', C: 5'-aaagtcttgattgattggcca-3') re‐ duce de tumorigenesis generated by the injection of BEL-7402 cells in an athymic mouse. Also, the levels of cytokines IL-6 and IL-8 were found to be markedly depressed [77]. In this

ment was delayed for several hours after endotoxemia of the onset of sepsis.

al from toxic shock induced death-limiting hyper-inflammation [76].

line, Lei Ming et al have designed four siRNA:

siRNA-180, 5'-GCCUGGAAUACCUUCUAAATT-3';

siRNA-224, 5'-GGGCAGUUCACUGAUAUUATT-3';

siRNA-341, 5'-CAGGAACUGACUCUUGAAATT-3';

on alternative therapeutic approaches [68].

636 Gene Therapy - Tools and Potential Applications

**5.1. Pattern associated membrane receptors**

of NFκB activation.

Severely burned patients are greatly susceptible to infection with various pathogens [79]. Macrophages (MΦs) have an important role in antibacterial innate immunity. In methicillinresistant *Staphylococcus aureus* infection (MRSA), MΦs (IL-12- IL-10- ) differentiate in two dif‐ ferent subpopulations, M1MΦ (IL-12<sup>+</sup> IL-10- ) and M2MΦ (IL-12- IL-10<sup>+</sup> ). The former are converted by the TLRs stimulation and has the ability to kill bacteria, to produce reactive nitrogen intermediates, and to release antimicrobial peptides [80], playing a pivotal role in host microbial resistance. M2MΦ have reduced ability to kill bacteria; IL-10 and CCL7 re‐ leased by M2MΦ are inhibitory molecules on the pathogen-stimulated MΦ conversion to M1MΦ. IL-10 is also a deactivator of antibacterial immunocompetent cells [81] and an inhib‐ itory molecule on various immunocompetent cell functions. Asai et al have demonstrated that IL-10 antisense oligonucleotides in a severely burned mice prevents the burn associated conversion of MΦ to M2MΦ and infectious complications stemming for MRSA local infec‐ tion did not develop [82].

CCL2 is a chemokine that attracts and activates mononuclear cells. The necessity of this che‐ mokine for Th2-cell generation has been demonstrated. In a study Shigematsu K et al [83] tried to protect thermally injured mice orally infected with a lethal dose of *E. faecalis* by gene therapy utilizing phosphorothioate-CCL2 antisense oligodeoxynucleotides. They demon‐ strate that sepsis stemming from *E. faecalis* translocation in severely burned mice is control‐ lable by the gene therapy using CCL2 antisense ODNs, through the elimination of mesenteric lymph node macrophages (MLN- MΦ)-M2aMΦs and M2cMΦs subtypes. [83].

IL-1β binds the type-1 IL-1 receptor, while LPS binds to TLR4, both activates intracellular pathways by phosphorylation of IRAK family members including IRAK-1, which involve the MyD88 adaptor protein [84]. The group of Johns RE et al developed a family of "smart" polymeric carriers, termed encrypted polymers that enhance the cytoplasmic delivery of therapeutic antisense oligonucleotides (ASONs). This group has demonstrated that these ASONs block LPS activation of the transcription factor NFκB reducing the LPS-induced ex‐ pression of cytokines and chemokines. IL-6 shows a 2-fold decrease whereas TNFα expres‐ sion trended to decrease. There was a 2-fold decrease in expression of several genes including MCP1, MCP3, eotaxin and IP10 [85].

#### **5.3. Apoptosis**

Caspases are pro-enzymes of the aspartate-specific cysteine protease family and its activa‐ tion plays a central role in the execution of apoptosis [86]. Depending of the stimuli, two cas‐ pase-activation pathways have been described, the mitochondria-initiated caspase-8 dependent pathway and mitochondria-initiated caspase-9-mediated pathway. Activation of these pathways initiates a downstream cascade of effector caspases, such as caspase-3 that cleaves substrates such as D4-GDI leading to cell death [87]. The group of Ayala A et al in 2005 demonstrated that suppression of Fas or caspase-8 gene expression with hydrodynamic administration of siRNA conferred a survival advantage in septic mice model after caecal ligation and perforation (CLP) [88]. In a work of Matsuda N et al, they examined the thera‐ peutic efficacy of caspase-8 and caspase-3 gene silencing with siRNAs delivered by systemic injection in a CLP endotoxic shock mouse model. They demonstrate that *in vivo* delivery of caspase-8/caspase-3 siRNAs conferred a dramatic survival advantage to CLP mice as com‐ pared to controls. Also they demonstrated that the survival benefit was observed despite ad‐ ministration of siRNA as late as 10h after CLP [88].

exudate floods the alveolar spaces, impairs gas exchange and precipitates respiratory failure [92]. Several studies indicate that CLP (cecal ligation and puncture) sepsis model, sepsis and endotoxemia impair the expression of heat shock protein (HSP-70). Data shown that HSP-70 can limit inflammatory responses protect proteins from damage, restore function to proteins that are damaged and prevent cellular destruction, key processes of ALI/ARDS [93]. Weiss et al have demonstrated that the use of an adenoviral vector (AdHSP, an adenovirus carry‐ ing the gene for HSP-70) correcting the relative defect in HSP-70 expression prevents neutro‐ phil accumulation, reduce protein rich edema fluid and improve the outcome in ARDS

Injury of the alveolo-capillary barrier alters active Na+ transport, leading to impaired edema fluid clearance from the alveolar spaces. Failure to return to normal clearance is associated with poor prognosis [95]. The primary force driving fluid reabsorption from the alveolar space into the interstitium and the pulmonary circulation is active Na+ transport. Sodium is taken up on the apical surface of the alveolar epithelium by amiloride-sensitive and -insensi‐

channels [96] and is subsequently pumped out of the cell by the Na+

the β1-subunit gene by utilizing a replication-incompetent human type-5 adenovirus ex‐

using electroporation increased alveolar fluid reabsorption [98]. Furthermore, while rats ex‐ posed to 100% oxygen develop ALI and impaired alveolar fluid clearance; overexpression of

100% survival over 14 days of hyperoxia (compared with 25-31% survival in the non-treated

In this line, Stern M et al used a cationic liposome to transfer cDNA encoding both α and β

thiourea; they observe a significant resolution of pulmonary edema *in vivo*. Also, overex‐ pression of the β2-adrenergic receptor leads to increased alveolar fluid clearance in rats by

ATPase function, probably enhancing responsiveness to endogenous catecholamines in the

The regulation of alveolar transport proteins is vital in the maintenance of alveolar fluid bal‐

and protein abundance at the plasma membrane by promoting the endocytosis of the pump, which contributes to a decrease in alveolar fluid reabsorption in both *in vivo* an *ex vivo* mod‐ els of hypoxia. Also, the overexpression of the reactive oxygen species scavenger, SOD2,


/K+





/K+

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 639


/K+



/K+


/K+ -


/K+


secondary to CLP [94].

triphosphatase (Na+

ed the importance of Na+

/K+

/K+

or null virus-treated control groups) [99].

/K+

alveolar epithelium [100].

/K+

ance and, most importantly, overexpression of the Na+

increasing both membrane-bound amiloride-sensitive Na+

ance in patients [101]. The exposure to hypoxia results in decreased Na+

prevents this hypoxia-mediated decrease in alveolar fluid reabsorption and Na+

fold compared with controls [97]. Similarly, gene transfer of the Na+

tive Na+

pressing Na+

/K+

subunits of Na+

function [102].

the Na+

BRCA1 is a critical regulator of DNA damage repair and cell survival. In a recent article, Teoh H et al demonstrated a reduction in 24 hours post caecal ligation and perforation and thioglycollate stimulation mortality with pretreatment with human BRCA1 adenovirus (AdBRCA1). Treatment with AdBRCA1, a human adenovirus type-5 (dE1/E3), blunted CLPassociated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed re‐ duced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to their Ad-null-treated counterparts [89].

#### **6. Gene therapy in ARDS/ALI**

Over the past 20 years, the feasibility of using gene transfer to treat ALI/ARDS has been demonstrated using a variety of viral and non-viral vectors to deliver various transgenes to the lung [90].

#### **6.1. Strategies to increase pulmonary surfactant**

ALI/ARDS is a surfactant-deficient state. *Pseudomonas aeruginosa* infection is a cause of pul‐ monary infection and ARDS with surfactant deficient phenotype. Zhou J et al have demon‐ strated the attenuation of the deleterious effects of *Pseudomonas aeruginosa* infection by adenoviral gene transfer overexpressing CCTpenta (a mutant form of the regulatory enzyme CCTα required for the biosynthesis of dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid of surfactant) with a significant increase of the biosynthesis of surfactant. This study suggests that augmentation of DPPC synthesis via gene delivery of CCTα can attenu‐ ate impaired lung function in surfactant-deficient states such as bacterial sepsis [91].

#### **6.2. Strategies to improve pulmonary edema**

The physiological hallmark of ARDS is disruption of the alveolar-capillary membrane barri‐ er, leading to development of non-cardiogenic pulmonary edema, in which proteinaceous exudate floods the alveolar spaces, impairs gas exchange and precipitates respiratory failure [92]. Several studies indicate that CLP (cecal ligation and puncture) sepsis model, sepsis and endotoxemia impair the expression of heat shock protein (HSP-70). Data shown that HSP-70 can limit inflammatory responses protect proteins from damage, restore function to proteins that are damaged and prevent cellular destruction, key processes of ALI/ARDS [93]. Weiss et al have demonstrated that the use of an adenoviral vector (AdHSP, an adenovirus carry‐ ing the gene for HSP-70) correcting the relative defect in HSP-70 expression prevents neutro‐ phil accumulation, reduce protein rich edema fluid and improve the outcome in ARDS secondary to CLP [94].

cleaves substrates such as D4-GDI leading to cell death [87]. The group of Ayala A et al in 2005 demonstrated that suppression of Fas or caspase-8 gene expression with hydrodynamic administration of siRNA conferred a survival advantage in septic mice model after caecal ligation and perforation (CLP) [88]. In a work of Matsuda N et al, they examined the thera‐ peutic efficacy of caspase-8 and caspase-3 gene silencing with siRNAs delivered by systemic injection in a CLP endotoxic shock mouse model. They demonstrate that *in vivo* delivery of caspase-8/caspase-3 siRNAs conferred a dramatic survival advantage to CLP mice as com‐ pared to controls. Also they demonstrated that the survival benefit was observed despite ad‐

BRCA1 is a critical regulator of DNA damage repair and cell survival. In a recent article, Teoh H et al demonstrated a reduction in 24 hours post caecal ligation and perforation and thioglycollate stimulation mortality with pretreatment with human BRCA1 adenovirus (AdBRCA1). Treatment with AdBRCA1, a human adenovirus type-5 (dE1/E3), blunted CLPassociated cardiac, pulmonary, hepatic and renal dysfunction and also reduced CLP-elicited double strand breaks and apoptosis in the liver. BRCA1 gene therapy was associated with lower CLP-evoked cardiac and hepatic superoxide generation that in the liver was in part due to improved reactive oxygen species removal. CLP also elevated mesenteric arteriolar and serum intercellular adhesion molecule-1, both of which were partially abrogated with AdBRCA1 administration. Thioglycollate-challenged AdBRCA1-treated mice displayed re‐ duced peritoneal neutrophil recruitment and dampened cytokine elaboration relative to

Over the past 20 years, the feasibility of using gene transfer to treat ALI/ARDS has been demonstrated using a variety of viral and non-viral vectors to deliver various transgenes to

ALI/ARDS is a surfactant-deficient state. *Pseudomonas aeruginosa* infection is a cause of pul‐ monary infection and ARDS with surfactant deficient phenotype. Zhou J et al have demon‐ strated the attenuation of the deleterious effects of *Pseudomonas aeruginosa* infection by adenoviral gene transfer overexpressing CCTpenta (a mutant form of the regulatory enzyme CCTα required for the biosynthesis of dipalmitoyl phosphatidylcholine (DPPC), the major phospholipid of surfactant) with a significant increase of the biosynthesis of surfactant. This study suggests that augmentation of DPPC synthesis via gene delivery of CCTα can attenu‐

The physiological hallmark of ARDS is disruption of the alveolar-capillary membrane barri‐ er, leading to development of non-cardiogenic pulmonary edema, in which proteinaceous

ate impaired lung function in surfactant-deficient states such as bacterial sepsis [91].

ministration of siRNA as late as 10h after CLP [88].

638 Gene Therapy - Tools and Potential Applications

their Ad-null-treated counterparts [89].

**6. Gene therapy in ARDS/ALI**

**6.1. Strategies to increase pulmonary surfactant**

**6.2. Strategies to improve pulmonary edema**

the lung [90].

Injury of the alveolo-capillary barrier alters active Na+ transport, leading to impaired edema fluid clearance from the alveolar spaces. Failure to return to normal clearance is associated with poor prognosis [95]. The primary force driving fluid reabsorption from the alveolar space into the interstitium and the pulmonary circulation is active Na+ transport. Sodium is taken up on the apical surface of the alveolar epithelium by amiloride-sensitive and -insensi‐ tive Na+ channels [96] and is subsequently pumped out of the cell by the Na+ /K+ -adenosine triphosphatase (Na+ /K+ -ATPase) on the baso-lateral side [96]. Some studies have demonstrat‐ ed the importance of Na+ /K+ -ATPase in ALI/ARDS. In normal adults rats, overexpression of the β1-subunit gene by utilizing a replication-incompetent human type-5 adenovirus ex‐ pressing Na+ /K+ -ATPase-β1 subunit cDNA increased alveolar edema clearance over twofold compared with controls [97]. Similarly, gene transfer of the Na+ /K+ -ATPase-β1 subunit using electroporation increased alveolar fluid reabsorption [98]. Furthermore, while rats ex‐ posed to 100% oxygen develop ALI and impaired alveolar fluid clearance; overexpression of the Na+ /K+ -ATPase-β1 subunit in the alveolar epithelium of rats increased lung liquid clear‐ ance and, most importantly, overexpression of the Na+ /K+ -ATPase-β1 subunit resulted in 100% survival over 14 days of hyperoxia (compared with 25-31% survival in the non-treated or null virus-treated control groups) [99].

In this line, Stern M et al used a cationic liposome to transfer cDNA encoding both α and β subunits of Na+ /K+ -ATPase to the lung of a mouse model of pulmonary edema induced by thiourea; they observe a significant resolution of pulmonary edema *in vivo*. Also, overex‐ pression of the β2-adrenergic receptor leads to increased alveolar fluid clearance in rats by increasing both membrane-bound amiloride-sensitive Na+ -channel expression and Na+ /K+ - ATPase function, probably enhancing responsiveness to endogenous catecholamines in the alveolar epithelium [100].

The regulation of alveolar transport proteins is vital in the maintenance of alveolar fluid bal‐ ance in patients [101]. The exposure to hypoxia results in decreased Na+ /K+ -ATPase activity and protein abundance at the plasma membrane by promoting the endocytosis of the pump, which contributes to a decrease in alveolar fluid reabsorption in both *in vivo* an *ex vivo* mod‐ els of hypoxia. Also, the overexpression of the reactive oxygen species scavenger, SOD2, prevents this hypoxia-mediated decrease in alveolar fluid reabsorption and Na+ /K+ -ATPase function [102].

#### **6.3. Strategies to afford oxidant injury-related injury, apoptosis and inflammation**

Keratinocyte growth factor (KGF) is an epithelial-specific growth factor secreted by fibro‐ blast and vascular smooth muscle cells and a main mitogen for alveolar type II cells [103]. Baba et al have demonstrated that transient over-expression of KGF in the lungs attenuate pathophysiological impairments in hyperoxia-induced acute lung injury by increasing Ki67 and surfactant protein C (Sp-C)-positive cells and proliferation of epithelial cuboidal cells [104]. There is an abundance of evidence regarding the protective effect of pre-treatment with KGF on lung injury induced by hyperoxia, acid instillation, radiation, bleomycin, αnaphthylthiourea, ventilator and bacterial pneumonia there are some studies that supports the potential clinical application of KGF-2 in the treatment of ALI/ARDS [105].

hyperoxia models and have shown significant reductions in inflammation and subsequent

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 641

Sepsis and acute lung injury/acute respiratory distress syndrome are important pathologies in critical care medicine. There are increasing evidence from relevant pre-clinical studies that support the efficacy of gene-based therapies. Multiple barriers exist to the successful use of gene therapy in critical care medicine and particularly in sepsis and ALI/ARDS. Fu‐ ture research approaches are necessary to overcome these barriers by developing better viral and non-viral vectors, enhanced and specific gene expression strategies, improved cellular

Although the treatment by transference of genetic material still presents many challenges, the technology is rapidly evolving and the possible use in clinical trials could be in a near future. So, the aim of this chapter was to understand the molecular mechanisms involved in acute respiratory distress syndrome and sepsis, to review the viral and non-viral gene thera‐ pies that have been developed to improve survival and to address the challenges of gene

This work was supported by CONACYT grant 164413 and PAPIIT-UNAM grant IN204410.

and Angel Zarain-Herzberg2

1 Intensive Care Unit, Hospital Universitari de Bellvitge, L'Hospitalet Llobregat, Barcelona,

2 Biochemistry Department, School of Medicine, Universidad Nacional Autónoma de Méxi‐

[1] Vincent JL. Evidence-based medicine in the ICU: important advances and limitations.

Chest. 2004;126(2):592-600. Epub 2004/08/11.

therapy in critical care patients using these two life-threating conditions as a model.

lung injury [90].

**Acknowledgments**

**Author details**

Spain

Gabriel J. Moreno-González1

co, Mexico City, México

**References**

**7. Future directions and conclusion**

uptake of vectors and better therapeutic targets.

Human angiopoeitin-1 (ANGPT1), a ligand for the endothelial-restricted receptor TEK tyro‐ sine kinase, plays an essential role in blood vessel maturation and stabilization during em‐ bryonic development. In postnatal, ANGPT1 maintains the normal quiescent phenotype of vascular ECs, protecting against vascular inflammation reducing permeability and promot‐ ing ECs survival. In a study of Mei SH and co-workers carried out in an ALI mice model (by intra-tracheal instillation of LPS), they have demonstrated that mesenchymal stem cells (MSCs) administration alone into the pulmonary circulation partially prevents LPS-induced lung inflammation. However, cell-based gene transfer using pANGPT1-transfected MSCs resulted in further improvement in both alveolar inflammation and membrane permeability. Also, MSCs-pANGPT1 dramatically reduced cytokine levels (IFNγ, TNFα, IL-6 and IL1-β) to the baseline values observed in naïve mice, suggesting a potential therapeutic approach to ALI/ARDS [106].

Pearl M and colleagues in a 2005 study using Fas- and caspase-8 siRNA intra-tracheal ad‐ ministration in a CLP mice model of sepsis demonstrated that the main targets of siRNA de‐ livery are the epithelial cells. Also, that down-regulation of Fas but not caspase-8 reduces pulmonary apoptosis and lung inflammation, decreases neutrophil influx and attenuates ALI [107].

Overexpression of interleukin IL-10 trough recombinant adeno-associated virus type-5 (AAV5) vector expressing murine IL-10 into pulmonary, tissue proinflammatory cytokines IL-1β and TNFα, macrophage inhibitory protein-1αand keratinocyte chemoattractant in the epithelial lining fluid and lung homogenate were decreased and neutrophil infiltration was less pronounced and more localized neutrophil infiltration in lung section [108].

Finally, Hemoxygenase-1 (HO-1) is an inducible isoform of the first and rate-controlling en‐ zyme of the degradation of heme into iron, carbon monoxide, and biliverdin, the latter being subsequently converted into bilirubin. Several positive biological effects exerted by this en‐ zyme have gained attention, as anti-inflammatory, antiapoptotic, angiogenic, and cytopro‐ tective functions are attributable to carbon monoxide and/or bilirubin Also, the enzyme has been involved in controlling infiltration of neutrophils into the injured lung and in the reso‐ lution of inflammation by modulating apoptotic cell death and cytokine expression. Several groups have delivered HO-1 expressing adenoviruses to the lungs in both pneumonia and hyperoxia models and have shown significant reductions in inflammation and subsequent lung injury [90].

#### **7. Future directions and conclusion**

**6.3. Strategies to afford oxidant injury-related injury, apoptosis and inflammation**

the potential clinical application of KGF-2 in the treatment of ALI/ARDS [105].

to ALI/ARDS [106].

640 Gene Therapy - Tools and Potential Applications

ALI [107].

Keratinocyte growth factor (KGF) is an epithelial-specific growth factor secreted by fibro‐ blast and vascular smooth muscle cells and a main mitogen for alveolar type II cells [103]. Baba et al have demonstrated that transient over-expression of KGF in the lungs attenuate pathophysiological impairments in hyperoxia-induced acute lung injury by increasing Ki67 and surfactant protein C (Sp-C)-positive cells and proliferation of epithelial cuboidal cells [104]. There is an abundance of evidence regarding the protective effect of pre-treatment with KGF on lung injury induced by hyperoxia, acid instillation, radiation, bleomycin, αnaphthylthiourea, ventilator and bacterial pneumonia there are some studies that supports

Human angiopoeitin-1 (ANGPT1), a ligand for the endothelial-restricted receptor TEK tyro‐ sine kinase, plays an essential role in blood vessel maturation and stabilization during em‐ bryonic development. In postnatal, ANGPT1 maintains the normal quiescent phenotype of vascular ECs, protecting against vascular inflammation reducing permeability and promot‐ ing ECs survival. In a study of Mei SH and co-workers carried out in an ALI mice model (by intra-tracheal instillation of LPS), they have demonstrated that mesenchymal stem cells (MSCs) administration alone into the pulmonary circulation partially prevents LPS-induced lung inflammation. However, cell-based gene transfer using pANGPT1-transfected MSCs resulted in further improvement in both alveolar inflammation and membrane permeability. Also, MSCs-pANGPT1 dramatically reduced cytokine levels (IFNγ, TNFα, IL-6 and IL1-β) to the baseline values observed in naïve mice, suggesting a potential therapeutic approach

Pearl M and colleagues in a 2005 study using Fas- and caspase-8 siRNA intra-tracheal ad‐ ministration in a CLP mice model of sepsis demonstrated that the main targets of siRNA de‐ livery are the epithelial cells. Also, that down-regulation of Fas but not caspase-8 reduces pulmonary apoptosis and lung inflammation, decreases neutrophil influx and attenuates

Overexpression of interleukin IL-10 trough recombinant adeno-associated virus type-5 (AAV5) vector expressing murine IL-10 into pulmonary, tissue proinflammatory cytokines IL-1β and TNFα, macrophage inhibitory protein-1αand keratinocyte chemoattractant in the epithelial lining fluid and lung homogenate were decreased and neutrophil infiltration was

Finally, Hemoxygenase-1 (HO-1) is an inducible isoform of the first and rate-controlling en‐ zyme of the degradation of heme into iron, carbon monoxide, and biliverdin, the latter being subsequently converted into bilirubin. Several positive biological effects exerted by this en‐ zyme have gained attention, as anti-inflammatory, antiapoptotic, angiogenic, and cytopro‐ tective functions are attributable to carbon monoxide and/or bilirubin Also, the enzyme has been involved in controlling infiltration of neutrophils into the injured lung and in the reso‐ lution of inflammation by modulating apoptotic cell death and cytokine expression. Several groups have delivered HO-1 expressing adenoviruses to the lungs in both pneumonia and

less pronounced and more localized neutrophil infiltration in lung section [108].

Sepsis and acute lung injury/acute respiratory distress syndrome are important pathologies in critical care medicine. There are increasing evidence from relevant pre-clinical studies that support the efficacy of gene-based therapies. Multiple barriers exist to the successful use of gene therapy in critical care medicine and particularly in sepsis and ALI/ARDS. Fu‐ ture research approaches are necessary to overcome these barriers by developing better viral and non-viral vectors, enhanced and specific gene expression strategies, improved cellular uptake of vectors and better therapeutic targets.

Although the treatment by transference of genetic material still presents many challenges, the technology is rapidly evolving and the possible use in clinical trials could be in a near future. So, the aim of this chapter was to understand the molecular mechanisms involved in acute respiratory distress syndrome and sepsis, to review the viral and non-viral gene thera‐ pies that have been developed to improve survival and to address the challenges of gene therapy in critical care patients using these two life-threating conditions as a model.

#### **Acknowledgments**

This work was supported by CONACYT grant 164413 and PAPIIT-UNAM grant IN204410.

#### **Author details**

Gabriel J. Moreno-González1 and Angel Zarain-Herzberg2

1 Intensive Care Unit, Hospital Universitari de Bellvitge, L'Hospitalet Llobregat, Barcelona, Spain

2 Biochemistry Department, School of Medicine, Universidad Nacional Autónoma de Méxi‐ co, Mexico City, México

#### **References**

[1] Vincent JL. Evidence-based medicine in the ICU: important advances and limitations. Chest. 2004;126(2):592-600. Epub 2004/08/11.

[2] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. The New England journal of medicine. 2005;353(16):1685-93. Epub 2005/10/21.

[15] Villar J, Perez-Mendez L, Lopez J, Belda J, Blanco J, Saralegui I, et al. An early PEEP/ FIO2 trial identifies different degrees of lung injury in patients with acute respiratory distress syndrome. American journal of respiratory and critical care medicine.

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 643

[16] Raghavendran K, Napolitano LM. Definition of ALI/ARDS. Critical care clinics.

[17] Martin GS. Temporal changes in clinical outcomes with ARDS. Chest. 2005;128(2):

[18] Frutos-Vivar F, Ferguson ND, Esteban A. Epidemiology of acute lung injury and acute respiratory distress syndrome. Seminars in respiratory and critical care medi‐

[19] Rubenfeld GD. Epidemiology of acute lung injury. Crit Care Med. 2003;31(4

[20] MacCallum NS, Evans TW. Epidemiology of acute lung injury. Current opinion in

[21] Martin GS, Bernard GR. Airway and lung in sepsis. Intensive care medicine. 2001;27

[22] Mogensen TH. Pathogen recognition and inflammatory signaling in innate immune defenses. Clinical microbiology reviews. 2009;22(2):240-73, Table of Contents. Epub

[23] Bowie A, O'Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: sig‐ nal generators for pro-inflammatory interleukins and microbial products. Journal of

[24] Akira S, Uematsu S, Takeuchi O. Pathogen recognition and innate immunity. Cell.

[25] Inohara, Chamaillard, McDonald C, Nunez G. NOD-LRR proteins: role in host-mi‐ crobial interactions and inflammatory disease. Annual review of biochemistry.

[26] Yoneyama M, Kikuchi M, Natsukawa T, Shinobu N, Imaizumi T, Miyagishi M, et al. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nature immunology. 2004;5(7):730-7. Epub 2004/06/23.

[27] Wagner H. Endogenous TLR ligands and autoimmunity. Advances in immunology.

[28] Barton GM, Kagan JC. A cell biological view of Toll-like receptor function: regulation through compartmentalization. Nature reviews Immunology. 2009;9(8):535-42. Epub

2007;176(8):795-804. Epub 2007/06/23.

2011;27(3):429-37. Epub 2011/07/12.

cine. 2006;27(4):327-36. Epub 2006/08/16.

critical care. 2005;11(1):43-9. Epub 2005/01/22.

leukocyte biology. 2000;67(4):508-14. Epub 2000/04/19.

Suppl):S276-84. Epub 2003/04/12.

Suppl 1:S63-79. Epub 2001/04/20.

2006;124(4):783-801. Epub 2006/02/25.

2005;74:355-83. Epub 2005/06/15.

2006;91:159-73. Epub 2006/08/30.

2009/04/16.

2009/06/27.

479-81. Epub 2005/08/16.


[15] Villar J, Perez-Mendez L, Lopez J, Belda J, Blanco J, Saralegui I, et al. An early PEEP/ FIO2 trial identifies different degrees of lung injury in patients with acute respiratory distress syndrome. American journal of respiratory and critical care medicine. 2007;176(8):795-804. Epub 2007/06/23.

[2] Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M, et al. Incidence and outcomes of acute lung injury. The New England journal of medicine.

[3] Linde-Zwirble WT, Angus DC. Severe sepsis epidemiology: sampling, selection, and

[4] American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis and organ failure and guidelines for the use of in‐ novative therapies in sepsis. Crit Care Med. 1992;20(6):864-74. Epub 1992/06/01.

[5] Levy MM, Fink MP, Marshall JC, Abraham E, Angus D, Cook D, et al. 2001 SCCM/ ESICM/ACCP/ATS/SIS International Sepsis Definitions Conference. Intensive care

[6] Johnson SB, Lissauer M, Bochicchio GV, Moore R, Cross AS, Scalea TM. Gene expres‐ sion profiles differentiate between sterile SIRS and early sepsis. Annals of surgery.

[7] Guidelines for the management of severe sepsis and septic shock. The International Sepsis Forum. Intensive care medicine. 2001;27 Suppl 1:S1-134. Epub 2001/08/25.

[8] Angus DC, Linde-Zwirble WT, Lidicker J, Clermont G, Carcillo J, Pinsky MR. Epi‐ demiology of severe sepsis in the United States: analysis of incidence, outcome, and

[9] Angus DC, Wax RS. Epidemiology of sepsis: an update. Crit Care Med. 2001;29(7

[10] Bone RC, Balk RA, Cerra FB, Dellinger RP, Fein AM, Knaus WA, et al. Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. The ACCP/SCCM Consensus Conference Committee. American College of Chest Physicians/Society of Critical Care Medicine. Chest. 1992;101(6):1644-55. Epub

[11] Rivers E, Nguyen B, Havstad S, Ressler J, Muzzin A, Knoblich B, et al. Early goaldirected therapy in the treatment of severe sepsis and septic shock. The New Eng‐

[12] Bernard GR, Vincent JL, Laterre PF, LaRosa SP, Dhainaut JF, Lopez-Rodriguez A, et al. Efficacy and safety of recombinant human activated protein C for severe sepsis.

The New England journal of medicine. 2001;344(10):699-709. Epub 2001/03/10.

[13] Natanson C, Esposito CJ, Banks SM. The sirens' songs of confirmatory sepsis trials: selection bias and sampling error. Crit Care Med. 1998;26(12):1927-31. Epub

[14] Phua J, Stewart TE, Ferguson ND. Acute respiratory distress syndrome 40 years later: time to revisit its definition. Crit Care Med. 2008;36(10):2912-21. Epub 2008/09/04.

land journal of medicine. 2001;345(19):1368-77. Epub 2002/01/17.

associated costs of care. Crit Care Med. 2001;29(7):1303-10. Epub 2001/07/11.

2005;353(16):1685-93. Epub 2005/10/21.

642 Gene Therapy - Tools and Potential Applications

society. Crit Care. 2004;8(4):222-6. Epub 2004/08/18.

medicine. 2003;29(4):530-8. Epub 2003/03/29.

2007;245(4):611-21. Epub 2007/04/07.

Suppl):S109-16. Epub 2001/07/11.

1992/06/01.

1999/01/06.


[29] Blasius AL, Beutler B. Intracellular toll-like receptors. Immunity. 2010;32(3):305-15. Epub 2010/03/30.

[43] Liu G, Park YJ, Tsuruta Y, Lorne E, Abraham E. p53 Attenuates lipopolysaccharideinduced NF-kappaB activation and acute lung injury. J Immunol. 2009;182(8):

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 645

[44] Albertine KH, Soulier MF, Wang Z, Ishizaka A, Hashimoto S, Zimmerman GA, et al. Fas and fas ligand are up-regulated in pulmonary edema fluid and lung tissue of pa‐ tients with acute lung injury and the acute respiratory distress syndrome. The Amer‐

[45] Gropper MA, Wiener-Kronish J. The epithelium in acute lung injury/acute respirato‐ ry distress syndrome. Current opinion in critical care. 2008;14(1):11-5. Epub

[46] Maniatis NA, Kotanidou A, Catravas JD, Orfanos SE. Endothelial pathomechanisms in acute lung injury. Vascular pharmacology. 2008;49(4-6):119-33. Epub 2008/08/30.

[47] Tsushima K, King LS, Aggarwal NR, De Gorordo A, D'Alessio FR, Kubo K. Acute

[48] Pene F, Zuber B, Courtine E, Rousseau C, Ouaaz F, Toubiana J, et al. Dendritic cells modulate lung response to *Pseudomonas aeruginosa* in a murine model of sepsis-in‐ duced immune dysfunction. J Immunol. 2008;181(12):8513-20. Epub 2008/12/04.

[49] Benjamim CF, Lundy SK, Lukacs NW, Hogaboam CM, Kunkel SL. Reversal of longterm sepsis-induced immunosuppression by dendritic cells. Blood. 2005;105(9):

[50] Kay MA, Liu D, Hoogerbrugge PM. Gene therapy. Proc Natl Acad Sci U S A.

[51] Yla-Herttuala S, Alitalo K. Gene transfer as a tool to induce therapeutic vascular

[52] Kamimura K, Suda T, Zhang G, Liu D. Advances in Gene Delivery Systems. Pharma‐

[53] Thomas CE, Ehrhardt A, Kay MA. Progress and problems with the use of viral vec‐ tors for gene therapy. Nature reviews Genetics. 2003;4(5):346-58. Epub 2003/05/03.

[54] Barquinero J, Eixarch H, Perez-Melgosa M. Retroviral vectors: new applications for

[55] Daniel R, Smith JA. Integration site selection by retroviral vectors: molecular mecha‐ nism and clinical consequences. Human gene therapy. 2008;19(6):557-68. Epub

[56] Khare R, Chen CY, Weaver EA, Barry MA. Advances and future challenges in adeno‐ viral vector pharmacology and targeting. Current gene therapy. 2011;11(4):241-58.

growth. Nature medicine. 2003;9(6):694-701. Epub 2003/06/05.

an old tool. Gene therapy. 2004;11 Suppl 1:S3-9. Epub 2004/09/30.

lung injury review. Intern Med. 2009;48(9):621-30. Epub 2009/05/08.

ican journal of pathology. 2002;161(5):1783-96. Epub 2002/11/05.

5063-71. Epub 2009/04/04.

3588-95. Epub 2004/12/18.

2008/06/07.

Epub 2011/04/02.

1997;94(24):12744-6. Epub 1997/12/05.

ceut Med. 2011;25(5):293-306. Epub 2011/12/28.

2008/01/16.


[43] Liu G, Park YJ, Tsuruta Y, Lorne E, Abraham E. p53 Attenuates lipopolysaccharideinduced NF-kappaB activation and acute lung injury. J Immunol. 2009;182(8): 5063-71. Epub 2009/04/04.

[29] Blasius AL, Beutler B. Intracellular toll-like receptors. Immunity. 2010;32(3):305-15.

[30] Li X, Jiang S, Tapping RI. Toll-like receptor signaling in cell proliferation and surviv‐

[31] McGettrick AF, O'Neill LA. Localisation and trafficking of Toll-like receptors: an im‐ portant mode of regulation. Current opinion in immunology. 2010;22(1):20-7. Epub

[32] Miggin SM, O'Neill LA. New insights into the regulation of TLR signaling. Journal of

[33] Pasare C, Medzhitov R. Toll-like receptors: linking innate and adaptive immunity. Advances in experimental medicine and biology. 2005;560:11-8. Epub 2005/06/04. [34] Martinon F, Burns K, Tschopp J. The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Molecular cell.

[35] Russell JA. Management of sepsis. The New England journal of medicine.

[36] Hotchkiss RS, Swanson PE, Freeman BD, Tinsley KW, Cobb JP, Matuschak GM, et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction.

[37] Ayala A, Herdon CD, Lehman DL, DeMaso CM, Ayala CA, Chaudry IH. The induc‐ tion of accelerated thymic programmed cell death during polymicrobial sepsis: con‐ trol by corticosteroids but not tumor necrosis factor. Shock. 1995;3(4):259-67. Epub

[38] Perl M, Lomas-Neira J, Venet F, Chung CS, Ayala A. Pathogenesis of indirect (secon‐ dary) acute lung injury. Expert review of respiratory medicine. 2011;5(1):115-26.

[39] Suzuki T, Moraes TJ, Vachon E, Ginzberg HH, Huang TT, Matthay MA, et al. Protei‐ nase-activated receptor-1 mediates elastase-induced apoptosis of human lung epithe‐ lial cells. American journal of respiratory cell and molecular biology. 2005;33(3):

[40] Woodfin A, Voisin MB, Nourshargh S. PECAM-1: a multi-functional molecule in in‐ flammation and vascular biology. Arteriosclerosis, thrombosis, and vascular biology.

[41] Dunican AL, Leuenroth SJ, Grutkoski P, Ayala A, Simms HH. TNFalpha-induced suppression of PMN apoptosis is mediated through interleukin-8 production. Shock.

[42] Nakanishi C, Toi M. Nuclear factor-kappaB inhibitors as sensitizers to anticancer

drugs. Nature reviews Cancer. 2005;5(4):297-309. Epub 2005/04/02.

Epub 2010/03/30.

644 Gene Therapy - Tools and Potential Applications

2010/01/12.

1995/04/01.

Epub 2011/02/26.

231-47. Epub 2005/05/14.

2007;27(12):2514-23. Epub 2007/09/18.

2000;14(3):284-8; discussion 8-9. Epub 2000/10/12.

al. Cytokine. 2010;49(1):1-9. Epub 2009/09/25.

2002;10(2):417-26. Epub 2002/08/23.

2006;355(16):1699-713. Epub 2006/10/20.

leukocyte biology. 2006;80(2):220-6. Epub 2006/05/16.

Crit Care Med. 1999;27(7):1230-51. Epub 1999/08/14.


[57] Friedman GK, Pressey JG, Reddy AT, Markert JM, Gillespie GY. Herpes simplex vi‐ rus oncolytic therapy for pediatric malignancies. Molecular therapy : the journal of the American Society of Gene Therapy. 2009;17(7):1125-35. Epub 2009/04/16.

[70] Gallay P, Heumann D, Le Roy D, Barras C, Glauser MP. Lipopolysaccharide-binding protein as a major plasma protein responsible for endotoxemic shock. Proc Natl Acad

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 647

[71] Le Roy D, Di Padova F, Adachi Y, Glauser MP, Calandra T, Heumann D. Critical role of lipopolysaccharide-binding protein and CD14 in immune responses against gram-

[72] Roger T, Froidevaux C, Le Roy D, Reymond MK, Chanson AL, Mauri D, et al. Protec‐ tion from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4. Proc

[73] Schmidt AM, Yan SD, Yan SF, Stern DM. The multiligand receptor RAGE as a pro‐ gression factor amplifying immune and inflammatory responses. J Clin Invest.

[74] Christaki E, Opal SM, Keith JC, Jr., Kessimian N, Palardy JE, Parejo NA, et al. A mon‐ oclonal antibody against RAGE alters gene expression and is protective in experi‐ mental models of sepsis and pneumococcal pneumonia. Shock. 2011;35(5):492-8.

[75] Beno DW, Uhing MR, Goto M, Chen Y, Jiyamapa-Serna VA, Kimura RE. Staphylo‐ coccal enterotoxin B potentiates LPS-induced hepatic dysfunction in chronically catheterized rats. American journal of physiology Gastrointestinal and liver physiol‐

[76] Kissner TL, Moisan L, Mann E, Alam S, Ruthel G, Ulrich RG, et al. A small molecule that mimics the BB-loop in the Toll interleukin-1 (IL-1) receptor domain of MyD88 attenuates staphylococcal enterotoxin B-induced pro-inflammatory cytokine produc‐ tion and toxicity in mice. The Journal of biological chemistry. 2011;286(36):31385-96.

[77] Huang Y, Cai B, Xu M, Qiu Z, Tao Y, Zhang Y, et al. Gene silencing of toll-like recep‐ tor 2 inhibits proliferation of human liver cancer cells and secretion of inflammatory

[78] Lei M, Jiao H, Liu T, Du L, Cheng Y, Zhang D, et al. siRNA targeting mCD14 inhibits TNF-alpha, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells. Applied microbiology and biotechnology. 2011;92(1):115-24. Epub

[79] Vostrugina K, Gudaviciene D, Vitkauskiene A. Bacteremias in patients with severe

[80] Houghton AM, Hartzell WO, Robbins CS, Gomis-Ruth FX, Shapiro SD. Macrophage elastase kills bacteria within murine macrophages. Nature. 2009;460(7255):637-41.

burn trauma. Medicina (Kaunas). 2006;42(7):576-9. Epub 2006/07/25.

negative bacteria. J Immunol. 2001;167(5):2759-65. Epub 2001/08/18.

Natl Acad Sci U S A. 2009;106(7):2348-52. Epub 2009/02/03.

Sci U S A. 1993;90(21):9935-8. Epub 1993/11/01.

2001;108(7):949-55. Epub 2001/10/03.

ogy. 2001;280(5):G866-72. Epub 2001/04/09.

cytokines. PloS one. 2012;7(7):e38890. Epub 2012/07/21.

Epub 2011/01/26.

Epub 2011/06/23.

2011/06/28.

Epub 2009/06/19.


[70] Gallay P, Heumann D, Le Roy D, Barras C, Glauser MP. Lipopolysaccharide-binding protein as a major plasma protein responsible for endotoxemic shock. Proc Natl Acad Sci U S A. 1993;90(21):9935-8. Epub 1993/11/01.

[57] Friedman GK, Pressey JG, Reddy AT, Markert JM, Gillespie GY. Herpes simplex vi‐ rus oncolytic therapy for pediatric malignancies. Molecular therapy : the journal of

[58] Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent prog‐

[59] Wasungu L, Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. Journal of controlled release : official journal of the Controlled Release Society.

[60] Sokolova V, Epple M. Inorganic nanoparticles as carriers of nucleic acids into cells.

[61] Wolff JA, Malone RW, Williams P, Chong W, Acsadi G, Jani A, et al. Direct gene transfer into mouse muscle in vivo. Science. 1990;247(4949 Pt 1):1465-8. Epub

[62] Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic ad‐ ministration of plasmid DNA. Gene therapy. 1999;6(7):1258-66. Epub 1999/08/24.

[63] O'Brien J, Lummis SC. An improved method of preparing microcarriers for biolistic transfection. Brain research Brain research protocols. 2002;10(1):12-5. Epub

[64] Titomirov AV, Sukharev S, Kistanova E. In vivo electroporation and stable transfor‐ mation of skin cells of newborn mice by plasmid DNA. Biochimica et biophysica ac‐

[65] Kim HJ, Greenleaf JF, Kinnick RR, Bronk JT, Bolander ME. Ultrasound-mediated transfection of mammalian cells. Human gene therapy. 1996;7(11):1339-46. Epub

[66] Dellinger RP, Levy MM, Carlet JM, Bion J, Parker MM, Jaeschke R, et al. Surviving Sepsis Campaign: international guidelines for management of severe sepsis and sep‐

[67] Loza Vazquez A, Leon Gil C, Leon Regidor A. [New therapeutic alternatives for se‐ vere sepsis in the critical patient. A review]. Medicina intensiva / Sociedad Espanola de Medicina Intensiva y Unidades Coronarias. 2011;35(4):236-45. Epub 2011/01/07. Nuevas alternativas terapeuticas para la sepsis grave en el paciente critico. Revision.

[68] Matsuda A, Jacob A, Wu R, Aziz M, Yang WL, Matsutani T, et al. Novel therapeutic targets for sepsis: regulation of exaggerated inflammatory responses. Journal of Ni‐ hon Medical School = Nihon Ika Daigaku zasshi. 2012;79(1):4-18. Epub 2012/03/09.

[69] Frevert CW, Matute-Bello G, Skerrett SJ, Goodman RB, Kajikawa O, Sittipunt C, et al. Effect of CD14 blockade in rabbits with Escherichia coli pneumonia and sepsis. J Im‐

tic shock: 2008. Crit Care Med. 2008;36(1):296-327. Epub 2007/12/26.

the American Society of Gene Therapy. 2009;17(7):1125-35. Epub 2009/04/16.

ress. The AAPS journal. 2009;11(4):671-81. Epub 2009/10/17.

Angew Chem Int Ed Engl. 2008;47(8):1382-95. Epub 2007/12/22.

2006;116(2):255-64. Epub 2006/08/18.

ta. 1991;1088(1):131-4. Epub 1991/01/17.

munol. 2000;164(10):5439-45. Epub 2000/05/09.

1990/03/23.

646 Gene Therapy - Tools and Potential Applications

2002/10/16.

1996/07/10.


[81] Katakura T, Miyazaki M, Kobayashi M, Herndon DN, Suzuki F. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol. 2004;172(3):1407-13. Epub 2004/01/22.

[94] Weiss YG, Maloyan A, Tazelaar J, Raj N, Deutschman CS. Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory dis‐

Gene Therapy in Critical Care Medicine http://dx.doi.org/10.5772/52701 649

[95] Comellas AP, Briva A. Role of endothelin-1 in acute lung injury. Translational re‐ search : the journal of laboratory and clinical medicine. 2009;153(6):263-71. Epub

[96] Matthay MA, Robriquet L, Fang X. Alveolar epithelium: role in lung fluid balance and acute lung injury. Proceedings of the American Thoracic Society. 2005;2(3):

[97] Factor P, Saldias F, Ridge K, Dumasius V, Zabner J, Jaffe HA, et al. Augmentation of lung liquid clearance via adenovirus-mediated transfer of a Na,K-ATPase beta1 sub‐

[98] Machado-Aranda D, Adir Y, Young JL, Briva A, Budinger GR, Yeldandi AV, et al. Gene transfer of the Na+,K+-ATPase beta1 subunit using electroporation increases lung liquid clearance. American journal of respiratory and critical care medicine.

[99] Factor P, Dumasius V, Saldias F, Brown LA, Sznajder JI. Adenovirus-mediated trans‐ fer of an Na+/K+-ATPase beta1 subunit gene improves alveolar fluid clearance and survival in hyperoxic rats. Human gene therapy. 2000;11(16):2231-42. Epub

[100] Dumasius V, Sznajder JI, Azzam ZS, Boja J, Mutlu GM, Maron MB, et al. beta(2) adrenergic receptor overexpression increases alveolar fluid clearance and respon‐ siveness to endogenous catecholamines in rats. Circulation research. 2001;89(10):

[101] Ware LB, Matthay MA. Alveolar fluid clearance is impaired in the majority of pa‐ tients with acute lung injury and the acute respiratory distress syndrome. American journal of respiratory and critical care medicine. 2001;163(6):1376-83. Epub

[102] Litvan J, Briva A, Wilson MS, Budinger GR, Sznajder JI, Ridge KM. Beta-adrenergic receptor stimulation and adenoviral overexpression of superoxide dismutase prevent the hypoxia-mediated decrease in Na,K-ATPase and alveolar fluid reabsorption. The

[103] Ware LB, Matthay MA. Keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair. American journal of physiology Lung cellular and molecular physiology. 2002;282(5):L924-40. Epub 2002/04/12.

[104] Baba Y, Yazawa T, Kanegae Y, Sakamoto S, Saito I, Morimura N, et al. Keratinocyte growth factor gene transduction ameliorates acute lung injury and mortality in mice.

Journal of biological chemistry. 2006;281(29):19892-8. Epub 2006/04/26.

Human gene therapy. 2007;18(2):130-41. Epub 2007/03/03.

tress syndrome. J Clin Invest. 2002;110(6):801-6. Epub 2002/09/18.

unit gene. J Clin Invest. 1998;102(7):1421-30. Epub 1998/10/14.

2009/05/19.

2000/11/21.

2001/05/24.

907-14. Epub 2001/11/10.

206-13. Epub 2005/10/14.

2005;171(3):204-11. Epub 2004/11/02.


[94] Weiss YG, Maloyan A, Tazelaar J, Raj N, Deutschman CS. Adenoviral transfer of HSP-70 into pulmonary epithelium ameliorates experimental acute respiratory dis‐ tress syndrome. J Clin Invest. 2002;110(6):801-6. Epub 2002/09/18.

[81] Katakura T, Miyazaki M, Kobayashi M, Herndon DN, Suzuki F. CCL17 and IL-10 as effectors that enable alternatively activated macrophages to inhibit the generation of classically activated macrophages. J Immunol. 2004;172(3):1407-13. Epub 2004/01/22.

[82] Asai A, Kogiso M, Kobayashi M, Herndon DN, Suzuki F. Effect of IL-10 antisense gene therapy in severely burned mice intradermally infected with MRSA. Immunobi‐

[83] Shigematsu K, Kogiso M, Kobayashi M, Herndon DN, Suzuki F. Effect of CCL2 anti‐ sense oligodeoxynucleotides on bacterial translocation and subsequent sepsis in se‐ verely burned mice orally infected with Enterococcus faecalis. European journal of

[84] Dunne A, O'Neill LA. The interleukin-1 receptor/Toll-like receptor superfamily: sig‐ nal transduction during inflammation and host defense. Science's STKE : signal

[85] Johns RE, El-Sayed ME, Bulmus V, Cuschieri J, Maier R, Hoffman AS, et al. Mecha‐ nistic analysis of macrophage response to IRAK-1 gene knockdown by a smart poly‐ mer-antisense oligonucleotide therapeutic. Journal of biomaterials science Polymer

[86] Cohen GM. Caspases: the executioners of apoptosis. The Biochemical journal.

[87] Goyal L. Cell death inhibition: keeping caspases in check. Cell. 2001;104(6):805-8.

[88] Wesche-Soldato DE, Chung CS, Lomas-Neira J, Doughty LA, Gregory SH, Ayala A. In vivo delivery of caspase-8 or Fas siRNA improves the survival of septic mice.

[89] Teoh H, Quan A, Creighton AK, Annie Bang KW, Singh KK, Shukla PC, et al. BRCA1 gene therapy reduces systemic inflammatory response and multiple organ failure and improves survival in experimental sepsis. Gene therapy. 2012. Epub 2012/01/20.

[90] Lin X, Dean DA. Gene therapy for ALI/ARDS. Critical care clinics. 2011;27(3):705-18.

[91] Zhou J, Wu Y, Henderson F, McCoy DM, Salome RG, McGowan SE, et al. Adenoviral gene transfer of a mutant surfactant enzyme ameliorates pseudomonas-induced lung

[92] Ware LB, Matthay MA. The acute respiratory distress syndrome. The New England

[93] Ryter SW, Alam J, Choi AM. Heme oxygenase-1/carbon monoxide: from basic sci‐ ence to therapeutic applications. Physiological reviews. 2006;86(2):583-650. Epub

injury. Gene therapy. 2006;13(12):974-85. Epub 2006/03/03.

journal of medicine. 2000;342(18):1334-49. Epub 2000/05/04.

transduction knowledge environment. 2003;2003(171):re3. Epub 2003/02/28.

ology. 2012;217(7):711-8. Epub 2012/01/03.

immunology. 2012;42(1):158-64. Epub 2011/10/18.

edition. 2008;19(10):1333-46. Epub 2008/10/16.

Blood. 2005;106(7):2295-301. Epub 2005/06/09.

1997;326 ( Pt 1):1-16. Epub 1997/08/15.

Epub 2001/04/06.

648 Gene Therapy - Tools and Potential Applications

Epub 2011/07/12.

2006/04/08.


[105] Fang X, Bai C, Wang X. Potential clinical application of KGF-2 (FGF-10) for acute lung injury/acute respiratory distress syndrome. Expert review of clinical pharmacol‐ ogy. 2010;3(6):797-805. Epub 2011/11/25.

**Chapter 27**

**Clinical and Translational Challenges in**

Divya Pankajakshan and Devendra K. Agrawal

Additional information is available at the end of the chapter

apeutic neo-vascularization to counteract ischemia.

http://dx.doi.org/10.5772/54075

**1. Introduction**

**Gene Therapy of Cardiovascular Diseases**

Cardiovascular (CV) disease is the most prevalent life-threatening clinical problem and is a major cause of disability and economic burden worldwide [1]. Despite extensive phar‐ macotherapies, there remain many vascular conditions for which pharmacological inter‐ ventions are either non-existent or largely ineffective. CV gene therapy offers the benefit of sustained and/or controlled expression of desired proteins in cell types, which makes it more beneficial in providing durable clinical benefits [2]. The therapeutic gene works by either over-expressing therapeutically beneficial proteins, replacing a deficient gene or its expression proteins, or silencing a particular gene whose expression is not beneficial in the clinical scenario [3]. In addition, success of gene therapy also depends on the choice of the vector and the delivery approach. Blood vessels are among the most feasible tar‐ gets for gene therapy because of ease of access using a catheter or by systemic delivery. The new genetic material should enter the cells in the vasculature overcoming the ana‐ tomical, cellular and physiological barriers and induce the expression of the transfected gene in the target tissue. The target cells in the arteries are endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts, which constitute the intimal, medial and adventitial layers, respectively [4]. In the case of atherosclerotic lesions, macrophages also become a target cell. For the treatment of cardiovascular diseases, gene therapy strategies have been designed to enhance re-endothelialization and EC function to reduce thrombosis, inhibit SMC proliferation and migration to prevent neointimal hyperplasia, and to improve ther‐

Viral and non-viral vector systems have been evaluated for gene transfer to the vasculature. Lipoplexes, polyplexes and lipopolyplexes as well as naked DNA have been used as nonviral vectors for gene delivery to vascular tissues. Retroviruses, lentiviruses, adenoviruses

> © 2013 Pankajakshan and Agrawal; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


### **Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases**

Divya Pankajakshan and Devendra K. Agrawal

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54075

#### **1. Introduction**

[105] Fang X, Bai C, Wang X. Potential clinical application of KGF-2 (FGF-10) for acute lung injury/acute respiratory distress syndrome. Expert review of clinical pharmacol‐

[106] Mei SH, McCarter SD, Deng Y, Parker CH, Liles WC, Stewart DJ. Prevention of LPSinduced acute lung injury in mice by mesenchymal stem cells overexpressing angio‐

[107] Perl M, Chung CS, Lomas-Neira J, Rachel TM, Biffl WL, Cioffi WG, et al. Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, in‐ flammation, and neutrophil influx after hemorrhagic shock and sepsis. The American

[108] Buff SM, Yu H, McCall JN, Caldwell SM, Ferkol TW, Flotte TR, et al. IL-10 delivery by AAV5 vector attenuates inflammation in mice with *Pseudomonas pneumonia*. Gene

ogy. 2010;3(6):797-805. Epub 2011/11/25.

650 Gene Therapy - Tools and Potential Applications

therapy. 2010;17(5):567-76. Epub 2010/04/02.

poietin 1. PLoS medicine. 2007;4(9):e269. Epub 2007/09/07.

journal of pathology. 2005;167(6):1545-59. Epub 2005/11/30.

Cardiovascular (CV) disease is the most prevalent life-threatening clinical problem and is a major cause of disability and economic burden worldwide [1]. Despite extensive phar‐ macotherapies, there remain many vascular conditions for which pharmacological inter‐ ventions are either non-existent or largely ineffective. CV gene therapy offers the benefit of sustained and/or controlled expression of desired proteins in cell types, which makes it more beneficial in providing durable clinical benefits [2]. The therapeutic gene works by either over-expressing therapeutically beneficial proteins, replacing a deficient gene or its expression proteins, or silencing a particular gene whose expression is not beneficial in the clinical scenario [3]. In addition, success of gene therapy also depends on the choice of the vector and the delivery approach. Blood vessels are among the most feasible tar‐ gets for gene therapy because of ease of access using a catheter or by systemic delivery. The new genetic material should enter the cells in the vasculature overcoming the ana‐ tomical, cellular and physiological barriers and induce the expression of the transfected gene in the target tissue. The target cells in the arteries are endothelial cells (EC), smooth muscle cells (SMC) and fibroblasts, which constitute the intimal, medial and adventitial layers, respectively [4]. In the case of atherosclerotic lesions, macrophages also become a target cell. For the treatment of cardiovascular diseases, gene therapy strategies have been designed to enhance re-endothelialization and EC function to reduce thrombosis, inhibit SMC proliferation and migration to prevent neointimal hyperplasia, and to improve ther‐ apeutic neo-vascularization to counteract ischemia.

Viral and non-viral vector systems have been evaluated for gene transfer to the vasculature. Lipoplexes, polyplexes and lipopolyplexes as well as naked DNA have been used as nonviral vectors for gene delivery to vascular tissues. Retroviruses, lentiviruses, adenoviruses

© 2013 Pankajakshan and Agrawal; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

and adeno-associated viruses have been tested as viral vectors. Both systems have their own advantages and disadvantages that determine its use for a particular subset of CV diseases. Another challenge is the development of delivery approaches that are clinically viable and are capable of achieving consistent therapy for diseased arterial tissues. The efficiency of lo‐ calization, restriction of systemic distribution and adequacy of permeation into the target tissue are required for the optimal delivery of the vector. It is also dependent on the require‐ ments of a given patho-physiological situation. Systemic, intravascular and perivascular ap‐ proaches are used for gene delivery to the vasculature.

tors demonstrate significantly broadened tropism and high stability and have been used to demonstrate efficient transgene delivery *in vitro* into SMCs and ECs from human sa‐ phenous vein [10], human coronary artery SMCs and ECs [11], and cardiomyocytes [12].

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653

Ad vectors are the most commonly used viral vectors in the CV system. They transfect non-dividing cells efficiently [Figure 1], but sustained gene expression is limited to ap‐ proximately 2 weeks because the gene is kept episomal [2]. The administration of the Ad vectors is almost invariably associated with the development of systemic neutralizing an‐ tibodies directed against the vector [13]. Therefore, lowering the immunogenicity of the Ad virus is desirable and can be achieved by deleting genes that encode viral proteins [14]. Another method of reducing the inflammatory reaction to gene transfer by Ad vec‐ tors is to preserve the E3 region, which is supposed to modulate the host immune re‐ sponse *in vivo* [15]. When systemically administered, Ad5 poorly transduced ECs but could effectively transduce medial SMCs during endothelial denudation [5]. Efficient my‐ ocardial transduction was observed following local delivery of Ad5 vectors in porcine

AAV vectors have emerged as versatile vehicles for gene delivery due to their efficient in‐ fection of dividing and non-dividing cells in the presence of helper virus, sustained main‐ tenance of viral genome leading to long-term expression of the transgene, and a strong clinical safety profile [17]. AAV is non-pathogenic since it cannot replicate without the as‐ sistance of a helper virus. Recombinant AAV (rAAV) vectors have almost the entire viral genome removed, thereby yielding a delivery vehicle with enhanced safety and reduced immunogenicity [18]. The AAV *Rep* and *Cap* genes, which are required for viral replica‐ tion and packaging, are supplied by a helper plasmid during the production process. Wild type AAV preferentially integrates to a specific locus of human chromosome 19. The rAAV has mechanisms for sustained episomal maintenance or semi-randomly integrates at a low rate [19]. Problems with AAV vectors include limited tissue tropism for sero‐ types that bind heparan sulphate, challenges with preexisting immunity due to prior ex‐ posure, and also substantially delayed onset of transgene expression compared to other

Even though the transfection efficiency of non-viral vectors are lower than that of their viral counterparts, they are associated with many advantages such as low immunogenic re‐ sponse, the capacity to carry large inserts of DNA (52Kb), the possibility of selective modifi‐ cation using ligand and large scale manufacture [20]. Ideal non-viral vectors should be degradable into low molecular weight components in response to biological stimuli for low‐ er toxicity and effective systemic clearance. They should also be efficient in overcoming ex‐ tracellular and intracellular barriers and tissue/cell-targeted for specific accumulations [21]. In this group of vectors, naked DNA, cationic liposomes and cationic polymers have been

heart, where almost 80% of cardiomyocytes were transduced [16].

vectors.

**2.2. Non-viral vectors**

used for vascular gene transfer.

In this chapter, our goal is to summarize the current understanding of gene therapy strat‐ egies used to treat CV diseases, specifically the therapies targeting thrombosis, atherogene‐ sis, SMC proliferation and migration, modification of extracellular matrix (ECM) and regeneration of the endothelial cell layer. We will discuss various vectors and delivery ap‐ proaches used in the CV gene therapy and describe, in detail, the challenges associated with each approach.

#### **2. Vectors in vascular gene therapy**

The ideal vector for clinical application would target the specific cell, offer the capacity to transfer large DNA sequences, result in therapeutic levels of transgene expression that are not attenuated by the host immune response, express transgene for a duration required to alleviate the clinical problem, pose no risk of toxicity either acutely (as a result of immuno‐ genicity or unregulated transgene expression) or in the long-term (such as oncogenesis), and be cost-effective and easy to produce in therapeutically applicable quantity [5]. Currently, no available vector fulfils all these criteria; therefore, a perfect vector for vascular gene thera‐ py does not exist. Nonetheless, viral and non-viral vector systems have been evaluated for gene transfer to the vasculature.

#### **2.1. Viral vectors**

Retroviruses, adenoviruses (Ad) and adeno-associated viruses (AAV) are used as viral vectors in vascular gene transfer. Recombinant retroviruses are RNA viruses that are ca‐ pable of integrating transgene into the target genome. Disadvantages of this vector in‐ clude instability, the requirement of cell division for gene transfer and the inability to attain high titers. Since the majority of vascular cells are not undergoing mitosis at the time of exposure to the viral vector, the efficiency of gene delivery to vascular cells by such vectors may be as low as 1% to 2% [6]. Attempts have been made to increase the transduction efficiency in endothelial cell using multiple viral exposures [7] or increasing viral titers by ultracentrifugation [8]. Murine leukemia retroviral vectors (MuLV) pseudo‐ typed with the vesicular stomatitis virus G glycoprotein (VSV-G) have the capacity to transfect human ECs and SMCs *in vitro* with significant improvement in stability and transduction efficiency [9]. Unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, which is an attractive characteristic for CV gene therapy. These vec‐ tors demonstrate significantly broadened tropism and high stability and have been used to demonstrate efficient transgene delivery *in vitro* into SMCs and ECs from human sa‐ phenous vein [10], human coronary artery SMCs and ECs [11], and cardiomyocytes [12].

Ad vectors are the most commonly used viral vectors in the CV system. They transfect non-dividing cells efficiently [Figure 1], but sustained gene expression is limited to ap‐ proximately 2 weeks because the gene is kept episomal [2]. The administration of the Ad vectors is almost invariably associated with the development of systemic neutralizing an‐ tibodies directed against the vector [13]. Therefore, lowering the immunogenicity of the Ad virus is desirable and can be achieved by deleting genes that encode viral proteins [14]. Another method of reducing the inflammatory reaction to gene transfer by Ad vec‐ tors is to preserve the E3 region, which is supposed to modulate the host immune re‐ sponse *in vivo* [15]. When systemically administered, Ad5 poorly transduced ECs but could effectively transduce medial SMCs during endothelial denudation [5]. Efficient my‐ ocardial transduction was observed following local delivery of Ad5 vectors in porcine heart, where almost 80% of cardiomyocytes were transduced [16].

AAV vectors have emerged as versatile vehicles for gene delivery due to their efficient in‐ fection of dividing and non-dividing cells in the presence of helper virus, sustained main‐ tenance of viral genome leading to long-term expression of the transgene, and a strong clinical safety profile [17]. AAV is non-pathogenic since it cannot replicate without the as‐ sistance of a helper virus. Recombinant AAV (rAAV) vectors have almost the entire viral genome removed, thereby yielding a delivery vehicle with enhanced safety and reduced immunogenicity [18]. The AAV *Rep* and *Cap* genes, which are required for viral replica‐ tion and packaging, are supplied by a helper plasmid during the production process. Wild type AAV preferentially integrates to a specific locus of human chromosome 19. The rAAV has mechanisms for sustained episomal maintenance or semi-randomly integrates at a low rate [19]. Problems with AAV vectors include limited tissue tropism for sero‐ types that bind heparan sulphate, challenges with preexisting immunity due to prior ex‐ posure, and also substantially delayed onset of transgene expression compared to other vectors.

#### **2.2. Non-viral vectors**

and adeno-associated viruses have been tested as viral vectors. Both systems have their own advantages and disadvantages that determine its use for a particular subset of CV diseases. Another challenge is the development of delivery approaches that are clinically viable and are capable of achieving consistent therapy for diseased arterial tissues. The efficiency of lo‐ calization, restriction of systemic distribution and adequacy of permeation into the target tissue are required for the optimal delivery of the vector. It is also dependent on the require‐ ments of a given patho-physiological situation. Systemic, intravascular and perivascular ap‐

In this chapter, our goal is to summarize the current understanding of gene therapy strat‐ egies used to treat CV diseases, specifically the therapies targeting thrombosis, atherogene‐ sis, SMC proliferation and migration, modification of extracellular matrix (ECM) and regeneration of the endothelial cell layer. We will discuss various vectors and delivery ap‐ proaches used in the CV gene therapy and describe, in detail, the challenges associated with

The ideal vector for clinical application would target the specific cell, offer the capacity to transfer large DNA sequences, result in therapeutic levels of transgene expression that are not attenuated by the host immune response, express transgene for a duration required to alleviate the clinical problem, pose no risk of toxicity either acutely (as a result of immuno‐ genicity or unregulated transgene expression) or in the long-term (such as oncogenesis), and be cost-effective and easy to produce in therapeutically applicable quantity [5]. Currently, no available vector fulfils all these criteria; therefore, a perfect vector for vascular gene thera‐ py does not exist. Nonetheless, viral and non-viral vector systems have been evaluated for

Retroviruses, adenoviruses (Ad) and adeno-associated viruses (AAV) are used as viral vectors in vascular gene transfer. Recombinant retroviruses are RNA viruses that are ca‐ pable of integrating transgene into the target genome. Disadvantages of this vector in‐ clude instability, the requirement of cell division for gene transfer and the inability to attain high titers. Since the majority of vascular cells are not undergoing mitosis at the time of exposure to the viral vector, the efficiency of gene delivery to vascular cells by such vectors may be as low as 1% to 2% [6]. Attempts have been made to increase the transduction efficiency in endothelial cell using multiple viral exposures [7] or increasing viral titers by ultracentrifugation [8]. Murine leukemia retroviral vectors (MuLV) pseudo‐ typed with the vesicular stomatitis virus G glycoprotein (VSV-G) have the capacity to transfect human ECs and SMCs *in vitro* with significant improvement in stability and transduction efficiency [9]. Unlike other retroviruses, lentiviruses are able to transduce non-dividing cells, which is an attractive characteristic for CV gene therapy. These vec‐

proaches are used for gene delivery to the vasculature.

652 Gene Therapy - Tools and Potential Applications

**2. Vectors in vascular gene therapy**

gene transfer to the vasculature.

**2.1. Viral vectors**

each approach.

Even though the transfection efficiency of non-viral vectors are lower than that of their viral counterparts, they are associated with many advantages such as low immunogenic re‐ sponse, the capacity to carry large inserts of DNA (52Kb), the possibility of selective modifi‐ cation using ligand and large scale manufacture [20]. Ideal non-viral vectors should be degradable into low molecular weight components in response to biological stimuli for low‐ er toxicity and effective systemic clearance. They should also be efficient in overcoming ex‐ tracellular and intracellular barriers and tissue/cell-targeted for specific accumulations [21]. In this group of vectors, naked DNA, cationic liposomes and cationic polymers have been used for vascular gene transfer.

thickening, reduced thrombogenicity, and restoration of endothelium-dependent vasomo‐ tor reactivity after injury due to balloon angioplasty in a rabbit model [23]. Physical ap‐ proaches have been explored for plasmid gene transfer into vascular cells *in vitro* and *in vivo*. Ultrasound exposure can induce transient pore formation in the cell membrane, thereby increasing the plasmid DNA uptake. Indeed, microbubble-enhanced ultrasound can achieve transgene expression levels *in vitro* at approximately 300-fold than that of naked plasmid DNA alone in porcine VSMCs [24]. The non-invasive nature of this techni‐ que makes it more feasible for clinical use. Local administration of plasmid DNA, cou‐ pled with application of brief electric pulses to cells or tissues to increase cellular permeability-- also called electroporation--yields high levels of transgene expression in the arteries [25]. However this technique is limited by its invasive nature and tissue damage

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

655

To increase the efficiency of gene transfer by naked DNA, they are complexed with cati‐ onic lipids (liposomes or lipoplexes) or polymers (polyplexes). The resulting net positive charge of the cationic lipid/polymer DNA complexes facilitates fusion with the negatively charged cell membrane and also reduces susceptibility to circulating nucleases. Transfec‐ tion efficiency of cationic lipoplexes varies dramatically depending on the structure of the cationic lipids (the overall geometric shape, the number of charged groups per molecules, the nature of lipid anchors, and linker bonds), the charge ratio used to form DNA–lipid complexes, and the properties of the co-lipid [22]. Although transfection efficiencies of liposomes are generally seen lower in vascular cells [22], the LID vector system, consist‐ ing of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), transfects primary porcine vascular SMCs and porcine aortic ECs with efficiency levels of 40% and 35%, respectively, under *in vitro* conditions [27]. Some of the cationic lipids have been found to negatively affect cell function. Cationic lipid-mediated transfection of bovine

The DNA packaging efficiency and *in vivo* stability are higher for cationic polymers com‐ pared to cationic lipids. Furthermore, these complexes can be surface-modified with anti‐ bodies or other targeting ligands to deliver nucleic acids to specific cells [29]. Several cationic polymers have been evaluated for their ability to form complexes with DNA, the most significant being poly-lysine (PLL) and polyethylene-imine (PEI) [30]. PEI affects EC function [31]; however, when conjugated with fractured polyamidoamine (PAMAM) den‐ drimers, less toxic effects were observed on vascular cells in addition to the enhanced transfection efficiencies [32]. Brito *et al*. [33] developed lipo-polyplex nanovector systems that can transfect EC and SMCs with reasonably high efficiency. They used a combina‐ tion of a cationic biodegradable polymer, poly(beta-amino ester) (PBAE), and a cationic phospholipid, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and obtained 20% and 33% transfection efficiencies *in vitro* in SMC and ECs, respectively. Molecular tuning of non-viral vectors via stimuli responsive degradation is another novel approach that can be adopted in vascular gene transfer [21]. Schematic representation of non-viral gene

associated with high voltages applied [26].

aortic ECs inhibits their attachment [28].

delivery is given in Figure 2.

**Figure 1. Transduction using adenoviral vectors**. Recombinant adenovirus enters cells via CAR-mediated binding allowing internalization via receptor-mediated endocytosis through clathrin-coated vesicles. Inside the cytoplasm, the endocytosed adenoviral vector escapes from the endosomes, disassembles the capsid and the viral DNA enter into the nucleus through the nuclear envelope pore complex. The viral DNA is not incorporated into the host cell genome, but rather assumes an epichromosomal location, where it can still use the transcriptional and translational machinery of the host cell to synthesize recombinant protein. [CAR; Coxsackievirus and adenovirus receptor]

Gene transfer with naked DNA is attractive because of its simplicity and lack of toxicity [22]. However, the efficiency of gene transfer with naked DNA is low due to its negative charge conferred by the phosphate groups, making cellular uptake difficult by the nega‐ tively charged cell surface, rapid degradation by nucleases in the serum and clearance by the mononuclear phagocyte system in the systemic circulation. However, site-specific arte‐ rial gene transfer of vascular endothelial growth factor (VEGF)-165 could yield efficient gene transfection resulting in accelerated re-endothelialization, inhibition of neointimal thickening, reduced thrombogenicity, and restoration of endothelium-dependent vasomo‐ tor reactivity after injury due to balloon angioplasty in a rabbit model [23]. Physical ap‐ proaches have been explored for plasmid gene transfer into vascular cells *in vitro* and *in vivo*. Ultrasound exposure can induce transient pore formation in the cell membrane, thereby increasing the plasmid DNA uptake. Indeed, microbubble-enhanced ultrasound can achieve transgene expression levels *in vitro* at approximately 300-fold than that of naked plasmid DNA alone in porcine VSMCs [24]. The non-invasive nature of this techni‐ que makes it more feasible for clinical use. Local administration of plasmid DNA, cou‐ pled with application of brief electric pulses to cells or tissues to increase cellular permeability-- also called electroporation--yields high levels of transgene expression in the arteries [25]. However this technique is limited by its invasive nature and tissue damage associated with high voltages applied [26].

To increase the efficiency of gene transfer by naked DNA, they are complexed with cati‐ onic lipids (liposomes or lipoplexes) or polymers (polyplexes). The resulting net positive charge of the cationic lipid/polymer DNA complexes facilitates fusion with the negatively charged cell membrane and also reduces susceptibility to circulating nucleases. Transfec‐ tion efficiency of cationic lipoplexes varies dramatically depending on the structure of the cationic lipids (the overall geometric shape, the number of charged groups per molecules, the nature of lipid anchors, and linker bonds), the charge ratio used to form DNA–lipid complexes, and the properties of the co-lipid [22]. Although transfection efficiencies of liposomes are generally seen lower in vascular cells [22], the LID vector system, consist‐ ing of a liposome (L), an integrin targeting peptide (I), and plasmid DNA (D), transfects primary porcine vascular SMCs and porcine aortic ECs with efficiency levels of 40% and 35%, respectively, under *in vitro* conditions [27]. Some of the cationic lipids have been found to negatively affect cell function. Cationic lipid-mediated transfection of bovine aortic ECs inhibits their attachment [28].

The DNA packaging efficiency and *in vivo* stability are higher for cationic polymers com‐ pared to cationic lipids. Furthermore, these complexes can be surface-modified with anti‐ bodies or other targeting ligands to deliver nucleic acids to specific cells [29]. Several cationic polymers have been evaluated for their ability to form complexes with DNA, the most significant being poly-lysine (PLL) and polyethylene-imine (PEI) [30]. PEI affects EC function [31]; however, when conjugated with fractured polyamidoamine (PAMAM) den‐ drimers, less toxic effects were observed on vascular cells in addition to the enhanced transfection efficiencies [32]. Brito *et al*. [33] developed lipo-polyplex nanovector systems that can transfect EC and SMCs with reasonably high efficiency. They used a combina‐ tion of a cationic biodegradable polymer, poly(beta-amino ester) (PBAE), and a cationic phospholipid, 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) and obtained 20% and 33% transfection efficiencies *in vitro* in SMC and ECs, respectively. Molecular tuning of non-viral vectors via stimuli responsive degradation is another novel approach that can be adopted in vascular gene transfer [21]. Schematic representation of non-viral gene delivery is given in Figure 2.

Gene transfer with naked DNA is attractive because of its simplicity and lack of toxicity [22]. However, the efficiency of gene transfer with naked DNA is low due to its negative charge conferred by the phosphate groups, making cellular uptake difficult by the nega‐ tively charged cell surface, rapid degradation by nucleases in the serum and clearance by the mononuclear phagocyte system in the systemic circulation. However, site-specific arte‐ rial gene transfer of vascular endothelial growth factor (VEGF)-165 could yield efficient gene transfection resulting in accelerated re-endothelialization, inhibition of neointimal

the host cell to synthesize recombinant protein. [CAR; Coxsackievirus and adenovirus receptor]

654 Gene Therapy - Tools and Potential Applications

**Figure 1. Transduction using adenoviral vectors**. Recombinant adenovirus enters cells via CAR-mediated binding allowing internalization via receptor-mediated endocytosis through clathrin-coated vesicles. Inside the cytoplasm, the endocytosed adenoviral vector escapes from the endosomes, disassembles the capsid and the viral DNA enter into the nucleus through the nuclear envelope pore complex. The viral DNA is not incorporated into the host cell genome, but rather assumes an epichromosomal location, where it can still use the transcriptional and translational machinery of

VEGF and platelet-derived growth factor (PDGF) had a favorable impact on the improve‐ ment of rat myocardial function accompanied by upregulation of tissue connexin 43 and

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657

EC loss because of vascular injury is a major contributing factor to the local activation of patho-physiological events leading to the development of neo-intimal hyperplasia [36]. Pre‐ vious reports have shown that transplantation of autologous endothelial progenitor cells (EPCs) onto balloon-injured carotid artery leads to rapid re-endothelialization of the denud‐ ed vessels [37]. EPCs can be genetically manipulated *ex vivo*, expanded, and reintroduced *in vivo*, where at least a proportion will contribute to a long-lasting pool that can provide thera‐ peutically relevant levels of transgene expression. Chemokine receptor, CXCR4, is a key molecule in regulating EPC homing [38]. Chen *et al*. [38] reported that CXCR4 gene transfer to EPCs contributes to their enhanced *in vivo* re-endothelialization capacity. In another study, Ohno and colleagues over-expressed C-type natriuretic peptide by gene transfer in rabbit jugular vein grafts and observed accelerated re-endothelialization [39]. EPCs over-ex‐ pressing endothelial nitric oxide synthase (eNOS) further enhance the vasculo-protective properties of these cells [40]. Local intravascular and extra-vascular expression of VEGF, us‐ ing plasmid DNA, accelerated re-endothelialization and decreased intimal thickening after

Antithrombotic and anticoagulation therapy generally involves the systemic administra‐ tion of agents that target a small region of the vasculature. Localized and controlled deliv‐ ery of specific genes could allow sustained antithrombotic or anticoagulant treatment when prolonged systemic administration is undesirable. Antithrombotic gene therapy strategies could include inhibition of coagulation factors, over-expression of anticoagulant factors, or modulation of EC biology to make thrombus formation or propagation unfav‐ orable [42]. Ad gene transfer of thrombomodulin decreased arterial thrombosis to 28% compared to 86% in control rabbit model [43]. Hemagglutinating virus of Japan (HVJ)-lip‐ osome-mediated gene transfer of tissue factor pathway inhibitor (TFPI), a primary inhibi‐ tor of TF-induced coagulation, significantly reduced/inhibited thrombosis after angioplasty in atherosclerotic arteries without any significant adverse effects [44]. Ad gene transfer of many mediators, including hirudin to inhibit thrombin [45], tissue plas‐ minogen activator (tPA) to enhance fibrinolysis [43], cyclo-oxygenase to augment prosta‐ cyclin synthesis [46], prevents arterial thrombosis and promotes local thromboresistance. Vascular gene delivery of anticoagulants by local infusion of retrovirally-transduced EPCs

pro-angiogenic molecules after infarction [35].

**3.1. Promotion of re-endothelialization**

arterial injury in rabbit models [23, 41].

**3.2. Promotion of endothelial cell function**

with tPA and hirudin genes has also been attempted [37].

**3. Major targets in vascular gene therapy**

**Figure 2. Non-viral gene delivery using lipoplexes:** DNA is complexed with cationic liposomes and is internalized through receptor mediated endocytosis. After their internalization large amounts of complexes are degraded in the endolysosomal compartments. Only a small fraction enters into the nucleus and elicits desired gene expression.

#### **2.3. Stem cells**

One of the recent approaches is to use stem cells as gene delivery vehicles. Stem cell-based gene therapy approaches are currently being employed in recent studies as an alternative strategy to promote myocardial angiogenesis and regeneration. Indeed, the injection of ge‐ netically modified bone marrow-derived mesenchymal stem cells to express angiopoietin-1 improved arteriogenesis and increased collateral blood flow in porcine model of chronic myocardial ischemia [34]. Nanofiber-expanded hematopoietic stem cells over-expressing VEGF and platelet-derived growth factor (PDGF) had a favorable impact on the improve‐ ment of rat myocardial function accompanied by upregulation of tissue connexin 43 and pro-angiogenic molecules after infarction [35].

#### **3. Major targets in vascular gene therapy**

#### **3.1. Promotion of re-endothelialization**

EC loss because of vascular injury is a major contributing factor to the local activation of patho-physiological events leading to the development of neo-intimal hyperplasia [36]. Pre‐ vious reports have shown that transplantation of autologous endothelial progenitor cells (EPCs) onto balloon-injured carotid artery leads to rapid re-endothelialization of the denud‐ ed vessels [37]. EPCs can be genetically manipulated *ex vivo*, expanded, and reintroduced *in vivo*, where at least a proportion will contribute to a long-lasting pool that can provide thera‐ peutically relevant levels of transgene expression. Chemokine receptor, CXCR4, is a key molecule in regulating EPC homing [38]. Chen *et al*. [38] reported that CXCR4 gene transfer to EPCs contributes to their enhanced *in vivo* re-endothelialization capacity. In another study, Ohno and colleagues over-expressed C-type natriuretic peptide by gene transfer in rabbit jugular vein grafts and observed accelerated re-endothelialization [39]. EPCs over-ex‐ pressing endothelial nitric oxide synthase (eNOS) further enhance the vasculo-protective properties of these cells [40]. Local intravascular and extra-vascular expression of VEGF, us‐ ing plasmid DNA, accelerated re-endothelialization and decreased intimal thickening after arterial injury in rabbit models [23, 41].

#### **3.2. Promotion of endothelial cell function**

**Figure 2. Non-viral gene delivery using lipoplexes:** DNA is complexed with cationic liposomes and is internalized through receptor mediated endocytosis. After their internalization large amounts of complexes are degraded in the endolysosomal compartments. Only a small fraction enters into the nucleus and elicits desired gene expression.

One of the recent approaches is to use stem cells as gene delivery vehicles. Stem cell-based gene therapy approaches are currently being employed in recent studies as an alternative strategy to promote myocardial angiogenesis and regeneration. Indeed, the injection of ge‐ netically modified bone marrow-derived mesenchymal stem cells to express angiopoietin-1 improved arteriogenesis and increased collateral blood flow in porcine model of chronic myocardial ischemia [34]. Nanofiber-expanded hematopoietic stem cells over-expressing

**2.3. Stem cells**

656 Gene Therapy - Tools and Potential Applications

Antithrombotic and anticoagulation therapy generally involves the systemic administra‐ tion of agents that target a small region of the vasculature. Localized and controlled deliv‐ ery of specific genes could allow sustained antithrombotic or anticoagulant treatment when prolonged systemic administration is undesirable. Antithrombotic gene therapy strategies could include inhibition of coagulation factors, over-expression of anticoagulant factors, or modulation of EC biology to make thrombus formation or propagation unfav‐ orable [42]. Ad gene transfer of thrombomodulin decreased arterial thrombosis to 28% compared to 86% in control rabbit model [43]. Hemagglutinating virus of Japan (HVJ)-lip‐ osome-mediated gene transfer of tissue factor pathway inhibitor (TFPI), a primary inhibi‐ tor of TF-induced coagulation, significantly reduced/inhibited thrombosis after angioplasty in atherosclerotic arteries without any significant adverse effects [44]. Ad gene transfer of many mediators, including hirudin to inhibit thrombin [45], tissue plas‐ minogen activator (tPA) to enhance fibrinolysis [43], cyclo-oxygenase to augment prosta‐ cyclin synthesis [46], prevents arterial thrombosis and promotes local thromboresistance. Vascular gene delivery of anticoagulants by local infusion of retrovirally-transduced EPCs with tPA and hirudin genes has also been attempted [37].

#### **3.3. Inhibition of atherogenesis**

The extensive cross-talk between the immune system and vasculature leading to the infil‐ tration of immune cells into the vascular wall is a major step in atherogenesis. In this process, reactive oxygen species play a crucial role, by inducing the oxidation of low-den‐ sity lipoprotein (LDL) and the formation of foam cells, and by activating a number of re‐ dox-sensitive transcriptional factors, such as nuclear factor kappa B (NFκB), Nuclear factor E2-related factor-2 (Nrf2) [47], or activating protein 1 (AP1) that regulate the ex‐ pression of multiple pro-and anti-inflammatory genes involved in atherogenesis [48]. De‐ livery of genes encoding antioxidant defense enzymes, like extracellular superoxide dismutase [49, 50], catalase [51], glutathione peroxidase [51] or heme oxygenase-1 [52], suppresses atherogenesis in animal models.

for binding of a specific transcription factor that is relevant for the respective gene [64]. Ad‐ ministration of AP-1 decoy ODNs *in vivo* using HVJ-liposome method virtually abolished neointimal formation after balloon injury to the rat carotid artery [68]. Transfection of vein grafts with a decoy antisense oligonucleotide to block transcription factor E2F imparted long-term resistance to neointimal hyperplasia and atherosclerosis in rabbits on a cholesterol diet [69]. Another approach was to drive SMC into apoptosis during the process of prolifera‐ tion and migration. Transduction of rabbit iliac arteries with recombinant Ad vectors for Fas ligand (L) reduced neointima formation, which occurred through the killing of Fas express‐

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659

The regulation of SMC migration is mediated partly through the action of matrix metallo‐ proteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of matrix metallo‐ proteinases (TIMPs) [71]. AAV-mediated TIMP1 transduction in SMCs of injured rat carotid arteries significantly reduced the ratio of intima to media (52.4%) after two months of treatment [72]. Overexpression of TIMP-2 [73], TIMP-3 [74] and TIMP-4 [75] has also been demonstrated to inhibit SMC migration and neo-intimal proliferation in hu‐ man vein grafts and porcine vascular injury models. Gurjar *et al.* [76] demonstrated that eNOS gene transfer inhibits SMC migration and MMP-2 and MMP-9 activities in SMCs *in vitro*. A combination approach of TIMP-1 and plasminogen activator system inhibited vein graft thickening in hypercholesterolemic mice, when plasmids encoding TIMP-1-ATF (amino terminal fragment of urokinase) were incorporated to the vein graft by intravascu‐

Ischemic diseases, including acute myocardial infarction and chronic cardiac ischemia, are characterized by an impaired supply of blood resulting from narrowed or blocked arter‐ ies that starve tissues of needed nutrients and oxygen [78]. Delivery of genes encoding angiogenic factors or the whole protein has been shown to induce angiogenesis in numer‐ ous animal models with the expression of a functioning product [79]. The successful ap‐ plication of recombinant protein and gene transfer for the treatment of myocardial ischemia was reported by Losordo and colleagues [80] by direct intra-myocardial gene transfer of naked plasmid DNA encoding VEGF-165 in porcine model. These results were confirmed in phase 1 assessment of direct intra-myocardial administration of Ad vector expressing VEGF-121 cDNA in patients with severe coronary artery disease [81]. Admediated FGF-4 gene transfer improved cardiac contractile function and regional blood flow in the ischemic region during stress in pig model [82]. Placebo-controlled trials in humans with chronic stable angina indicate that Ad5FGF-4 increased treadmill exercise duration and improved stress-related ischemia [82]. In another study, following coronary artery occlusion, rabbits treated with Ad vector containing acidic FGF showed a 50% re‐

ing neighboring SMC by FasL-transduced cells [70].

**3.5. Enhancement of therapeutic angiogenesis**

duction in the risk region for myocardial infarction [83].

lar electroporation [77].

Apolipoprotein E (ApoE), a blood circulating protein with pleiotropic atheroprotective properties, has emerged as a strong candidate for treating hypercholesterolemia and CV dis‐ ease. The gene transfer of ApoE Ad vectors produced substantial amounts of plasma ApoE following intravenous injection into ApoE-/- mice, which lowered plasma cholesterol, and after 1 month, slowed aortic atherogenesis [53]. Hepatic expression of human ApoE3 using a second-generation recombinant Ad vector directly induced regression of pre-existing athe‐ rosclerotic lesions without reducing plasma cholesterol or altering lipoprotein distribution [54]. High concentrations of atherogenic apolipoprotein (apo) B100 could also be lowered by hepatic gene transfer with the catalytic subunit of apoB mRNA editing enzyme [55].

#### **3.4. Inhibition of SMC proliferation and migration**

SMC migration and proliferation as well as deposition and turnover of ECM proteins con‐ tribute to the process of Intimal hyperplasia. Several different approaches were introduced to inhibit SMC proliferation during restenosis. Most of the approaches targeted inhibition of cell cycle, where cell cycle inhibitor genes are over-expressed. Non-phosphorylated retino‐ blastoma gene (Rb) [56]; p21 [57, 58]; p27-p16 fusion gene [59, 60] ; cyclin-dependent kinase inhibitor p57Kip2 [61]; and the growth-arrest homeobox gene gax [62] are few of the genes over-expressed to inhibit cell proliferation and neo-intimal formation. Genes that have a beneficial influence on various aspects of vessel wall physiology also inhibit SMC prolifera‐ tion. Nitric oxide generation by endothelial nitric oxide synthase inhibits SMC proliferation *in vitro* and modulates vascular tone locally *in vivo* [63].

Another approach was to inhibit growth factor signaling by the introduction of nucleic acid constructs that interfere with mRNA stability, such as antisense oligonucleotides, hammer head ribozymes and siRNA [64]. Gene transfer of a truncated form of fibroblast growth fac‐ tor (FGF) receptor using Ad vector suppressed SMC proliferation *in vitro* [65]. Hammerhead ribozymes directed against PDGF-A chain [66] and transforming growth factor-β [67] inhib‐ ited SMC proliferation and neointima formation in rat carotid artery after balloon injury.

The regulation of a target gene can influence the level of transcription, either by decoy oligo‐ nucleotides, which are either short double-stranded oligonucleotides or dumb-bell shaped circular oligonucleotides that represent transcription factor binding sites, and thus compete for binding of a specific transcription factor that is relevant for the respective gene [64]. Ad‐ ministration of AP-1 decoy ODNs *in vivo* using HVJ-liposome method virtually abolished neointimal formation after balloon injury to the rat carotid artery [68]. Transfection of vein grafts with a decoy antisense oligonucleotide to block transcription factor E2F imparted long-term resistance to neointimal hyperplasia and atherosclerosis in rabbits on a cholesterol diet [69]. Another approach was to drive SMC into apoptosis during the process of prolifera‐ tion and migration. Transduction of rabbit iliac arteries with recombinant Ad vectors for Fas ligand (L) reduced neointima formation, which occurred through the killing of Fas express‐ ing neighboring SMC by FasL-transduced cells [70].

The regulation of SMC migration is mediated partly through the action of matrix metallo‐ proteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of matrix metallo‐ proteinases (TIMPs) [71]. AAV-mediated TIMP1 transduction in SMCs of injured rat carotid arteries significantly reduced the ratio of intima to media (52.4%) after two months of treatment [72]. Overexpression of TIMP-2 [73], TIMP-3 [74] and TIMP-4 [75] has also been demonstrated to inhibit SMC migration and neo-intimal proliferation in hu‐ man vein grafts and porcine vascular injury models. Gurjar *et al.* [76] demonstrated that eNOS gene transfer inhibits SMC migration and MMP-2 and MMP-9 activities in SMCs *in vitro*. A combination approach of TIMP-1 and plasminogen activator system inhibited vein graft thickening in hypercholesterolemic mice, when plasmids encoding TIMP-1-ATF (amino terminal fragment of urokinase) were incorporated to the vein graft by intravascu‐ lar electroporation [77].

#### **3.5. Enhancement of therapeutic angiogenesis**

**3.3. Inhibition of atherogenesis**

658 Gene Therapy - Tools and Potential Applications

suppresses atherogenesis in animal models.

**3.4. Inhibition of SMC proliferation and migration**

*in vitro* and modulates vascular tone locally *in vivo* [63].

The extensive cross-talk between the immune system and vasculature leading to the infil‐ tration of immune cells into the vascular wall is a major step in atherogenesis. In this process, reactive oxygen species play a crucial role, by inducing the oxidation of low-den‐ sity lipoprotein (LDL) and the formation of foam cells, and by activating a number of re‐ dox-sensitive transcriptional factors, such as nuclear factor kappa B (NFκB), Nuclear factor E2-related factor-2 (Nrf2) [47], or activating protein 1 (AP1) that regulate the ex‐ pression of multiple pro-and anti-inflammatory genes involved in atherogenesis [48]. De‐ livery of genes encoding antioxidant defense enzymes, like extracellular superoxide dismutase [49, 50], catalase [51], glutathione peroxidase [51] or heme oxygenase-1 [52],

Apolipoprotein E (ApoE), a blood circulating protein with pleiotropic atheroprotective properties, has emerged as a strong candidate for treating hypercholesterolemia and CV dis‐ ease. The gene transfer of ApoE Ad vectors produced substantial amounts of plasma ApoE following intravenous injection into ApoE-/- mice, which lowered plasma cholesterol, and after 1 month, slowed aortic atherogenesis [53]. Hepatic expression of human ApoE3 using a second-generation recombinant Ad vector directly induced regression of pre-existing athe‐ rosclerotic lesions without reducing plasma cholesterol or altering lipoprotein distribution [54]. High concentrations of atherogenic apolipoprotein (apo) B100 could also be lowered by

hepatic gene transfer with the catalytic subunit of apoB mRNA editing enzyme [55].

SMC migration and proliferation as well as deposition and turnover of ECM proteins con‐ tribute to the process of Intimal hyperplasia. Several different approaches were introduced to inhibit SMC proliferation during restenosis. Most of the approaches targeted inhibition of cell cycle, where cell cycle inhibitor genes are over-expressed. Non-phosphorylated retino‐ blastoma gene (Rb) [56]; p21 [57, 58]; p27-p16 fusion gene [59, 60] ; cyclin-dependent kinase inhibitor p57Kip2 [61]; and the growth-arrest homeobox gene gax [62] are few of the genes over-expressed to inhibit cell proliferation and neo-intimal formation. Genes that have a beneficial influence on various aspects of vessel wall physiology also inhibit SMC prolifera‐ tion. Nitric oxide generation by endothelial nitric oxide synthase inhibits SMC proliferation

Another approach was to inhibit growth factor signaling by the introduction of nucleic acid constructs that interfere with mRNA stability, such as antisense oligonucleotides, hammer head ribozymes and siRNA [64]. Gene transfer of a truncated form of fibroblast growth fac‐ tor (FGF) receptor using Ad vector suppressed SMC proliferation *in vitro* [65]. Hammerhead ribozymes directed against PDGF-A chain [66] and transforming growth factor-β [67] inhib‐ ited SMC proliferation and neointima formation in rat carotid artery after balloon injury.

The regulation of a target gene can influence the level of transcription, either by decoy oligo‐ nucleotides, which are either short double-stranded oligonucleotides or dumb-bell shaped circular oligonucleotides that represent transcription factor binding sites, and thus compete Ischemic diseases, including acute myocardial infarction and chronic cardiac ischemia, are characterized by an impaired supply of blood resulting from narrowed or blocked arter‐ ies that starve tissues of needed nutrients and oxygen [78]. Delivery of genes encoding angiogenic factors or the whole protein has been shown to induce angiogenesis in numer‐ ous animal models with the expression of a functioning product [79]. The successful ap‐ plication of recombinant protein and gene transfer for the treatment of myocardial ischemia was reported by Losordo and colleagues [80] by direct intra-myocardial gene transfer of naked plasmid DNA encoding VEGF-165 in porcine model. These results were confirmed in phase 1 assessment of direct intra-myocardial administration of Ad vector expressing VEGF-121 cDNA in patients with severe coronary artery disease [81]. Admediated FGF-4 gene transfer improved cardiac contractile function and regional blood flow in the ischemic region during stress in pig model [82]. Placebo-controlled trials in humans with chronic stable angina indicate that Ad5FGF-4 increased treadmill exercise duration and improved stress-related ischemia [82]. In another study, following coronary artery occlusion, rabbits treated with Ad vector containing acidic FGF showed a 50% re‐ duction in the risk region for myocardial infarction [83].

#### **4. Challenges in gene therapy**

#### **4.1. Cellular and extracellular barriers in gene delivery**

Viruses have highly evolved mechanisms for obtaining optimized receptor-mediated inter‐ nalization, efficient cytosolic release, directed and fast intracellular transport towards com‐ partments and readily disassemble. In contrast, non-viral vectors must overcome multiple extracellular and intracellular barriers [21]. These barriers include binding to the cell surface, traversing the plasma membrane, escaping lysosomal degradation, and overcoming the nu‐ clear envelope. To overcome the delivery barriers in non-viral gene transfer, various strat‐ egies have been employed to enhance the circulation time, improve intracellular delivery, and enhance endosomal escape and nuclear import. Lipoplexes have shown rapid hepatic clearance during systemic administration. Modification of lipoplexes with hydrophilic mole‐ cules like polyethylene glycol (PEG) and polyethyleneimine (PEI) causes steric hinderance between opsonins and the delivery vectors, increasing their circulation time in the blood. PEGylation of PLL decreases interparticle aggregation, resulting in high transfection effi‐ ciency in the presence of serum [29]. One study has demonstrated that when artery wall binding peptide (AWBP), a core peptide of apo B100 -- a major protein component of LDL - was conjugated to PLL with PEG as the linker, the PLL-PEG-AWBP protected the plasmid DNA from nucleases for more than 120 min in circulation and also showed 100 times higher transfection efficiency when compared to PLL and PLL-g-PEG in bovine aortic ECs and SMCs [84]. In an innovative approach, micellar nanovectors made of PEG-block-polycation, carrying ethylenediamine units in the side chain [PEG-PAsp(DET)], complexed with plas‐ mid DNA to form polyplex micelle effectively transfected vascular smooth muscle cells in vascular lesions without any vessel occlusion by thrombus [85] in rabbit carotid arteries. However, PEI-mediated gene delivery can affect EC function and viability [31].

tibody was used for gene targeting to activated vascular ECs [88]. The lectin-like oxidized LDL receptor (LOX-1) is expressed selectively at low levels on ECs but is strongly upregulat‐ ed in dysfunctional ECs associated with hypertension and atherogenesis. White and collea‐ gues [89] confirmed the selectivity to LOX-1 for peptides LSIPPKA, FQTPPQL, and LTPATAI, which could be potential targets to dysfunctional ECs expressing LOX-1 receptor. Another approach to increase intracellular delivery is to use cell penetrating peptides (CPPs). CPPs consist of short peptide sequences that are able to translocate large molecules

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

661

Following internalization of lipoplexes and polyplexes via endocytosis, endosomal entrap‐ ment and subsequent lysosomal degradation are the major hurdles that limit transfection ef‐ ficiency [29]. Lipoplexes are modified with dioleoylphosphatidylethanolamine (DOPE) or other helper lipids due to its fusogenic functionality and its ability to destabilize endosomal membranes. Small PLLs with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoylspermine)-propane] enhanced gene transfer in primary porcine SMCs *in vitro* and *in vivo* in porcine femoral arteries [91]. Polyplexes, PEI and PAMAM are cationic polymers of high ef‐ ficiency partly because of their ability to burst the endosomal membrane due to 'proton

A promising new delivery strategy is to use synthetic peptide carriers containing a nuclear localization signal to facilitate nuclear uptake of plasmid DNA. Nuclear import of plasmid DNA is more challenging for transfecting non-dividing cells. Strategies to increase the nu‐ clear import of genes involve tagging the nuclear localization sequence (NLS) with DNA vectors. NLS is a major player that shuttles protein-plasmid complexes through the nuclear pore by interaction with importins and transportin [92, 93]. Incorporation of DNA nuclear targeting sequence SV40 into expression plasmids results in 10-40 fold increases in vascular gene expression in rat mesenteric arteries [94], confirming the function of DNA nuclear tar‐

Insertional mutagenesis is a major concern in gene therapy involving viral vectors. These vectors integrate randomly or quasi-randomly into the host cell's genome, to stably transfect the target cell. The variable site and frequency of integration of the transgene can induce mutagenesis in the host genome, resulting in devastating consequences for the cell and for the organism. [95, 96]. Another disadvantage of the random integration of a transgene is the unpredictability of its stability and its expression. The genomic locus in which the vector in‐ tegrates can have profound effects on the level of transgene expression, as it can completely silence the transgene, or it can increase or decrease its expression. These effects could not be avoided by sophisticated vector design or inclusion of the gene's own promoter and/or en‐ hancer region in the transgenic vector construct, as the surrounding chromatin can override the activity of the original regulatory regions. Gene targeting by homologous recombination, however, lacks many of these shortcomings [96]. In this process, the transgene recombines

into the cells and increase the transfection efficiency [90].

sponge effect'.

geting sequences *in vivo*.

*4.2.1. Insertional mutagenesis*

**4.2. Challenges associated with the vectors**

The size and charge of the lipoplex/polyplex play an important role in their intracellular de‐ livery. Lipoplexes and polyplexes are generally formulated into particles with net positive charges to trigger endocytosis by non-specific electrostatic interaction between the positive‐ ly charged complexes and negatively charged cell surface [29]. Since drug carriers with a smaller particle size have resulted in higher arterial uptake compared to carriers with larger size, the size of the complexes was expected to be a dominating factor in the arterial wall lesions because of the rapid blood flow which could wash out most of the drugs or thera‐ peutic chemical agents from the arterial wall lesions within 20–30 min. Song *et al*. [86] re‐ ported a potentially useful particle size of 70∼160 nm for local intraluminal therapy of restenosis.

By taking advantage of high expression levels of receptors or antigens in diseased condi‐ tions, gene complexes can be targeted using specific ligands, such as antibodies, peptides and proteins. Cyclic RGD (cRGD) peptide recognizes α(v)β(3) and α(v)β(5) integrins, which are abundantly expressed in vascular lesions. When cRGD was conjugated to PEG-PAsp(DET) to form polyplex micelles through complexing with plasmid DNA, the micelles achieved significantly more efficient gene expression and cellular uptake as compared to PEG-PAsp(DET) micelles in ECs and SMCs [87]. PAMAM dendrimers with E/P-selectin an‐ tibody was used for gene targeting to activated vascular ECs [88]. The lectin-like oxidized LDL receptor (LOX-1) is expressed selectively at low levels on ECs but is strongly upregulat‐ ed in dysfunctional ECs associated with hypertension and atherogenesis. White and collea‐ gues [89] confirmed the selectivity to LOX-1 for peptides LSIPPKA, FQTPPQL, and LTPATAI, which could be potential targets to dysfunctional ECs expressing LOX-1 receptor. Another approach to increase intracellular delivery is to use cell penetrating peptides (CPPs). CPPs consist of short peptide sequences that are able to translocate large molecules into the cells and increase the transfection efficiency [90].

Following internalization of lipoplexes and polyplexes via endocytosis, endosomal entrap‐ ment and subsequent lysosomal degradation are the major hurdles that limit transfection ef‐ ficiency [29]. Lipoplexes are modified with dioleoylphosphatidylethanolamine (DOPE) or other helper lipids due to its fusogenic functionality and its ability to destabilize endosomal membranes. Small PLLs with cationic lipid DOCSPER [1,3-dioleoyloxy-2-(N(5)-carbamoylspermine)-propane] enhanced gene transfer in primary porcine SMCs *in vitro* and *in vivo* in porcine femoral arteries [91]. Polyplexes, PEI and PAMAM are cationic polymers of high ef‐ ficiency partly because of their ability to burst the endosomal membrane due to 'proton sponge effect'.

A promising new delivery strategy is to use synthetic peptide carriers containing a nuclear localization signal to facilitate nuclear uptake of plasmid DNA. Nuclear import of plasmid DNA is more challenging for transfecting non-dividing cells. Strategies to increase the nu‐ clear import of genes involve tagging the nuclear localization sequence (NLS) with DNA vectors. NLS is a major player that shuttles protein-plasmid complexes through the nuclear pore by interaction with importins and transportin [92, 93]. Incorporation of DNA nuclear targeting sequence SV40 into expression plasmids results in 10-40 fold increases in vascular gene expression in rat mesenteric arteries [94], confirming the function of DNA nuclear tar‐ geting sequences *in vivo*.

#### **4.2. Challenges associated with the vectors**

#### *4.2.1. Insertional mutagenesis*

**4. Challenges in gene therapy**

660 Gene Therapy - Tools and Potential Applications

restenosis.

**4.1. Cellular and extracellular barriers in gene delivery**

Viruses have highly evolved mechanisms for obtaining optimized receptor-mediated inter‐ nalization, efficient cytosolic release, directed and fast intracellular transport towards com‐ partments and readily disassemble. In contrast, non-viral vectors must overcome multiple extracellular and intracellular barriers [21]. These barriers include binding to the cell surface, traversing the plasma membrane, escaping lysosomal degradation, and overcoming the nu‐ clear envelope. To overcome the delivery barriers in non-viral gene transfer, various strat‐ egies have been employed to enhance the circulation time, improve intracellular delivery, and enhance endosomal escape and nuclear import. Lipoplexes have shown rapid hepatic clearance during systemic administration. Modification of lipoplexes with hydrophilic mole‐ cules like polyethylene glycol (PEG) and polyethyleneimine (PEI) causes steric hinderance between opsonins and the delivery vectors, increasing their circulation time in the blood. PEGylation of PLL decreases interparticle aggregation, resulting in high transfection effi‐ ciency in the presence of serum [29]. One study has demonstrated that when artery wall binding peptide (AWBP), a core peptide of apo B100 -- a major protein component of LDL - was conjugated to PLL with PEG as the linker, the PLL-PEG-AWBP protected the plasmid DNA from nucleases for more than 120 min in circulation and also showed 100 times higher transfection efficiency when compared to PLL and PLL-g-PEG in bovine aortic ECs and SMCs [84]. In an innovative approach, micellar nanovectors made of PEG-block-polycation, carrying ethylenediamine units in the side chain [PEG-PAsp(DET)], complexed with plas‐ mid DNA to form polyplex micelle effectively transfected vascular smooth muscle cells in vascular lesions without any vessel occlusion by thrombus [85] in rabbit carotid arteries.

However, PEI-mediated gene delivery can affect EC function and viability [31].

The size and charge of the lipoplex/polyplex play an important role in their intracellular de‐ livery. Lipoplexes and polyplexes are generally formulated into particles with net positive charges to trigger endocytosis by non-specific electrostatic interaction between the positive‐ ly charged complexes and negatively charged cell surface [29]. Since drug carriers with a smaller particle size have resulted in higher arterial uptake compared to carriers with larger size, the size of the complexes was expected to be a dominating factor in the arterial wall lesions because of the rapid blood flow which could wash out most of the drugs or thera‐ peutic chemical agents from the arterial wall lesions within 20–30 min. Song *et al*. [86] re‐ ported a potentially useful particle size of 70∼160 nm for local intraluminal therapy of

By taking advantage of high expression levels of receptors or antigens in diseased condi‐ tions, gene complexes can be targeted using specific ligands, such as antibodies, peptides and proteins. Cyclic RGD (cRGD) peptide recognizes α(v)β(3) and α(v)β(5) integrins, which are abundantly expressed in vascular lesions. When cRGD was conjugated to PEG-PAsp(DET) to form polyplex micelles through complexing with plasmid DNA, the micelles achieved significantly more efficient gene expression and cellular uptake as compared to PEG-PAsp(DET) micelles in ECs and SMCs [87]. PAMAM dendrimers with E/P-selectin an‐ Insertional mutagenesis is a major concern in gene therapy involving viral vectors. These vectors integrate randomly or quasi-randomly into the host cell's genome, to stably transfect the target cell. The variable site and frequency of integration of the transgene can induce mutagenesis in the host genome, resulting in devastating consequences for the cell and for the organism. [95, 96]. Another disadvantage of the random integration of a transgene is the unpredictability of its stability and its expression. The genomic locus in which the vector in‐ tegrates can have profound effects on the level of transgene expression, as it can completely silence the transgene, or it can increase or decrease its expression. These effects could not be avoided by sophisticated vector design or inclusion of the gene's own promoter and/or en‐ hancer region in the transgenic vector construct, as the surrounding chromatin can override the activity of the original regulatory regions. Gene targeting by homologous recombination, however, lacks many of these shortcomings [96]. In this process, the transgene recombines with its natural locus in the host genome, thereby ensuring correct transcription. Also, after homologous recombination, the targeted modification of the chromosomal locus is stable, whereas randomly integrated sequences might be lost over time. In their seminal paper, Russel and Hirata [97] reported that DNA vectors based on the AAV could target homolo‐ gous chromosomal DNA sequences and allow high-fidelity, non-mutagenic gene repair in a host cell. Although the laborious vector design and low transfection efficiencies of AAV vec‐ tors compared to the other viral vectors still remains a concern, statistical information neatly outlines the advantage of rAAV gene replacement system over standard viral vectors, which induce strong immune response.

approach of the Tie2 promoter and enhancer (Tshort) by Minami and collegues [119] direct‐

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663

Another challenge was in generating an EC-specific promoter with comparable efficiency as the CMV promoter. White *et al.* [120] examined several novel Ad expression cassettes for EC-specific gene transfer with CMV, Tshort, ICAM-2, ICAM-1, FLT-1 promoters, respective‐ ly and found that LOX-1 promoter elements significantly increased reporter gene expression in carotid arteries compared to other promoters. The efficacy of these novel expression cas‐

An increasingly important area to in-tissue specific targeting is to engineer viral vectors Ads and AAVs with altered cell tropisms to narrow or broaden its efficiency in tissues refractory to infection [19, 121]. Non-genetic approaches typically utilize bispecific antibodies that both neutralize wild-type virus tropism and provide a new cell binding capacity [122]. For genet‐ ic targeting strategies, the virus capsid are engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the natural tropism of the virus [122, 123]. Ad homing to target endothelial cells at specific sites of the body can be achieved by deleting the ability of the virus to interact with its natural receptor, Coxsackievirus and adenovirus receptor (CAR), and a simultaneous addition of a ligand that directs the virus to the angiotensin converting enzyme on the ECs. Retargeting of AAV-2 with novel peptides could increase both transduction efficiency and selectivity [124]

The vascular system represents an ideal route of substance transport for reaching a specific site for therapeutic intervention. However, in the case of non-viral vectors, which are cation‐ ic polymers in most cases, it has been found that electrostatic interactions between the sulphated glycosaminoglycans in the serum as well as those expressed on the cell surface cause premature release of plasmid DNA leading to its inactivation and extracellular degra‐ dation by serum DNAses [21]. Also, after systemic vascular application, non-specific distri‐ bution of plasmid DNA throughout the vasculature would result in undesired side effects because of accumulation at non-specific sites. Intravenous administration of cationic poly‐ mers resulted in their localization to liver, lung, kidney, and spleen in pigs and rabbits [127-129]. Other barriers to systemic delivery include rapid clearance of the lipoplexes by

Most Ad vectors are trapped by the liver, hampering delivery to target CV tissues after sys‐ temic application. Systemic tail vein injection of Ad vector in mice resulted in virus DNA deposition liver, lung, kidney and testis [130]. Furthermore, the use of a heterologous viral promoter CMV in the majority of vascular gene transfers causes systemic organ toxicity re‐ sulting from unrestricted transgene expression [131]. Retargeting of vectors and use of tissue specific promoters offers an enhanced safety profile by reducing ectopic expression in vital

ed widespread EC expression *in vivo.*

settes in large animal models have yet to be established.

in vascular ECs [125] and SMCs [126] *in vitro.*

the reticulo-endothelial system and target specificity.

organs including the liver and lungs.

*4.3.1. Systemic gene delivery*

**4.3. Challenges associated with the mode and route of gene delivery**

#### *4.2.2. Tissue-specific targeting*

The promiscuous tropism of vectors resulting in high-level transgene expression in multiple tissues is another major challenge in vascular gene therapy. After systemic application, most viral vectors are trapped by the liver, hampering delivery to target CV tissues. Approaches to restrict gene delivery to desired cell types *in vivo* relied mostly on cell surface targeting or cell-specific promoters.

The *cis*-acting regulatory elements of the SM (smooth muscle)22α [98-100], telokin [101], smooth muscle myosin heavy chain [102], smooth muscle α- [100] and γ-actin [103], and desmin [104] genes have been shown to direct reporter gene expression to smooth muscle tissues in transgenic mice. In our studies, specific gene transfer to the SMC layer was ach‐ ieved in swine coronary and peripheral arteries using SM22α promoter in AAV [17]. Al‐ though the efficiency of transduction was low when compared to a similar study using AAV vectors with cytomegalovirus (CMV) promoter [105], the use of SM22α promoter caused specific transduction of SMCs *in vivo*. An interesting approach to enhance the transduction efficiency of SM22α -containing plasmid was to incorporate chimeric transcriptional cas‐ settes containing a SM-myosin heavy chain enhancer element combined with the SM22α promoter [106]. The transfection levels obtained using these chimeric constructs in Ad vec‐ tor were similar to that with CMV promoter when tested in rat carotid arteries. Certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types. Young *et al.* [107] improved the efficiency of transduction in SMCs of rat vasculature using a SMC-specific DNA nuclear targeting sequence.

EC specific gene expression was obtained when promoters of *fms*-like tyrosine kinase-1 (FLT-1) [108], intercellular adhesion molecule (ICAM) -2 [109], angiopoietin-2 [110], eNOS [111], vascular cell adhesion molecule-1 (VCAM-1) [112], von Willebrand factor [113], tyro‐ sine kinase with immunoglobulin and epidermal growth factor homology domains (Tie) [114], kinase-like domain receptor [115] were used in transgenic mouse models. Other ECspecific promoters include the oxidized LDL receptor LOX-1 [116] and ICAM-1 [117], which exhibit upregulation upon cytokine stimulation, a possible advantage depending on the ap‐ plication in inflammatory conditions [118]. With the possible exception of the mouse Tie-2 and human ICAM-2 genes, most of EC–specific promoters tested to-date have been shown to direct expression in distinct and restricted sites of the vascular tree [119]. A combination approach of the Tie2 promoter and enhancer (Tshort) by Minami and collegues [119] direct‐ ed widespread EC expression *in vivo.*

Another challenge was in generating an EC-specific promoter with comparable efficiency as the CMV promoter. White *et al.* [120] examined several novel Ad expression cassettes for EC-specific gene transfer with CMV, Tshort, ICAM-2, ICAM-1, FLT-1 promoters, respective‐ ly and found that LOX-1 promoter elements significantly increased reporter gene expression in carotid arteries compared to other promoters. The efficacy of these novel expression cas‐ settes in large animal models have yet to be established.

An increasingly important area to in-tissue specific targeting is to engineer viral vectors Ads and AAVs with altered cell tropisms to narrow or broaden its efficiency in tissues refractory to infection [19, 121]. Non-genetic approaches typically utilize bispecific antibodies that both neutralize wild-type virus tropism and provide a new cell binding capacity [122]. For genet‐ ic targeting strategies, the virus capsid are engineered to express foreign ligands that target selected receptors in the absence or presence of additional modification to ablate the natural tropism of the virus [122, 123]. Ad homing to target endothelial cells at specific sites of the body can be achieved by deleting the ability of the virus to interact with its natural receptor, Coxsackievirus and adenovirus receptor (CAR), and a simultaneous addition of a ligand that directs the virus to the angiotensin converting enzyme on the ECs. Retargeting of AAV-2 with novel peptides could increase both transduction efficiency and selectivity [124] in vascular ECs [125] and SMCs [126] *in vitro.*

#### **4.3. Challenges associated with the mode and route of gene delivery**

#### *4.3.1. Systemic gene delivery*

with its natural locus in the host genome, thereby ensuring correct transcription. Also, after homologous recombination, the targeted modification of the chromosomal locus is stable, whereas randomly integrated sequences might be lost over time. In their seminal paper, Russel and Hirata [97] reported that DNA vectors based on the AAV could target homolo‐ gous chromosomal DNA sequences and allow high-fidelity, non-mutagenic gene repair in a host cell. Although the laborious vector design and low transfection efficiencies of AAV vec‐ tors compared to the other viral vectors still remains a concern, statistical information neatly outlines the advantage of rAAV gene replacement system over standard viral vectors, which

The promiscuous tropism of vectors resulting in high-level transgene expression in multiple tissues is another major challenge in vascular gene therapy. After systemic application, most viral vectors are trapped by the liver, hampering delivery to target CV tissues. Approaches to restrict gene delivery to desired cell types *in vivo* relied mostly on cell surface targeting or

The *cis*-acting regulatory elements of the SM (smooth muscle)22α [98-100], telokin [101], smooth muscle myosin heavy chain [102], smooth muscle α- [100] and γ-actin [103], and desmin [104] genes have been shown to direct reporter gene expression to smooth muscle tissues in transgenic mice. In our studies, specific gene transfer to the SMC layer was ach‐ ieved in swine coronary and peripheral arteries using SM22α promoter in AAV [17]. Al‐ though the efficiency of transduction was low when compared to a similar study using AAV vectors with cytomegalovirus (CMV) promoter [105], the use of SM22α promoter caused specific transduction of SMCs *in vivo*. An interesting approach to enhance the transduction efficiency of SM22α -containing plasmid was to incorporate chimeric transcriptional cas‐ settes containing a SM-myosin heavy chain enhancer element combined with the SM22α promoter [106]. The transfection levels obtained using these chimeric constructs in Ad vec‐ tor were similar to that with CMV promoter when tested in rat carotid arteries. Certain DNA nuclear targeting sequences can be used to restrict DNA nuclear import to specific cell types. Young *et al.* [107] improved the efficiency of transduction in SMCs of rat vasculature

EC specific gene expression was obtained when promoters of *fms*-like tyrosine kinase-1 (FLT-1) [108], intercellular adhesion molecule (ICAM) -2 [109], angiopoietin-2 [110], eNOS [111], vascular cell adhesion molecule-1 (VCAM-1) [112], von Willebrand factor [113], tyro‐ sine kinase with immunoglobulin and epidermal growth factor homology domains (Tie) [114], kinase-like domain receptor [115] were used in transgenic mouse models. Other ECspecific promoters include the oxidized LDL receptor LOX-1 [116] and ICAM-1 [117], which exhibit upregulation upon cytokine stimulation, a possible advantage depending on the ap‐ plication in inflammatory conditions [118]. With the possible exception of the mouse Tie-2 and human ICAM-2 genes, most of EC–specific promoters tested to-date have been shown to direct expression in distinct and restricted sites of the vascular tree [119]. A combination

induce strong immune response.

662 Gene Therapy - Tools and Potential Applications

*4.2.2. Tissue-specific targeting*

cell-specific promoters.

using a SMC-specific DNA nuclear targeting sequence.

The vascular system represents an ideal route of substance transport for reaching a specific site for therapeutic intervention. However, in the case of non-viral vectors, which are cation‐ ic polymers in most cases, it has been found that electrostatic interactions between the sulphated glycosaminoglycans in the serum as well as those expressed on the cell surface cause premature release of plasmid DNA leading to its inactivation and extracellular degra‐ dation by serum DNAses [21]. Also, after systemic vascular application, non-specific distri‐ bution of plasmid DNA throughout the vasculature would result in undesired side effects because of accumulation at non-specific sites. Intravenous administration of cationic poly‐ mers resulted in their localization to liver, lung, kidney, and spleen in pigs and rabbits [127-129]. Other barriers to systemic delivery include rapid clearance of the lipoplexes by the reticulo-endothelial system and target specificity.

Most Ad vectors are trapped by the liver, hampering delivery to target CV tissues after sys‐ temic application. Systemic tail vein injection of Ad vector in mice resulted in virus DNA deposition liver, lung, kidney and testis [130]. Furthermore, the use of a heterologous viral promoter CMV in the majority of vascular gene transfers causes systemic organ toxicity re‐ sulting from unrestricted transgene expression [131]. Retargeting of vectors and use of tissue specific promoters offers an enhanced safety profile by reducing ectopic expression in vital organs including the liver and lungs.

#### *4.3.2. Endovascular gene delivery*

Endovascular catheter-based gene delivery allows localization of vectors to the vessel wall and has the advantage that smaller quantities of viral vectors can be used when compared to those used in systemic delivery. The localized delivery minimizes widespread bio-distribu‐ tion of vectors and simultaneously increases the local vector concentration. Several catheters are used for vascular gene delivery [132], and the efficiency of gene transfer depends on multiple physical parameters during the delivery process, including balloon pressure, vessel wall exposure time, concentration, and injection force [133]. Diffusive balloon catheters that include double balloon, channel, microporous and hydrogel balloons, facilitate passive dif‐ fusion of the vector to reach only the innermost layers of the artery (intima and inner media) [134]. Although this system has the advantage of causing relatively minor damage to the vessel media and intima, the major drawbacks include tissue ischemia caused due to blood flow blockage following balloon inflation and relatively low gene transfection rates owing to the short exposure time to the vessel wall. The pressure-driven balloon catheters [135], like the circumferential needle injection balloon catheter and the porous balloon catheter, are thought to efficiently delivery vectors to the deeper medial and adventitial layers of the ar‐ tery compared to passive diffusion catheters, but they increase the risk of vascular injury. Damage to the endothelial lining promotes SMC proliferation and may lead to restenosis. The localized vascular injury can also cause increased inflammatory response. Iontophoretic catheters, a mechanically assisted injection catheter, enhance the vector penetration across the EC lining by generating an electrical current gradient to drive charged or hydrophilic molecules as deep as the adventitial layer of the artery wall, but depends on the charge, size, and concentration of the delivered compound [136]. Despite the theoretical aspects, in most cases of catheter-based gene transfer the vector is not distributed to the target vessels but to the region of tissue surrounding the target vessel or into the systemic circulation.

rectly into the adventitia bypassing intima and media may facilitate relatively rapid and efficient delivery compared to endovascular approaches [132]. The advantages of perivascu‐ lar gene transfer are that the blood flow and endothelium are not disrupted and the place‐ ment of vector particles within tissues will result in enhanced local transduction efficiency compared to that achievable by endoluminal delivery [142]. Moreover, the local gene deliv‐ ery through this 'outside in' approach has received increased attention due to important findings on the capacity of adventitia to influence neointima formation and vascular remod‐ eling [143]. Localized adventitial delivery of a replication-deficient Ad construct containing a fibroblast-active promoter with the gp19ds portion of NADPH inhibitor was effective in


rat common carotid artery [144]. Shneider *et al.* [145] showed that the infusion of Ad vectors into the carotid artery adventitia achieved recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Further, perivascular approach has been reported to minimize the pro-inflammatory effects of Ad vectors [145]. Adventitial gene delivery are also reported to be performed with silastic or biodegradable collars [146]

The endovascular access is comparatively difficult in the case of coronary arteries, and the numerous side branches will also permit the run-off of the infused volume. An alternative delivery approach for coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries [147]. Transvascular needle injections of Ad vectors to the adventitia and perivascular tissue of coronary arteries have

The immune system has evolved to eliminate foreign material and therefore, constrains the successful use of gene-replacement therapy based on viral vectors. There are several reports that suggest innate and adaptive immune responses to gene transfer [149, 150]. The vector dose, the route of administration, the nature of the transgene, and host-related factors re‐ sponsible for inter-individual variability influence the immune response [151]. The early re‐ sponses involve mechanisms that include the detection of pathogen-associated molecular patterns (PAMPs) present on the viral structural proteins containing the transgene by pat‐ tern recognition receptors (PRRs) on cells of the innate immune system (i.e., macrophages and dendritic cells) and the subsequent elaboration of pro-inflammatory cytokines that can up-regulate later adaptive immune responses [152]. The most studied family of PRRs are the toll-like receptors (TLRs), of which TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 have been im‐ plicated in initiating inflammatory responses to viruses [153]. The adaptive responses can include: the generation of antibodies to the transgene delivery vehicle compromising vector administration, or the generation of antibodies to the transgene product which nullifies transgene expression, or cytotoxicity to vector and/or transgene product which leads to the loss of transduced cells. It also results in a CD8+ memory T cell response that thwarts further

and neointima formation after angioplasty in

http://dx.doi.org/10.5772/54075

665

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

reducing overall vascular superoxide anion O2

which act as reservoirs of the vector.

**4.4. Immunological barriers to gene transfer**

efforts to use the same vector or transgene.

also been reported [148].

Gene eluting stents are attractive alternatives for localized gene delivery as they provide a platform for prolonged gene elution and efficient transduction of opposed arterial walls, es‐ pecially in the treatment of in stent restenosis [132]. Local delivery of naked plasmid DNA encoding for human VEGF-2 via gene-eluting stent could decrease neointima formation while accelerating re-endothelialization in rabbit model [137]. Stents coated with lipoplexes containing eNOS plasmid accelerated re-endothelialization in hypercholesterolemic rabbits [138]. The same research group also demonstrated successful Ad and AAV delivery to the vessel wall by gene eluting stents with no systemic dissemination of the viral vectors [139]. Stents are often coated with synthetic or naturally occurring biopolymers for prolonged re‐ lease of the gene to the vessel wall [140]. Recently, fully biodegradable stents have shown great promise in the treatment of peripheral arterial disease [141]. A combination approach of therapeutic gene delivery and fully biodegradable stents would be a novel approach to gene therapy.

#### *4.3.3. Perivascular gene delivery*

In endovascular approach, most catheters require prolonged total vascular occlusion for effi‐ cient gene delivery to the vasculature increasing the risk of ischemia. Delivery of genes di‐ rectly into the adventitia bypassing intima and media may facilitate relatively rapid and efficient delivery compared to endovascular approaches [132]. The advantages of perivascu‐ lar gene transfer are that the blood flow and endothelium are not disrupted and the place‐ ment of vector particles within tissues will result in enhanced local transduction efficiency compared to that achievable by endoluminal delivery [142]. Moreover, the local gene deliv‐ ery through this 'outside in' approach has received increased attention due to important findings on the capacity of adventitia to influence neointima formation and vascular remod‐ eling [143]. Localized adventitial delivery of a replication-deficient Ad construct containing a fibroblast-active promoter with the gp19ds portion of NADPH inhibitor was effective in reducing overall vascular superoxide anion O2 and neointima formation after angioplasty in rat common carotid artery [144]. Shneider *et al.* [145] showed that the infusion of Ad vectors into the carotid artery adventitia achieved recombinant gene expression at a level equivalent to that achieved by means of intraluminal vector infusion. Further, perivascular approach has been reported to minimize the pro-inflammatory effects of Ad vectors [145]. Adventitial gene delivery are also reported to be performed with silastic or biodegradable collars [146] which act as reservoirs of the vector.

The endovascular access is comparatively difficult in the case of coronary arteries, and the numerous side branches will also permit the run-off of the infused volume. An alternative delivery approach for coronary arteries is the expression of diffusible gene products into the pericardial space surrounding the heart and coronary arteries [147]. Transvascular needle injections of Ad vectors to the adventitia and perivascular tissue of coronary arteries have also been reported [148].

#### **4.4. Immunological barriers to gene transfer**

*4.3.2. Endovascular gene delivery*

664 Gene Therapy - Tools and Potential Applications

gene therapy.

*4.3.3. Perivascular gene delivery*

Endovascular catheter-based gene delivery allows localization of vectors to the vessel wall and has the advantage that smaller quantities of viral vectors can be used when compared to those used in systemic delivery. The localized delivery minimizes widespread bio-distribu‐ tion of vectors and simultaneously increases the local vector concentration. Several catheters are used for vascular gene delivery [132], and the efficiency of gene transfer depends on multiple physical parameters during the delivery process, including balloon pressure, vessel wall exposure time, concentration, and injection force [133]. Diffusive balloon catheters that include double balloon, channel, microporous and hydrogel balloons, facilitate passive dif‐ fusion of the vector to reach only the innermost layers of the artery (intima and inner media) [134]. Although this system has the advantage of causing relatively minor damage to the vessel media and intima, the major drawbacks include tissue ischemia caused due to blood flow blockage following balloon inflation and relatively low gene transfection rates owing to the short exposure time to the vessel wall. The pressure-driven balloon catheters [135], like the circumferential needle injection balloon catheter and the porous balloon catheter, are thought to efficiently delivery vectors to the deeper medial and adventitial layers of the ar‐ tery compared to passive diffusion catheters, but they increase the risk of vascular injury. Damage to the endothelial lining promotes SMC proliferation and may lead to restenosis. The localized vascular injury can also cause increased inflammatory response. Iontophoretic catheters, a mechanically assisted injection catheter, enhance the vector penetration across the EC lining by generating an electrical current gradient to drive charged or hydrophilic molecules as deep as the adventitial layer of the artery wall, but depends on the charge, size, and concentration of the delivered compound [136]. Despite the theoretical aspects, in most cases of catheter-based gene transfer the vector is not distributed to the target vessels but to

the region of tissue surrounding the target vessel or into the systemic circulation.

Gene eluting stents are attractive alternatives for localized gene delivery as they provide a platform for prolonged gene elution and efficient transduction of opposed arterial walls, es‐ pecially in the treatment of in stent restenosis [132]. Local delivery of naked plasmid DNA encoding for human VEGF-2 via gene-eluting stent could decrease neointima formation while accelerating re-endothelialization in rabbit model [137]. Stents coated with lipoplexes containing eNOS plasmid accelerated re-endothelialization in hypercholesterolemic rabbits [138]. The same research group also demonstrated successful Ad and AAV delivery to the vessel wall by gene eluting stents with no systemic dissemination of the viral vectors [139]. Stents are often coated with synthetic or naturally occurring biopolymers for prolonged re‐ lease of the gene to the vessel wall [140]. Recently, fully biodegradable stents have shown great promise in the treatment of peripheral arterial disease [141]. A combination approach of therapeutic gene delivery and fully biodegradable stents would be a novel approach to

In endovascular approach, most catheters require prolonged total vascular occlusion for effi‐ cient gene delivery to the vasculature increasing the risk of ischemia. Delivery of genes di‐ The immune system has evolved to eliminate foreign material and therefore, constrains the successful use of gene-replacement therapy based on viral vectors. There are several reports that suggest innate and adaptive immune responses to gene transfer [149, 150]. The vector dose, the route of administration, the nature of the transgene, and host-related factors re‐ sponsible for inter-individual variability influence the immune response [151]. The early re‐ sponses involve mechanisms that include the detection of pathogen-associated molecular patterns (PAMPs) present on the viral structural proteins containing the transgene by pat‐ tern recognition receptors (PRRs) on cells of the innate immune system (i.e., macrophages and dendritic cells) and the subsequent elaboration of pro-inflammatory cytokines that can up-regulate later adaptive immune responses [152]. The most studied family of PRRs are the toll-like receptors (TLRs), of which TLR2, TLR3, TLR4, TLR7, TLR8 and TLR9 have been im‐ plicated in initiating inflammatory responses to viruses [153]. The adaptive responses can include: the generation of antibodies to the transgene delivery vehicle compromising vector administration, or the generation of antibodies to the transgene product which nullifies transgene expression, or cytotoxicity to vector and/or transgene product which leads to the loss of transduced cells. It also results in a CD8+ memory T cell response that thwarts further efforts to use the same vector or transgene.

Ad vector particles can elicit strong innate and adaptive immune responses. The interplay of both systems activates CD4+ and CD8+ T cells and B cells as well as facilitates the induction of transgene-specific immune responses. The innate immune responses after systemic ad‐ ministration of Ad vectors are due to several processes: complement system activation, ana‐ phylotoxin release, macrophage activation, release of cytokines and chemokines, including Interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, macrophage inhibitory protein-2, and RANTES (regulated and normal T cell expressed and secreted); EC activation, generalized transcriptome dysregulation in multiple tissues, activation of macrophages and dendritic cells, mobilization of granulocyte and mast cells, and thrombocytopenia [154]. These re‐ sponses are due to activation of multiple PRRs including RIG-I-like receptors and Toll-like receptors: TLR-2, TLR-4 and TLR-9 [155]. *In vivo* administration of higher doses of Ad vec‐ tors can result in one or all of these innate responses or may even lead to mortality in small animal models [156]. Ad infection of ECs is followed by expression of adhesion molecules such as ICAM-1 and VCAM-1 leading to increased leukocyte infiltration within transduced tissues [157]. Kupffer cells, the resident macrophages of the liver, rapidly scavenge and elim‐ inate Ad5-based vectors from the circulation in mice [158], and this interaction contributes to the induction of pro-inflammatory cytokines and chemokines [159]. It has been reported that increasing the dose of Ad vector would probably fail to increase transgene expression, as the CAR adenoviral receptors would become saturated; in addition, the higher dose would induce a stronger inflammatory response responsible for increased elimination of the infected cells expressing the transgene [151].

mune response is to modify the route of delivery of the vector. In the adventitial delivery of Ad vectors to rabbit carotid arteries, recombinant gene expression was achieved at a level equivalent to that achieved by intraluminal vector infusion. Despite the generation of a sys‐ temic immune response, adventitial infusion had no detectable pathologic effects on the vas‐

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

667

Pre-existing immunity due to neutralizing antibodies against endemic Ad serotypes in hu‐ man populations can contribute to pre-existing Ad specific adaptive immune responses [154]. These cellular responses may be more challenging than humoral immune responses, as these cellular adaptive immune responses to Ads have been shown to recognize multiple diverse, cross-clade Ad serotypes subsequent to exposure to only a single Ad serotype [154]. Arterial gene transfer with type 5 Ad vectors did not cause significant levels of gene expres‐ sion in the majority of humans. Both immune-suppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers

The innate immune response to the AAV capsid has received limited attention due to the minimal responses that AAV2 elicits [162]. According to recent reports by Herzog and oth‐ ers [165], innate immune system also plays important roles in activation of immunity by AAV mediated gene transfer, both in inducing the initial response to the vector and in pro‐ moting a deleterious adaptive immune responses. The initial innate immune responses were mediated by the TLR9-MyD88 pathway via a traditional NF-κB pathway to induce type 1 interferon production. Subsequently, alternative NF-κB pathway is triggered, prompting adaptive immune responses [166]. *In vivo*, intravenous injection of AAV-lacZ rapidly indu‐ ces the expression of messenger RNAs (mRNAs) for the cytokines TNF-α, RANTES, inter‐ feron-γ-induced protein 10, macrophage inflammatory protein(MIP)-1β, monocyte chemotactic protein-1, and MIP-2. However, this effect lasts only 6 h, compared to more than 24 h with Ad infection [151]. The adaptive cell-mediated response is far weaker with AAV vectors than with adenoviral vectors probably due to the inability of AAVs to efficient‐ ly infect APC, including dendritic cells and macrophages. AAV vectors may be capable of infecting immature dendritic cells, but only when large doses of vector are used. In addition, even though a modest amount of dendritic cells are present at sites of AAV infection *in vivo,* they usually fail to induce a T-cell response of sufficient magnitude to eliminate the infected

cells and, therefore, to decrease the duration of transgene expression [151].

Cytotoxic T-cell responses to AAV capsid antigen especially in patients with pre-existing neutralizing antibodies against AAV remain a major road block to achieve persistent thera‐ peutic correction for clinical application. Natural, asymptomatic AAV infection in humans is common, and it estimates that up to 80% of humans possess neutralizing antibodies to some AAV serotypes, especially AAV-2 [167]. Recently, multiple serotypes of AAV in addition to AAV2 have been developed; these serotypes carry different capsid proteins and exhibit dif‐ ferent tropism towards different organs [18]. However, changing serotypes may only lead to partial success due to the strong conservation of immune-dominant capsid epitopes in AAVs. In patients with high titers of neutralizing antibodies to gene therapy vectors such as AAV and Ad vectors, IgGs can be removed from blood by plasmapheresis, double filtration

cular intima or media [145]

to Ad-mediated arterial gene transfer [164].

Ad-based gene transfers can be hindered due to adaptive immune responses to the virus or the transgene it encodes. Ad viruses can induce a cytotoxic T-cell response as well as infil‐ tration by CD4+ and CD8+ T cells. The mechanism involves internalization and priming by dendritic cells of capsid antigens associated with Class II Major histocompatibility complex (MHC) antigens, presentation of these antigens to CD4+ T cells, which become activated, and in turn CD8+ T cell activation by these CD4+ T cells [151]. These adaptive immune re‐ sponses can limit the duration of transgene expression, and/or limit the ability to re-admin‐ ister the vector.

Development of new large capacity or gutless (devoid of all viral genes) vectors [160] or modification of capsid sequences [161] are a few of the various strategies devised to reduce the immunogenicity of the Ad viral vectors. Adaptive immunity against these vectors has been substantially reduced through the development of helper-dependent Ad vectors that contain no Ad genes. However, these gutless Ad vectors can efficiently transduce antigen presenting cells (APCs) [162], which readily triggered innate immune responses and further augmented the induction of adaptive immune responses to the transgene product. This problem led to the introduction of tissue-specific promoters in gutless Ad vectors to restrict transgene expression in target cells but not in APCs [162]. Genome modification, capsid modification by Ad capsid-display of immuno-evasive proteins, chimeric Ad vectors and Ad vectors derived from alternative Ad serotypes are few techniques adopted for eluding Ad vector immunity [161]. The tropism modification strategies for targeted gene delivery using Ad vectors have been extensively reviewed [163]. Another method to decrease the im‐ mune response is to modify the route of delivery of the vector. In the adventitial delivery of Ad vectors to rabbit carotid arteries, recombinant gene expression was achieved at a level equivalent to that achieved by intraluminal vector infusion. Despite the generation of a sys‐ temic immune response, adventitial infusion had no detectable pathologic effects on the vas‐ cular intima or media [145]

Ad vector particles can elicit strong innate and adaptive immune responses. The interplay of both systems activates CD4+ and CD8+ T cells and B cells as well as facilitates the induction of transgene-specific immune responses. The innate immune responses after systemic ad‐ ministration of Ad vectors are due to several processes: complement system activation, ana‐ phylotoxin release, macrophage activation, release of cytokines and chemokines, including Interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, macrophage inhibitory protein-2, and RANTES (regulated and normal T cell expressed and secreted); EC activation, generalized transcriptome dysregulation in multiple tissues, activation of macrophages and dendritic cells, mobilization of granulocyte and mast cells, and thrombocytopenia [154]. These re‐ sponses are due to activation of multiple PRRs including RIG-I-like receptors and Toll-like receptors: TLR-2, TLR-4 and TLR-9 [155]. *In vivo* administration of higher doses of Ad vec‐ tors can result in one or all of these innate responses or may even lead to mortality in small animal models [156]. Ad infection of ECs is followed by expression of adhesion molecules such as ICAM-1 and VCAM-1 leading to increased leukocyte infiltration within transduced tissues [157]. Kupffer cells, the resident macrophages of the liver, rapidly scavenge and elim‐ inate Ad5-based vectors from the circulation in mice [158], and this interaction contributes to the induction of pro-inflammatory cytokines and chemokines [159]. It has been reported that increasing the dose of Ad vector would probably fail to increase transgene expression, as the CAR adenoviral receptors would become saturated; in addition, the higher dose would induce a stronger inflammatory response responsible for increased elimination of the

Ad-based gene transfers can be hindered due to adaptive immune responses to the virus or the transgene it encodes. Ad viruses can induce a cytotoxic T-cell response as well as infil‐ tration by CD4+ and CD8+ T cells. The mechanism involves internalization and priming by dendritic cells of capsid antigens associated with Class II Major histocompatibility complex (MHC) antigens, presentation of these antigens to CD4+ T cells, which become activated, and in turn CD8+ T cell activation by these CD4+ T cells [151]. These adaptive immune re‐ sponses can limit the duration of transgene expression, and/or limit the ability to re-admin‐

Development of new large capacity or gutless (devoid of all viral genes) vectors [160] or modification of capsid sequences [161] are a few of the various strategies devised to reduce the immunogenicity of the Ad viral vectors. Adaptive immunity against these vectors has been substantially reduced through the development of helper-dependent Ad vectors that contain no Ad genes. However, these gutless Ad vectors can efficiently transduce antigen presenting cells (APCs) [162], which readily triggered innate immune responses and further augmented the induction of adaptive immune responses to the transgene product. This problem led to the introduction of tissue-specific promoters in gutless Ad vectors to restrict transgene expression in target cells but not in APCs [162]. Genome modification, capsid modification by Ad capsid-display of immuno-evasive proteins, chimeric Ad vectors and Ad vectors derived from alternative Ad serotypes are few techniques adopted for eluding Ad vector immunity [161]. The tropism modification strategies for targeted gene delivery using Ad vectors have been extensively reviewed [163]. Another method to decrease the im‐

infected cells expressing the transgene [151].

666 Gene Therapy - Tools and Potential Applications

ister the vector.

Pre-existing immunity due to neutralizing antibodies against endemic Ad serotypes in hu‐ man populations can contribute to pre-existing Ad specific adaptive immune responses [154]. These cellular responses may be more challenging than humoral immune responses, as these cellular adaptive immune responses to Ads have been shown to recognize multiple diverse, cross-clade Ad serotypes subsequent to exposure to only a single Ad serotype [154]. Arterial gene transfer with type 5 Ad vectors did not cause significant levels of gene expres‐ sion in the majority of humans. Both immune-suppression and further engineering of the vector genome to decrease expression of viral genes show promise in circumventing barriers to Ad-mediated arterial gene transfer [164].

The innate immune response to the AAV capsid has received limited attention due to the minimal responses that AAV2 elicits [162]. According to recent reports by Herzog and oth‐ ers [165], innate immune system also plays important roles in activation of immunity by AAV mediated gene transfer, both in inducing the initial response to the vector and in pro‐ moting a deleterious adaptive immune responses. The initial innate immune responses were mediated by the TLR9-MyD88 pathway via a traditional NF-κB pathway to induce type 1 interferon production. Subsequently, alternative NF-κB pathway is triggered, prompting adaptive immune responses [166]. *In vivo*, intravenous injection of AAV-lacZ rapidly indu‐ ces the expression of messenger RNAs (mRNAs) for the cytokines TNF-α, RANTES, inter‐ feron-γ-induced protein 10, macrophage inflammatory protein(MIP)-1β, monocyte chemotactic protein-1, and MIP-2. However, this effect lasts only 6 h, compared to more than 24 h with Ad infection [151]. The adaptive cell-mediated response is far weaker with AAV vectors than with adenoviral vectors probably due to the inability of AAVs to efficient‐ ly infect APC, including dendritic cells and macrophages. AAV vectors may be capable of infecting immature dendritic cells, but only when large doses of vector are used. In addition, even though a modest amount of dendritic cells are present at sites of AAV infection *in vivo,* they usually fail to induce a T-cell response of sufficient magnitude to eliminate the infected cells and, therefore, to decrease the duration of transgene expression [151].

Cytotoxic T-cell responses to AAV capsid antigen especially in patients with pre-existing neutralizing antibodies against AAV remain a major road block to achieve persistent thera‐ peutic correction for clinical application. Natural, asymptomatic AAV infection in humans is common, and it estimates that up to 80% of humans possess neutralizing antibodies to some AAV serotypes, especially AAV-2 [167]. Recently, multiple serotypes of AAV in addition to AAV2 have been developed; these serotypes carry different capsid proteins and exhibit dif‐ ferent tropism towards different organs [18]. However, changing serotypes may only lead to partial success due to the strong conservation of immune-dominant capsid epitopes in AAVs. In patients with high titers of neutralizing antibodies to gene therapy vectors such as AAV and Ad vectors, IgGs can be removed from blood by plasmapheresis, double filtration plasmapheresis and immune-absorbant plasmapheresis before gene transfer procedure to increase transduction rates of target tissues [168].

**Author details**

on.edu

**References**

213-222.

119-127.

1876-1883.

Divya Pankajakshan\*

and Devendra K. Agrawal

on University School of Medicine, Omaha, NE, USA

\*Address all correspondence to: DivyaPankajakshan@creighton.edu and dkagr@creight‐

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

669

Department of Biomedical Sciences and Center for Clinical & Translational Science Creight‐

[1] Roger VL, Go AS, Lloyd-Jones DM, Benjamin EJ, Berry JD, Borden WB, Bravata DM, Dai S, Ford ES, Fox CS, Fullerton HJ, Gillespie C, Hailpern SM, Heit JA, Howard VJ, Kissela BM, Kittner SJ, Lackland DT, Lichtman JH, Lisabeth LD, Makuc DM, Marcus GM, Marelli A, Matchar DB, Moy CS, Mozaffarian D, Mussolino ME, Nichol G, Paynter NP, Soliman EZ, Sorlie PD, Sotoodehnia N, Turan TN, Virani SS, Wong ND, Woo D, Turner MB. Heart disease and stroke statistics--2012 update: a report from

[2] Brewster LP, Brey EM, Greisler HP. Cardiovascular gene delivery: The good road is

[3] Yla-Herttuala S, Martin JF. Cardiovascular gene therapy. Lancet. 2000;355(9199)

[4] Pankajakshan D, Agrawal DK. Scaffolds in tissue engineering of blood vessels. Can J

[5] Williams PD, Ranjzad P, Kakar SJ, Kingston PA. Development of viral vectors for use

[6] Kahn ML, Lee SW, Dichek DA. Optimization of retroviral vector-mediated gene

[7] Inaba M, Toninelli E, Vanmeter G, Bender JR, Conte MS. Retroviral gene transfer: ef‐

[8] Zelenock JA, Welling TH, Sarkar R, Gordon DG, Messina LM. Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers. J Vasc Surg. 1997;26(1)

[9] Yu H, Eton D, Wang Y, Kumar SR, Tang L, Terramani TT, Benedict C, Hung G, An‐ derson WF. High efficiency in vitro gene transfer into vascular tissues using a pseu‐ dotyped retroviral vector without pseudotransduction. Gene Ther. 1999;6(11)

the American Heart Association. Circulation. 2012;125(1) e2-e220.

awaiting. Adv Drug Deliv Rev. 2006;58(4) 604-629.

in cardiovascular gene therapy. Viruses. 2010;2(2) 334-371.

transfer into endothelial cells in vitro. Circ Res. 1992;71(6) 1508-1517.

fects on endothelial cell phenotype. J Surg Res. 1998;78(1) 31-36.

Physiol Pharmacol. 2010;88(9) 855-873.

Plasmids alone or in combination with naked bacterial DNA can stimulate innate immune responses [152]. Plasmids, composed chiefly of bacterial DNA, contain far greater amounts of unmethylated CpG motifs than do the DNA in eukaryotic cells. DNA devoid of CpG mo‐ tifs does not induce proinflammatory cytokine synthesis by macrophages *in vitro*. TLR 9 rec‐ ognizes the unmethylated CpG motifs in immunostimulatory sequences of bacterial DNA which activate the cells responsible for innate immune responses (for example macrophag‐ es) after penetration of bacteria into the body [169]. Indeed, elimination or methylation of these sequences could be a method for suppressing the inflammatory response induced by unmethylated CpG sequences in plasmids [168].

#### **5. Conclusion**

An enormous amount of research has been done in the past few decades on the choice of the therapeutic gene, vectors and delivery approaches for effective vascular gene transfer. The low efficiency of gene transfer to vascular tissues still remains a major drawback.. Of the several approaches used so far, Ad-mediated gene transfer has been found to be the most efficient when compared to other methods. However, gene transfer using viral vectors has often caused ectopic expression and also an increased immunological response. The use of tropism modified vectors and plasmids with cell specific promoters are solutions for reduc‐ ing the ectopic expression. Using "gutless" viral vectors devoid of the immunogenic regions of viral plasmid is an attractive option to reduce the immunologic response, but we have to wait for more *in vivo* data using these third-generation vectors to reach a conclusive result [160]. Non-viral methods have more barriers to overcome to successfully transfect the cell; however, with the advent of innovative technologies like nanobots [170], stimuli responsive polymers [171], novel erythrocyte based carriers [172], magnetically targeted delivery [173] and focused *in vivo* plasmid DNA delivery to the vascular wall via intravascular ultrasound destruction of microbubbles [174]; we expect enhanced transgene expression in vascular cells in future studies. This will also be a possible solution to tackle with the immune re‐ sponse associated with the viral vectors. Site specific biodegradable stent based gene deliv‐ ery approach [175] and modified percutaneous gene delivery systems offer new opportunities for enhanced gene delivery to vascular cells.

#### **Acknowledgements**

This work was supported by research grants from the National Institute of Health, R01 HL104516, R01 HL112597 and R01 HL116042 to DKA. The content is solely the responsibili‐ ty of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health. Both authors declare no con‐ flict of any sort with the content in this paper.

#### **Author details**

plasmapheresis and immune-absorbant plasmapheresis before gene transfer procedure to

Plasmids alone or in combination with naked bacterial DNA can stimulate innate immune responses [152]. Plasmids, composed chiefly of bacterial DNA, contain far greater amounts of unmethylated CpG motifs than do the DNA in eukaryotic cells. DNA devoid of CpG mo‐ tifs does not induce proinflammatory cytokine synthesis by macrophages *in vitro*. TLR 9 rec‐ ognizes the unmethylated CpG motifs in immunostimulatory sequences of bacterial DNA which activate the cells responsible for innate immune responses (for example macrophag‐ es) after penetration of bacteria into the body [169]. Indeed, elimination or methylation of these sequences could be a method for suppressing the inflammatory response induced by

An enormous amount of research has been done in the past few decades on the choice of the therapeutic gene, vectors and delivery approaches for effective vascular gene transfer. The low efficiency of gene transfer to vascular tissues still remains a major drawback.. Of the several approaches used so far, Ad-mediated gene transfer has been found to be the most efficient when compared to other methods. However, gene transfer using viral vectors has often caused ectopic expression and also an increased immunological response. The use of tropism modified vectors and plasmids with cell specific promoters are solutions for reduc‐ ing the ectopic expression. Using "gutless" viral vectors devoid of the immunogenic regions of viral plasmid is an attractive option to reduce the immunologic response, but we have to wait for more *in vivo* data using these third-generation vectors to reach a conclusive result [160]. Non-viral methods have more barriers to overcome to successfully transfect the cell; however, with the advent of innovative technologies like nanobots [170], stimuli responsive polymers [171], novel erythrocyte based carriers [172], magnetically targeted delivery [173] and focused *in vivo* plasmid DNA delivery to the vascular wall via intravascular ultrasound destruction of microbubbles [174]; we expect enhanced transgene expression in vascular cells in future studies. This will also be a possible solution to tackle with the immune re‐ sponse associated with the viral vectors. Site specific biodegradable stent based gene deliv‐ ery approach [175] and modified percutaneous gene delivery systems offer new

This work was supported by research grants from the National Institute of Health, R01 HL104516, R01 HL112597 and R01 HL116042 to DKA. The content is solely the responsibili‐ ty of the authors and does not necessarily represent the official views of the National Heart, Lung, and Blood Institute or the National Institutes of Health. Both authors declare no con‐

increase transduction rates of target tissues [168].

668 Gene Therapy - Tools and Potential Applications

unmethylated CpG sequences in plasmids [168].

opportunities for enhanced gene delivery to vascular cells.

flict of any sort with the content in this paper.

**5. Conclusion**

**Acknowledgements**

Divya Pankajakshan\* and Devendra K. Agrawal

\*Address all correspondence to: DivyaPankajakshan@creighton.edu and dkagr@creight‐ on.edu

Department of Biomedical Sciences and Center for Clinical & Translational Science Creight‐ on University School of Medicine, Omaha, NE, USA

#### **References**


[10] Dishart KL, Denby L, George SJ, Nicklin SA, Yendluri S, Tuerk MJ, Kelley MP, Dona‐ hue BA, Newby AC, Harding T, Baker AH. Third-generation lentivirus vectors effi‐ ciently transduce and phenotypically modify vascular cells: implications for gene therapy. J Mol Cell Cardiol. 2003;35(7) 739-748.

[22] Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent prog‐

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

671

[23] Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM. Accelerated restitution of endothelial integrity and endothelium-dependent function

[24] Lawrie A, Brisken AF, Francis SE, Cumberland DC, Crossman DC, Newman CM. Microbubble-enhanced ultrasound for vascular gene delivery. Gene Ther. 2000;7(23)

[25] Nishi T, Yoshizato K, Yamashiro S, Takeshima H, Sato K, Hamada K, Kitamura I, Yoshimura T, Saya H, Kuratsu J, Ushio Y. High-efficiency in vivo gene transfer using intraarterial plasmid DNA injection following in vivo electroporation. Cancer Res.

[26] Williams PD, Kingston PA. Plasmid-mediated gene therapy for cardiovascular dis‐

[27] Parkes R, Meng QH, Siapati KE, McEwan JR, Hart SL. High efficiency transfection of porcine vascular cells in vitro with a synthetic vector system. J Gene Med. 2002;4(3)

[28] Kader KN, Sweany JM, Bellamkonda RV. Cationic lipid-mediated transfection of bo‐ vine aortic endothelial cells inhibits their attachment. J Biomed Mater Res. 2002;60(3)

[29] Wang T, Upponi JR, Torchilin VP. Design of multifunctional non-viral gene vectors to overcome physiological barriers: dilemmas and strategies. Int J Pharm. 2012;427(1)

[30] Zaric V, Weltin D, Erbacher P, Remy JS, Behr JP, Stephan D. Effective polyethyleni‐ mine-mediated gene transfer into human endothelial cells. J Gene Med. 2004;6(2)

[31] Godbey WT, Wu KK, Mikos AG. Poly(ethylenimine)-mediated gene delivery affects

[32] Turunen MP, Hiltunen MO, Ruponen M, Virkamaki L, Szoka FC, Jr., Urtti A, Yla-Herttuala S. Efficient adventitial gene delivery to rabbit carotid artery with cationic

[33] Brito L, Little S, Langer R, Amiji M. Poly(beta-amino ester) and cationic phospholi‐ pid-based lipopolyplexes for gene delivery and transfection in human aortic endo‐

[34] Chen SL, Zhu CC, Liu YQ, Tang LJ, Yi L, Yu BJ, Wang DJ. Mesenchymal stem cells genetically modified with the angiopoietin-1 gene enhanced arteriogenesis in a por‐

cine model of chronic myocardial ischaemia. J Int Med Res. 2009;37(1) 68-78.

thelial and smooth muscle cells. Biomacromolecules. 2008;9(4) 1179-1187.

endothelial cell function and viability. Biomaterials. 2001;22(5) 471-480.

polymer-plasmid complexes. Gene Ther. 1999;6(1) 6-11.

after phVEGF165 gene transfer. Circulation. 1996;94(12) 3291-3302.

ress. Aaps J. 2009;11(4) 671-681.

2023-2027.

292-299.

405-410.

3-20.

176-184.

1996;56(5) 1050-1055.

ease. Cardiovasc Res. 2011;91(4) 565-576.


[22] Al-Dosari MS, Gao X. Nonviral gene delivery: principle, limitations, and recent prog‐ ress. Aaps J. 2009;11(4) 671-681.

[10] Dishart KL, Denby L, George SJ, Nicklin SA, Yendluri S, Tuerk MJ, Kelley MP, Dona‐ hue BA, Newby AC, Harding T, Baker AH. Third-generation lentivirus vectors effi‐ ciently transduce and phenotypically modify vascular cells: implications for gene

[11] Cefai D, Simeoni E, Ludunge KM, Driscoll R, von Segesser LK, Kappenberger L, Vas‐ salli G. Multiply attenuated, self-inactivating lentiviral vectors efficiently transduce human coronary artery cells in vitro and rat arteries in vivo. J Mol Cell Cardiol.

[12] Bonci D, Cittadini A, Latronico MV, Borello U, Aycock JK, Drusco A, Innocenzi A, Follenzi A, Lavitrano M, Monti MG, Ross J, Jr., Naldini L, Peschle C, Cossu G, Con‐ dorelli G. 'Advanced' generation lentiviruses as efficient vectors for cardiomyocyte

[13] Harvey BG, Hackett NR, El-Sawy T, Rosengart TK, Hirschowitz EA, Lieberman MD, Lesser ML, Crystal RG. Variability of human systemic humoral immune responses to adenovirus gene transfer vectors administered to different organs. J Virol. 1999;73(8)

[14] Mastrangeli A, Harvey BG, Yao J, Wolff G, Kovesdi I, Crystal RG, Falck-Pedersen E. "Sero-switch" adenovirus-mediated in vivo gene transfer: circumvention of anti-ade‐ novirus humoral immune defenses against repeat adenovirus vector administration

[15] Wen S, Driscoll RM, Schneider DB, Dichek DA. Inclusion of the E3 region in an ade‐ noviral vector decreases inflammation and neointima formation after arterial gene

[16] Sasano T, Kikuchi K, McDonald AD, Lai S, Donahue JK. Targeted high-efficiency, ho‐ mogeneous myocardial gene transfer. J Mol Cell Cardiol. 2007;42(5) 954-961.

[17] Pankajakshan D, Makinde TO, Gaurav R, Del Core M, Hatzoudis G, Pipinos I, Agrawal DK. Successful transfection of genes using AAV-2/9 vector in swine coro‐

[18] Choi VW, McCarty DM, Samulski RJ. AAV hybrid serotypes: improved vectors for

[19] Kwon I, Schaffer DV. Designer gene delivery vectors: molecular engineering and evolution of adeno-associated viral vectors for enhanced gene transfer. Pharm Res.

[20] Morris VB, Sharma CP. Folate mediated in vitro targeting of depolymerised trime‐ thylated chitosan having arginine functionality. J Colloid Interface Sci. 2010;348(2)

[21] Shim MS, Kwon YJ. Stimuli-responsive polymers and nanomaterials for gene deliv‐ ery and imaging applications. Adv Drug Deliv Rev. 2012;64(11) 1046-1059.

gene transduction in vitro and in vivo. Gene Ther. 2003;10(8) 630-636.

by changing the adenovirus serotype. Hum Gene Ther. 1996;7(1) 79-87.

transfer. Arterioscler Thromb Vasc Biol. 2001;21(11) 1777-1782.

nary and peripheral arteries. J Surg Res. 2012;175(1) 169-175.

gene delivery. Curr Gene Ther. 2005;5(3) 299-310.

therapy. J Mol Cell Cardiol. 2003;35(7) 739-748.

2005;38(2) 333-344.

670 Gene Therapy - Tools and Potential Applications

6729-6742.

2008;25(3) 489-499.

360-368.


[35] Das H, George JC, Joseph M, Das M, Abdulhameed N, Blitz A, Khan M, Sakthivel R, Mao HQ, Hoit BD, Kuppusamy P, Pompili VJ. Stem cell therapy with overexpressed VEGF and PDGF genes improves cardiac function in a rat infarct model. PLoS One. 2009;4(10) e7325.

[47] Levonen AL, Inkala M, Heikura T, Jauhiainen S, Jyrkkanen HK, Kansanen E, Maatta K, Romppanen E, Turunen P, Rutanen J, Yla-Herttuala S. Nrf2 gene transfer induces antioxidant enzymes and suppresses smooth muscle cell growth in vitro and reduces oxidative stress in rabbit aorta in vivo. Arterioscler Thromb Vasc Biol. 2007;27(4)

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

673

[48] Levonen AL, Vahakangas E, Koponen JK, Yla-Herttuala S. Antioxidant gene therapy for cardiovascular disease: current status and future perspectives. Circulation.

[49] Li Q, Bolli R, Qiu Y, Tang XL, Guo Y, French BA. Gene therapy with extracellular su‐ peroxide dismutase protects conscious rabbits against myocardial infarction. Circula‐

[50] Laukkanen MO, Kivela A, Rissanen T, Rutanen J, Karkkainen MK, Leppanen O, Bra‐ sen JH, Yla-Herttuala S. Adenovirus-mediated extracellular superoxide dismutase gene therapy reduces neointima formation in balloon-denuded rabbit aorta. Circula‐

[51] Woo YJ, Zhang JC, Vijayasarathy C, Zwacka RM, Englehardt JF, Gardner TJ, Swee‐ ney HL. Recombinant adenovirus-mediated cardiac gene transfer of superoxide dis‐ mutase and catalase attenuates postischemic contractile dysfunction. Circulation.

[52] Duckers HJ, Boehm M, True AL, Yet SF, San H, Park JL, Clinton Webb R, Lee ME, Nabel GJ, Nabel EG. Heme oxygenase-1 protects against vascular constriction and

[53] Kashyap VS, Santamarina-Fojo S, Brown DR, Parrott CL, Applebaum-Bowden D, Meyn S, Talley G, Paigen B, Maeda N, Brewer HB, Jr. Apolipoprotein E deficiency in mice: gene replacement and prevention of atherosclerosis using adenovirus vectors. J

[54] Tsukamoto K, Tangirala R, Chun SH, Pure E, Rader DJ. Rapid regression of athero‐ sclerosis induced by liver-directed gene transfer of ApoE in ApoE-deficient mice. Ar‐

[55] Greeve J, Jona VK, Chowdhury NR, Horwitz MS, Chowdhury JR. Hepatic gene transfer of the catalytic subunit of the apolipoprotein B mRNA editing enzyme re‐ sults in a reduction of plasma LDL levels in normal and watanabe heritable hyperli‐

[56] Khurana R, Martin JF, Zachary I. Gene therapy for cardiovascular disease: a case for

[57] Granada JF, Ensenat D, Keswani AN, Kaluza GL, Raizner AE, Liu XM, Peyton KJ, Azam MA, Wang H, Durante W. Single perivascular delivery of mitomycin C stimu‐ lates p21 expression and inhibits neointima formation in rat arteries. Arterioscler

741-747.

2008;117(16) 2142-2150.

tion. 2001;103(14) 1893-1898.

tion. 2002;106(15) 1999-2003.

1998;98(19 Suppl) II255-260; discussion II260-251.

terioscler Thromb Vasc Biol. 1999;19(9) 2162-2170.

pidemic rabbits. J Lipid Res. 1996;37(9) 2001-2017.

Thromb Vasc Biol. 2005;25(11) 2343-2348.

cautious optimism. Hypertension. 2001;38(5) 1210-1216.

proliferation. Nat Med. 2001;7(6) 693-698.

Clin Invest. 1995;96(3) 1612-1620.


[47] Levonen AL, Inkala M, Heikura T, Jauhiainen S, Jyrkkanen HK, Kansanen E, Maatta K, Romppanen E, Turunen P, Rutanen J, Yla-Herttuala S. Nrf2 gene transfer induces antioxidant enzymes and suppresses smooth muscle cell growth in vitro and reduces oxidative stress in rabbit aorta in vivo. Arterioscler Thromb Vasc Biol. 2007;27(4) 741-747.

[35] Das H, George JC, Joseph M, Das M, Abdulhameed N, Blitz A, Khan M, Sakthivel R, Mao HQ, Hoit BD, Kuppusamy P, Pompili VJ. Stem cell therapy with overexpressed VEGF and PDGF genes improves cardiac function in a rat infarct model. PLoS One.

[36] Behrendt D, Ganz P. Endothelial function. From vascular biology to clinical applica‐

[37] Griese DP, Achatz S, Batzlsperger CA, Strauch UG, Grumbeck B, Weil J, Riegger GA. Vascular gene delivery of anticoagulants by transplantation of retrovirally-trans‐

[38] Chen L, Wu F, Xia WH, Zhang YY, Xu SY, Cheng F, Liu X, Zhang XY, Wang SM, Tao J. CXCR4 gene transfer contributes to in vivo reendothelialization capacity of endo‐

[39] Ohno N, Itoh H, Ikeda T, Ueyama K, Yamahara K, Doi K, Yamashita J, Inoue M, Ma‐ satsugu K, Sawada N, Fukunaga Y, Sakaguchi S, Sone M, Yurugi T, Kook H, Komeda M, Nakao K. Accelerated reendothelialization with suppressed thrombogenic prop‐ erty and neointimal hyperplasia of rabbit jugular vein grafts by adenovirus-mediated gene transfer of C-type natriuretic peptide. Circulation. 2002;105(14) 1623-1626. [40] Kong D, Melo LG, Mangi AA, Zhang L, Lopez-Ilasaca M, Perrella MA, Liew CC, Pratt RE, Dzau VJ. Enhanced inhibition of neointimal hyperplasia by genetically en‐

gineered endothelial progenitor cells. Circulation. 2004;109(14) 1769-1775.

arteries after angioplasty. Hum Gene Ther. 2000;11(2) 263-270.

arterial thrombosis in a rabbit model. Circ Res. 1999;84(1) 84-92.

transfer of cyclooxygenase gene. Circulation. 1996;93(1) 10-17.

[41] Laitinen M, Hartikainen J, Hiltunen MO, Eranen J, Kiviniemi M, Narvanen O, Maki‐ nen K, Manninen H, Syvanne M, Martin JF, Laakso M, Yla-Herttuala S. Cathetermediated vascular endothelial growth factor gene transfer to human coronary

[42] Channon KM, Annex BH. Antithrombotic strategies in gene therapy. Curr Cardiol

[43] Waugh JM, Yuksel E, Li J, Kuo MD, Kattash M, Saxena R, Geske R, Thung SN, She‐ naq SM, Woo SL. Local overexpression of thrombomodulin for in vivo prevention of

[44] Yin X, Yutani C, Ikeda Y, Enjyoji K, Ishibashi-Ueda H, Yasuda S, Tsukamoto Y, Non‐ ogi H, Kaneda Y, Kato H. Tissue factor pathway inhibitor gene delivery using HVJ-AVE liposomes markedly reduces restenosis in atherosclerotic arteries. Cardiovasc

[45] Rade JJ, Schulick AH, Virmani R, Dichek DA. Local adenoviral-mediated expression of recombinant hirudin reduces neointima formation after arterial injury. Nat Med.

[46] Zoldhelyi P, McNatt J, Xu XM, Loose-Mitchell D, Meidell RS, Clubb FJ, Jr., Buja LM, Willerson JT, Wu KK. Prevention of arterial thrombosis by adenovirus-mediated

duced endothelial progenitor cells. Cardiovasc Res. 2003;58(2) 469-477.

thelial progenitor cells. Cardiovasc Res. 2010;88(3) 462-470.

2009;4(10) e7325.

672 Gene Therapy - Tools and Potential Applications

Rep. 2000;2(1) 34-38.

Res. 2002;56(3) 454-463.

1996;2(3) 293-298.

tions. Am J Cardiol. 2002;90(10C) 40L-48L.


[58] Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-ex‐ pression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery model of balloon angioplasty. J Clin Invest. 1995;96(5) 2260-2268.

[69] Ehsan A, Mann MJ, Dell'Acqua G, Dzau VJ. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

675

[71] Newby AC. Matrix metalloproteinases regulate migration, proliferation, and death of vascular smooth muscle cells by degrading matrix and non-matrix substrates. Cardi‐

[72] Ramirez Correa GA, Zacchigna S, Arsic N, Zentilin L, Salvi A, Sinagra G, Giacca M. Potent inhibition of arterial intimal hyperplasia by TIMP1 gene transfer using AAV

[73] George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in

[74] Johnson TW, Wu YX, Herdeg C, Baumbach A, Newby AC, Karsch KR, Oberhoff M. Stent-based delivery of tissue inhibitor of metalloproteinase-3 adenovirus inhibits ne‐ ointimal formation in porcine coronary arteries. Arterioscler Thromb Vasc Biol.

[75] Guo YH, Gao W, Li Q, Li PF, Yao PY, Chen K. Tissue inhibitor of metalloproteinas‐ es-4 suppresses vascular smooth muscle cell migration and induces cell apoptosis.

[76] Gurjar MV, Sharma RV, Bhalla RC. eNOS gene transfer inhibits smooth muscle cell migration and MMP-2 and MMP-9 activity. Arterioscler Thromb Vasc Biol.

[77] Eefting D, de Vries MR, Grimbergen JM, Karper JC, van Bockel JH, Quax PH. In vivo suppression of vein graft disease by nonviral, electroporation-mediated, gene trans‐ fer of tissue inhibitor of metalloproteinase-1 linked to the amino terminal fragment of urokinase (TIMP-1.ATF), a cell-surface directed matrix metalloproteinase inhibitor. J

[78] Emanueli C, Madeddu P. Angiogenesis gene therapy to rescue ischaemic tissues:

[79] Syed IS, Sanborn TA, Rosengart TK. Therapeutic angiogenesis: a biologic bypass.

[80] Losordo DW, Vale PR, Isner JM. Gene therapy for myocardial angiogenesis. Am

[81] Rosengart TK, Lee LY, Patel SR, Kligfield PD, Okin PM, Hackett NR, Isom OW, Crys‐ tal RG. Six-month assessment of a phase I trial of angiogenic gene therapy for the

achievements and future directions. Br J Pharmacol. 2001;133(7) 951-958.

oligonucleotide gene therapy. J Thorac Cardiovasc Surg. 2001;121(4) 714-722. [70] Luo Z, Garron T, Palasis M, Lu H, Belanger AJ, Scaria A, Vincent KA, Date T, Akita GY, Cheng SH, Barry J, Gregory RJ, Jiang C. Enhancement of Fas ligand-induced in‐ hibition of neointimal formation in rabbit femoral and iliac arteries by coexpression

of p35. Hum Gene Ther. 2001;12(18) 2191-2202.

human saphenous veins. Gene Ther. 1998;5(11) 1552-1560.

ovasc Res. 2006;69(3) 614-624.

2005;25(4) 754-759.

1999;19(12) 2871-2877.

Life Sci. 2004;75(20) 2483-2493.

Vasc Surg. 2010;51(2) 429-437.

Cardiology. 2004;101(1-3) 131-143.

Heart J. 1999;138(2 Pt 2) S132-141.

vectors. Mol Ther. 2004;9(6) 876-884.


[69] Ehsan A, Mann MJ, Dell'Acqua G, Dzau VJ. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg. 2001;121(4) 714-722.

[58] Chang MW, Barr E, Lu MM, Barton K, Leiden JM. Adenovirus-mediated over-ex‐ pression of the cyclin/cyclin-dependent kinase inhibitor, p21 inhibits vascular smooth muscle cell proliferation and neointima formation in the rat carotid artery

[59] McArthur JG, Qian H, Citron D, Banik GG, Lamphere L, Gyuris J, Tsui L, George SE. p27-p16 Chimera: a superior antiproliferative for the prevention of neointimal hyper‐

[60] Tsui LV, Camrud A, Mondesire J, Carlson P, Zayek N, Camrud L, Donahue B, Bauer S, Lin A, Frey D, Rivkin M, Subramanian A, Falotico R, Gyuris J, Schwartz R, McAr‐ thur JG. p27-p16 fusion gene inhibits angioplasty-induced neointimal hyperplasia

[61] Takagi Y. Adenovirus-mediated overexpression of a cyclin-dependent kinase inhibi‐ tor, p57Kip2, suppressed vascular smooth muscle cell proliferation. Hokkaido Igaku

[62] Maillard L, Van Belle E, Smith RC, Le Roux A, Denefle P, Steg G, Barry JJ, Branellec D, Isner JM, Walsh K. Percutaneous delivery of the gax gene inhibits vessel stenosis

[63] Chen L, Daum G, Forough R, Clowes M, Walter U, Clowes AW. Overexpression of human endothelial nitric oxide synthase in rat vascular smooth muscle cells and in

[64] Kopp CW, de Martin R. Gene therapy approaches for the prevention of restenosis.

[65] Yukawa H, Miyatake SI, Saiki M, Takahashi JC, Mima T, Ueno H, Nagata I, Kikuchi H, Hashimoto N. In vitro growth suppression of vascular smooth muscle cells using adenovirus-mediated gene transfer of a truncated form of fibroblast growth factor re‐

[66] Kotani M, Fukuda N, Ando H, Hu WY, Kunimoto S, Saito S, Kanmatsuse K. Chimer‐ ic DNA-RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically in‐ hibits neointima formation in rat carotid artery after balloon injury. Cardiovasc Res.

[67] Ando H, Fukuda N, Kotani M, Yokoyama S, Kunimoto S, Matsumoto K, Saito S, Kanmatsuse K, Mugishima H. Chimeric DNA-RNA hammerhead ribozyme target‐ ing transforming growth factor-beta 1 mRNA inhibits neointima formation in rat car‐

[68] Ahn JD, Morishita R, Kaneda Y, Lee SJ, Kwon KY, Choi SY, Lee KU, Park JY, Moon IJ, Park JG, Yoshizumi M, Ouchi Y, Lee IK. Inhibitory effects of novel AP-1 decoy oli‐ godeoxynucleotides on vascular smooth muscle cell proliferation in vitro and neoin‐

otid artery after balloon injury. Eur J Pharmacol. 2004;483(2-3) 207-214.

timal formation in vivo. Circ Res. 2002;90(12) 1325-1332.

in a rabbit model of balloon angioplasty. Cardiovasc Res. 1997;35(3) 536-546.

model of balloon angioplasty. J Clin Invest. 1995;96(5) 2260-2268.

and coronary artery occlusion. Circ Res. 2001;89(4) 323-328.

balloon-injured carotid artery. Circ Res. 1998;82(8) 862-870.

Curr Vasc Pharmacol. 2004;2(2) 183-189.

ceptor. Atherosclerosis. 1998;141(1) 125-132.

2003;57(1) 265-276.

plasia. Mol Ther. 2001;3(1) 8-13.

674 Gene Therapy - Tools and Potential Applications

Zasshi. 2002;77(3) 221-230.


treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA. Ann Surg. 1999;230(4) 466-470; discussion 470-462.

[94] Young JL, Benoit JN, Dean DA. Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature. Gene Ther. 2003;10(17)

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

677

[95] Vasileva A, Jessberger R. Precise hit: adeno-associated virus in gene targeting. Nat

[96] Vasileva A, Linden RM, Jessberger R. Homologous recombination is required for

[97] Russell DW, Hirata RK. Human gene targeting by viral vectors. Nat Genet. 1998;18(4)

[98] Li L, Miano JM, Mercer B, Olson EN. Expression of the SM22alpha promoter in trans‐ genic mice provides evidence for distinct transcriptional regulatory programs in vas‐

[99] Mack CP, Owens GK. Regulation of smooth muscle alpha-actin expression in vivo is dependent on CArG elements within the 5' and first intron promoter regions. Circ

[100] Mack CP, Thompson MM, Lawrenz-Smith S, Owens GK. Smooth muscle alpha-actin CArG elements coordinate formation of a smooth muscle cell-selective, serum re‐

[101] Hoggatt AM, Simon GM, Herring BP. Cell-specific regulatory modules control ex‐ pression of genes in vascular and visceral smooth muscle tissues. Circ Res.

[102] Madsen CS, Regan CP, Hungerford JE, White SL, Manabe I, Owens GK. Smooth muscle-specific expression of the smooth muscle myosin heavy chain gene in trans‐ genic mice requires 5'-flanking and first intronic DNA sequence. Circ Res. 1998;82(8)

[103] Qian J, Kumar A, Szucsik JC, Lessard JL. Tissue and developmental specific expres‐ sion of murine smooth muscle gamma-actin fusion genes in transgenic mice. Dev

[104] Mericskay M, Parlakian A, Porteu A, Dandre F, Bonnet J, Paulin D, Li Z. An overlap‐ ping CArG/octamer element is required for regulation of desmin gene transcription

[105] Su H, Yeghiazarians Y, Lee A, Huang Y, Arakawa-Hoyt J, Ye J, Orcino G, Grossman W, Kan YW. AAV serotype 1 mediates more efficient gene transfer to pig myocardi‐

[106] Ribault S, Neuville P, Mechine-Neuville A, Auge F, Parlakian A, Gabbiani G, Paulin D, Calenda V. Chimeric smooth muscle-specific enhancer/promoters: valuable tools for adenovirus-mediated cardiovascular gene therapy. Circ Res. 2001;88(5) 468-475.

in arterial smooth muscle cells. Dev Biol. 2000;226(2) 192-208.

um than AAV serotype 2 and plasmid. J Gene Med. 2008;10(1) 33-41.

sponse factor-containing activation complex. Circ Res. 2000;86(2) 221-232.

AAV-mediated gene targeting. Nucleic Acids Res. 2006;34(11) 3345-3360.

cular and visceral smooth muscle cells. J Cell Biol. 1996;132(5) 849-859.

1465-1470.

325-330.

Res. 1999;84(7) 852-861.

2002;91(12) 1151-1159.

Dyn. 1996;207(2) 135-144.

908-917.

Rev Microbiol. 2005;3(11) 837-847.


[94] Young JL, Benoit JN, Dean DA. Effect of a DNA nuclear targeting sequence on gene transfer and expression of plasmids in the intact vasculature. Gene Ther. 2003;10(17) 1465-1470.

treatment of coronary artery disease using direct intramyocardial administration of an adenovirus vector expressing the VEGF121 cDNA. Ann Surg. 1999;230(4) 466-470;

[82] Grines C, Rubanyi GM, Kleiman NS, Marrott P, Watkins MW. Angiogenic gene ther‐ apy with adenovirus 5 fibroblast growth factor-4 (Ad5FGF-4): a new option for the

[83] Safi J, Jr., DiPaula AF, Jr., Riccioni T, Kajstura J, Ambrosio G, Becker LC, Anversa P, Capogrossi MC. Adenovirus-mediated acidic fibroblast growth factor gene transfer induces angiogenesis in the nonischemic rabbit heart. Microvasc Res. 1999;58(3)

[84] Nah JW, Yu L, Han SO, Ahn CH, Kim SW. Artery wall binding peptide-poly(ethyl‐ ene glycol)-grafted-poly(L-lysine)-based gene delivery to artery wall cells. J Control

[85] Akagi D, Oba M, Koyama H, Nishiyama N, Fukushima S, Miyata T, Nagawa H, Ka‐ taoka K. Biocompatible micellar nanovectors achieve efficient gene transfer to vascu‐ lar lesions without cytotoxicity and thrombus formation. Gene Ther. 2007;14(13)

[86] Song C, Labhasetwar V, Cui X, Underwood T, Levy RJ. Arterial uptake of biodegrad‐ able nanoparticles for intravascular local drug delivery: results with an acute dog

[87] Kagaya H, Oba M, Miura Y, Koyama H, Ishii T, Shimada T, Takato T, Kataoka K, Miyata T. Impact of polyplex micelles installed with cyclic RGD peptide as ligand on

[88] Theoharis S, Krueger U, Tan PH, Haskard DO, Weber M, George AJ. Targeting gene delivery to activated vascular endothelium using anti E/P-Selectin antibody linked to

[89] White SJ, Nicklin SA, Sawamura T, Baker AH. Identification of peptides that target the endothelial cell-specific LOX-1 receptor. Hypertension. 2001;37(2 Part 2) 449-455.

[90] Jarver P, Langel K, El-Andaloussi S, Langel U. Applications of cell-penetrating pepti‐ des in regulation of gene expression. Biochem Soc Trans. 2007;35(Pt 4) 770-774. [91] Golda A, Pelisek J, Klocke R, Engelmann MG, Rolland PH, Mekkaoui C, Nikol S. Small poly-L-lysines improve cationic lipid-mediated gene transfer in vascular cells

[92] Cartier R, Reszka R. Utilization of synthetic peptides containing nuclear localization

[93] Hebert E. Improvement of exogenous DNA nuclear importation by nuclear localiza‐ tion signal-bearing vectors: a promising way for non-viral gene therapy? Biol Cell.

signals for nonviral gene transfer systems. Gene Ther. 2002;9(3) 157-167.

gene delivery to vascular lesions. Gene Ther. 2012;19(1) 61-69.

PAMAM dendrimers. J Immunol Methods. 2009;343(2) 79-90.

in vitro and in vivo. J Vasc Res. 2007;44(4) 273-282.

treatment of coronary artery disease. Am J Cardiol. 2003;92(9B) 24N-31N.

discussion 470-462.

676 Gene Therapy - Tools and Potential Applications

Release. 2002;78(1-3) 273-284.

model. J Control Release. 1998;54(2) 201-211.

238-249.

1029-1038.

2003;95(2) 59-68.


[107] Young JL, Zimmer WE, Dean DA. Smooth muscle-specific gene delivery in the vas‐ culature based on restriction of DNA nuclear import. Exp Biol Med (Maywood). 2008;233(7) 840-848.

enhancer in Hprt targeted transgenic mice. Arterioscler Thromb Vasc Biol.

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

679

[120] White SJ, Papadakis ED, Rogers CA, Johnson JL, Biessen EA, Newby AC. In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer. Gene

[121] Gigout L, Rebollo P, Clement N, Warrington KH, Jr., Muzyczka N, Linden RM, Web‐ er T. Altering AAV tropism with mosaic viral capsids. Mol Ther. 2005;11(6) 856-865.

[122] Nicklin SA, Baker AH. Tropism-modified adenoviral and adeno-associated viral vec‐

[123] Baker AH. Designing gene delivery vectors for cardiovascular gene therapy. Prog Bi‐

[124] White SJ, Nicklin SA, Buning H, Brosnan MJ, Leike K, Papadakis ED, Hallek M, Bak‐ er AH. Targeted gene delivery to vascular tissue in vivo by tropism-modified adeno-

[125] Nicklin SA, Buening H, Dishart KL, de Alwis M, Girod A, Hacker U, Thrasher AJ, Ali RR, Hallek M, Baker AH. Efficient and selective AAV2-mediated gene transfer direct‐

[126] Work LM, Nicklin SA, Brain NJ, Dishart KL, Von Seggern DJ, Hallek M, Buning H, Baker AH. Development of efficient viral vectors selective for vascular smooth mus‐

[127] Takakura Y, Nishikawa M, Yamashita F, Hashida M. Influence of physicochemical properties on pharmacokinetics of non-viral vectors for gene delivery. J Drug Target.

[128] Mahato RI, Kawabata K, Takakura Y, Hashida M. In vivo disposition characteristics of plasmid DNA complexed with cationic liposomes. J Drug Target. 1995;3(2)

[129] Gonin P, Gaillard C. Gene transfer vector biodistribution: pivotal safety studies in clinical gene therapy development. Gene Ther. 2004;11 Suppl 1 S98-S108.

[130] Ye X, Gao GP, Pabin C, Raper SE, Wilson JM. Evaluating the potential of germ line transmission after intravenous administration of recombinant adenovirus in the C3H

[131] Beck C, Uramoto H, Boren J, Akyurek LM. Tissue-specific targeting for cardiovascu‐ lar gene transfer. Potential vectors and future challenges. Curr Gene Ther. 2004;4(4)

[132] Sharif F, Daly K, Crowley J, O'Brien T. Current status of catheter- and stent-based

[133] Fram DB, Aretz T, Azrin MA, Mitchel JF, Samady H, Gillam LD, Sahatjian R, Waters D, McKay RG. Localized intramural drug delivery during balloon angioplasty using

ed to human vascular endothelial cells. Mol Ther. 2001;4(3) 174-181.

tors for gene therapy. Curr Gene Ther. 2002;2(3) 273-293.

associated virus vectors. Circulation. 2004;109(4) 513-519.

2003;23(11) 2041-2047.

Ther. 2008;15(5) 340-346.

ophys Mol Biol. 2004;84(2-3) 279-299.

cle cells. Mol Ther. 2004;9(2) 198-208.

mouse. Hum Gene Ther. 1998;9(14) 2135-2142.

gene therapy. Cardiovasc Res. 2004;64(2) 208-216.

2002;10(2) 99-104.

149-157.

457-467.


enhancer in Hprt targeted transgenic mice. Arterioscler Thromb Vasc Biol. 2003;23(11) 2041-2047.

[120] White SJ, Papadakis ED, Rogers CA, Johnson JL, Biessen EA, Newby AC. In vitro and in vivo analysis of expression cassettes designed for vascular gene transfer. Gene Ther. 2008;15(5) 340-346.

[107] Young JL, Zimmer WE, Dean DA. Smooth muscle-specific gene delivery in the vas‐ culature based on restriction of DNA nuclear import. Exp Biol Med (Maywood).

[108] Morishita K, Johnson DE, Williams LT. A novel promoter for vascular endothelial growth factor receptor (flt-1) that confers endothelial-specific gene expression. J Biol

[109] Cowan PJ, Shinkel TA, Witort EJ, Barlow H, Pearse MJ, d'Apice AJ. Targeting gene expression to endothelial cells in transgenic mice using the human intercellular adhe‐

[110] Hegen A, Koidl S, Weindel K, Marme D, Augustin HG, Fiedler U. Expression of an‐ giopoietin-2 in endothelial cells is controlled by positive and negative regulatory pro‐

[111] Karantzoulis-Fegaras F, Antoniou H, Lai SL, Kulkarni G, D'Abreo C, Wong GK, Mill‐ er TL, Chan Y, Atkins J, Wang Y, Marsden PA. Characterization of the human endo‐

[112] Neish AS, Williams AJ, Palmer HJ, Whitley MZ, Collins T. Functional analysis of the human vascular cell adhesion molecule 1 promoter. J Exp Med. 1992;176(6)

[113] Jahroudi N, Lynch DC. Endothelial-cell-specific regulation of von Willebrand factor

[114] Korhonen J, Lahtinen I, Halmekyto M, Alhonen L, Janne J, Dumont D, Alitalo K. En‐ dothelial-specific gene expression directed by the tie gene promoter in vivo. Blood.

[115] Patterson C, Perrella MA, Hsieh CM, Yoshizumi M, Lee ME, Haber E. Cloning and functional analysis of the promoter for KDR/flk-1, a receptor for vascular endothelial

[116] Aoyama T, Sawamura T, Furutani Y, Matsuoka R, Yoshida MC, Fujiwara H, Masaki T. Structure and chromosomal assignment of the human lectin-like oxidized lowdensity-lipoprotein receptor-1 (LOX-1) gene. Biochem J. 1999;339 ( Pt 1) 177-184. [117] Hou J, Baichwal V, Cao Z. Regulatory elements and transcription factors controlling basal and cytokine-induced expression of the gene encoding intercellular adhesion

[118] Tessitore A, Pastore L, Rispoli A, Cilenti L, Toniato E, Flati V, Farina AR, Frati L, Gu‐ lino A, Martinotti S. Two gamma-interferon-activation sites (GAS) on the promoter of the human intercellular adhesion molecule (ICAM-1) gene are required for induc‐

[119] Minami T, Kuivenhoven JA, Evans V, Kodama T, Rosenberg RD, Aird WC. Ets mo‐ tifs are necessary for endothelial cell-specific expression of a 723-bp Tie-2 promoter/

tion of transcription by IFN-gamma. Eur J Biochem. 1998;258(3) 968-975.

thelial nitric-oxide synthase promoter. J Biol Chem. 1999;274(5) 3076-3093.

moter elements. Arterioscler Thromb Vasc Biol. 2004;24(10) 1803-1809.

sion molecule 2 promoter. Transplantation. 1996;62(2) 155-160.

gene expression. Mol Cell Biol. 1994;14(2) 999-1008.

growth factor. J Biol Chem. 1995;270(39) 23111-23118.

molecule 1. Proc Natl Acad Sci U S A. 1994;91(24) 11641-11645.

2008;233(7) 840-848.

678 Gene Therapy - Tools and Potential Applications

1583-1593.

1995;86(5) 1828-1835.

Chem. 1995;270(46) 27948-27953.


hydrogel-coated balloons and pressure-augmented diffusion. J Am Coll Cardiol. 1994;23(7) 1570-1577.

ointimal proliferation of rat carotid artery. Am J Physiol Heart Circ Physiol.

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

681

[145] Schneider DB, Sassani AB, Vassalli G, Driscoll RM, Dichek DA. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999;29(3)

[146] Laitinen M, Pakkanen T, Donetti E, Baetta R, Luoma J, Lehtolainen P, Viita H, Agrawal R, Miyanohara A, Friedmann T, Risau W, Martin JF, Soma M, Yla-Herttuala S. Gene transfer into the carotid artery using an adventitial collar: comparison of the effectiveness of the plasmid-liposome complexes, retroviruses, pseudotyped retrovi‐

[147] March KL, Woody M, Mehdi K, Zipes DP, Brantly M, Trapnell BC. Efficient in vivo catheter-based pericardial gene transfer mediated by adenoviral vectors. Clin Cardi‐

[148] Baek S, March KL. Gene therapy for restenosis: getting nearer the heart of the matter.

[149] Marshall E. Gene therapy death prompts review of adenovirus vector. Science.

[150] Manno CS, Pierce GF, Arruda VR, Glader B, Ragni M, Rasko JJ, Ozelo MC, Hoots K, Blatt P, Konkle B, Dake M, Kaye R, Razavi M, Zajko A, Zehnder J, Rustagi PK, Nakai H, Chew A, Leonard D, Wright JF, Lessard RR, Sommer JM, Tigges M, Sabatino D, Luk A, Jiang H, Mingozzi F, Couto L, Ertl HC, High KA, Kay MA. Successful trans‐ duction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host

[151] Bessis N, GarciaCozar FJ, Boissier MC. Immune responses to gene therapy vectors: influence on vector function and effector mechanisms. Gene Ther. 2004;11 Suppl 1

[152] Waters B, Lillicrap D. The molecular mechanisms of immunomodulation and toler‐

[153] Hartman ZC, Appledorn DM, Amalfitano A. Adenovirus vector induced innate im‐ mune responses: impact upon efficacy and toxicity in gene therapy and vaccine ap‐

[154] Seregin SS, Amalfitano A. Improving adenovirus based gene transfer: strategies to

[155] Kawai T, Akira S. Toll-like receptor and RIG-I-like receptor signaling. Ann N Y Acad

[156] Varnavski AN, Calcedo R, Bove M, Gao G, Wilson JM. Evaluation of toxicity from high-dose systemic administration of recombinant adenovirus vector in vector-naive

ance induction to factor VIII. J Thromb Haemost. 2009;7(9) 1446-1456.

accomplish immune evasion. Viruses. 2010;2(9) 2013-2036.

and pre-immunized mice. Gene Ther. 2005;12(5) 427-436.

ruses, and adenoviruses. Hum Gene Ther. 1997;8(14) 1645-1650.

2005;288(2) H946-953.

ol. 1999;22(1 Suppl 1) I23-29.

Circ Res. 1998;82(3) 295-305.

1999;286(5448) 2244-2245.

immune response. Nat Med. 2006;12(3) 342-347.

plications. Virus Res. 2008;132(1-2) 1-14.

543-550.

S10-17.

Sci. 2008;1143 1-20.


ointimal proliferation of rat carotid artery. Am J Physiol Heart Circ Physiol. 2005;288(2) H946-953.

[145] Schneider DB, Sassani AB, Vassalli G, Driscoll RM, Dichek DA. Adventitial delivery minimizes the proinflammatory effects of adenoviral vectors. J Vasc Surg. 1999;29(3) 543-550.

hydrogel-coated balloons and pressure-augmented diffusion. J Am Coll Cardiol.

[134] Opie SR, Dib N. Local endovascular delivery, gene therapy, and cell transplantation for peripheral arterial disease. J Endovasc Ther. 2004;11 Suppl 2 II151-162.

[135] Barath P, Popov A, Dillehay GL, Matos G, McKiernan T. Infiltrator Angioplasty Bal‐ loon Catheter: a device for combined angioplasty and intramural site-specific treat‐

[136] Fernandez-Ortiz A, Meyer BJ, Mailhac A, Falk E, Badimon L, Fallon JT, Fuster V, Chesebro JH, Badimon JJ. A new approach for local intravascular drug delivery. Ion‐

[137] Walter DH, Cejna M, Diaz-Sandoval L, Willis S, Kirkwood L, Stratford PW, Tietz AB, Kirchmair R, Silver M, Curry C, Wecker A, Yoon YS, Heidenreich R, Hanley A, Kear‐ ney M, Tio FO, Kuenzler P, Isner JM, Losordo DW. Local gene transfer of phVEGF-2 plasmid by gene-eluting stents: an alternative strategy for inhibition of restenosis.

[138] Sharif F, Hynes SO, McCullagh KJ, Ganley S, Greiser U, McHugh P, Crowley J, Barry F, O'Brien T. Gene-eluting stents: non-viral, liposome-based gene delivery of eNOS to the blood vessel wall in vivo results in enhanced endothelialization but does not reduce restenosis in a hypercholesterolemic model. Gene Ther. 2012;19(3) 321-328.

[139] Sharif F, Hynes SO, McMahon J, Cooney R, Conroy S, Dockery P, Duffy G, Daly K, Crowley J, Bartlett JS, O'Brien T. Gene-eluting stents: comparison of adenoviral and adeno- associated viral gene delivery to the blood vessel wall in vivo. Hum Gene

[140] Klugherz BD, Song C, DeFelice S, Cui X, Lu Z, Connolly J, Hinson JT, Wilensky RL, Levy RJ. Gene delivery to pig coronary arteries from stents carrying antibody-teth‐

[141] Nishio S, Kosuga K, Igaki K, Okada M, Kyo E, Tsuji T, Takeuchi E, Inuzuka Y, Take‐ da S, Hata T, Takeuchi Y, Kawada Y, Harita T, Seki J, Akamatsu S, Hasegawa S, Bruining N, Brugaletta S, de Winter S, Muramatsu T, Onuma Y, Serruys PW, Ikegu‐ chi S. Long-Term (>10 Years) clinical outcomes of first-in-human biodegradable polyl-lactic acid coronary stents: Igaki-Tamai stents. Circulation. 2012;125(19) 2343-2353.

[142] George SJ, Baker AH. Gene transfer to the vasculature: historical perspective and im‐ plication for future research objectives. Mol Biotechnol. 2002;22(2) 153-164.

[143] Siow RC, Churchman AT. Adventitial growth factor signalling and vascular remod‐ elling: potential of perivascular gene transfer from the outside-in. Cardiovasc Res.

[144] Dourron HM, Jacobson GM, Park JL, Liu J, Reddy DJ, Scheel ML, Pagano PJ. Perivas‐ cular gene transfer of NADPH oxidase inhibitor suppresses angioplasty-induced ne‐

ment. Cathet Cardiovasc Diagn. 1997;41(3) 333-341.

tophoretic balloon. Circulation. 1994;89(4) 1518-1522.

ered adenovirus. Hum Gene Ther. 2002;13(3) 443-454.

1994;23(7) 1570-1577.

680 Gene Therapy - Tools and Potential Applications

Circulation. 2004;110(1) 36-45.

Ther. 2006;17(7) 741-750.

2007;75(4) 659-668.


[157] Rafii S, Dias S, Meeus S, Hattori K, Ramachandran R, Feuerback F, Worgall S, Hack‐ ett NR, Crystal RG. Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), ade‐ novirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression. Circ Res. 2001;88(9) 903-910.

[170] Jacob T, Hemavathy K, Jacob J, Hingorani A, Marks N, Ascher E. A nanotechnologybased delivery system: Nanobots. Novel vehicles for molecular medicine. J Cardio‐

Clinical and Translational Challenges in Gene Therapy of Cardiovascular Diseases

http://dx.doi.org/10.5772/54075

683

[171] Piskin E. Stimuli-responsive polymers in gene delivery. Expert Rev Med Devices.

[172] Lande C, Cecchettini A, Tedeschi L, Taranta M, Naldi I, Citti L, Trivella MG, Grimal‐ di S, Cinti C. Innovative Erythrocyte-based Carriers for Gene Delivery in Porcine Vascular Smooth Muscle Cells: Basis for Local Therapy to Prevent Restenosis. Cardi‐

[173] Chorny M, Fishbein I, Adamo RF, Forbes SP, Folchman-Wagner Z, Alferiev IS. Mag‐ netically targeted delivery of therapeutic agents to injured blood vessels for preven‐

[174] Phillips LC, Klibanov AL, Bowles DK, Ragosta M, Hossack JA, Wamhoff BR. Focused in vivo delivery of plasmid DNA to the porcine vascular wall via intravascular ultra‐

[175] Fishbein I, Chorny M, Levy RJ. Site-specific gene therapy for cardiovascular disease.

tion of in-stent stenosis. Methodist Debakey Cardiovasc J. 2012;8(1) 23-27.

sound destruction of microbubbles. J Vasc Res. 2010;47(3) 270-274.

vasc Surg (Torino). 2011;52(2) 159-167.

ovasc Hematol Disord Drug Targets. 2012;12(1) 68-75.

Curr Opin Drug Discov Devel. 2010;13(2) 203-213.

2005;2(4) 501-509.


[170] Jacob T, Hemavathy K, Jacob J, Hingorani A, Marks N, Ascher E. A nanotechnologybased delivery system: Nanobots. Novel vehicles for molecular medicine. J Cardio‐ vasc Surg (Torino). 2011;52(2) 159-167.

[157] Rafii S, Dias S, Meeus S, Hattori K, Ramachandran R, Feuerback F, Worgall S, Hack‐ ett NR, Crystal RG. Infection of endothelium with E1(-)E4(+), but not E1(-)E4(-), ade‐ novirus gene transfer vectors enhances leukocyte adhesion and migration by modulation of ICAM-1, VCAM-1, CD34, and chemokine expression. Circ Res.

[158] Lieber A, He CY, Meuse L, Schowalter D, Kirillova I, Winther B, Kay MA. The role of Kupffer cell activation and viral gene expression in early liver toxicity after infusion

[159] Shayakhmetov DM, Gaggar A, Ni S, Li ZY, Lieber A. Adenovirus binding to blood factors results in liver cell infection and hepatotoxicity. J Virol. 2005;79(12) 7478-7491.

[160] Alba R, Bosch A, Chillon M. Gutless adenovirus: last-generation adenovirus for gene

[161] Bangari DS, Mittal SK. Current strategies and future directions for eluding adenovi‐

[162] Miao CH. Advances in Overcoming Immune Responses following Hemophilia Gene

[163] Coughlan L, Alba R, Parker AL, Bradshaw AC, McNeish IA, Nicklin SA, Baker AH. Tropism-modification strategies for targeted gene delivery using adenoviral vectors.

[164] Schulick AH, Vassalli G, Dunn PF, Dong G, Rade JJ, Zamarron C, Dichek DA. Estab‐ lished immunity precludes adenovirus-mediated gene transfer in rat carotid arteries. Potential for immunosuppression and vector engineering to overcome barriers of im‐

[165] Herzog RW, Dobrzynski E. Immune implications of gene therapy for hemophilia.

[166] Jayandharan GR, Aslanidi G, Martino AT, Jahn SC, Perrin GQ, Herzog RW, Srivasta‐ va A. Activation of the NF-kappaB pathway by adeno-associated virus (AAV) vec‐ tors and its implications in immune response and gene therapy. Proc Natl Acad Sci U

[167] Moskalenko M, Chen L, van Roey M, Donahue BA, Snyder RO, McArthur JG, Patel SD. Epitope mapping of human anti-adeno-associated virus type 2 neutralizing anti‐ bodies: implications for gene therapy and virus structure. J Virol. 2000;74(4)

[168] Wu TL, Ertl HC. Immune barriers to successful gene therapy. Trends Mol Med.

[169] Verthelyi D. Adjuvant properties of CpG oligonucleotides in primates. Methods Mol

of recombinant adenovirus vectors. J Virol. 1997;71(11) 8798-8807.

therapy. Gene Ther. 2005;12 Suppl 1 S18-27.

Therapy. J Genet Syndr Gene Ther. 2011;S1.

munity. J Clin Invest. 1997;99(2) 209-219.

Semin Thromb Hemost. 2004;30(2) 215-226.

Viruses. 2010;2(10) 2290-2355.

S A. 2011;108(9) 3743-3748.

1761-1766.

2009;15(1) 32-39.

Med. 2006;127 139-158.

ral vector immunity. Curr Gene Ther. 2006;6(2) 215-226.

2001;88(9) 903-910.

682 Gene Therapy - Tools and Potential Applications


**Chapter 28**

**Gene Therapy for Chronic Pain Management**

This chapter provides an overview of the main current applications of gene therapy for chronic pain in what concerns animal studies and putative clinical applications. The value of gene therapy in unravelling neuronal brain circuits involved in pain modulation is also analysed. After alerting to the huge socioeconomic impact of chronic pain in modern societies and justifying the need to develop new avenues in pain management, we review the most common animal studies using gene therapy, which consisted on deliveries of replication-defective viral vectors at the periphery with the aim to block nociceptive transmission at the spinal cord. Departing from the data of these animal studies, we present the latest results of clinical trials using gene therapy for pain management in cancer patients. The animal studies dealing with gene delivery in pain control centres of the brain are analysed in what concerns their com‐ plexity and interest in unravelling the neurobiological mechanisms of descending pain modulation. The chapter will finish by analysing possible futures of gene therapy for chronic pain management based on the development of vectors which are safer and more specific for

Pain is not easy to define since it is a highly subjective experience. The more consensual definition of pain was provided by the International Association for the Study of Pain (IASP) and states that "*Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage*" [1]. Acute pain is important as an alert signal to potentially threaten situations (internal or external to the organism) and it is important for survival. Acute pain may progress to chronic pain which, according to IASP, is the pain that lasts more than 3 months and persists beyond the normal tissue healing time [2].

> © 2013 Tavares and Martins; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Isaura Tavares and Isabel Martins

http://dx.doi.org/10.5772/55050

the different types of chronic pain.

**2. Chronic pain: A burden for modern societies**

**1. Introduction**

Additional information is available at the end of the chapter

### **Gene Therapy for Chronic Pain Management**

Isaura Tavares and Isabel Martins

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55050

#### **1. Introduction**

This chapter provides an overview of the main current applications of gene therapy for chronic pain in what concerns animal studies and putative clinical applications. The value of gene therapy in unravelling neuronal brain circuits involved in pain modulation is also analysed. After alerting to the huge socioeconomic impact of chronic pain in modern societies and justifying the need to develop new avenues in pain management, we review the most common animal studies using gene therapy, which consisted on deliveries of replication-defective viral vectors at the periphery with the aim to block nociceptive transmission at the spinal cord. Departing from the data of these animal studies, we present the latest results of clinical trials using gene therapy for pain management in cancer patients. The animal studies dealing with gene delivery in pain control centres of the brain are analysed in what concerns their com‐ plexity and interest in unravelling the neurobiological mechanisms of descending pain modulation. The chapter will finish by analysing possible futures of gene therapy for chronic pain management based on the development of vectors which are safer and more specific for the different types of chronic pain.

#### **2. Chronic pain: A burden for modern societies**

Pain is not easy to define since it is a highly subjective experience. The more consensual definition of pain was provided by the International Association for the Study of Pain (IASP) and states that "*Pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage*" [1]. Acute pain is important as an alert signal to potentially threaten situations (internal or external to the organism) and it is important for survival. Acute pain may progress to chronic pain which, according to IASP, is the pain that lasts more than 3 months and persists beyond the normal tissue healing time [2].

© 2013 Tavares and Martins; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Chronic pain may be divided into "nociceptive" and "neuropathic" [3]. Nociceptive pain is caused by activation of nociceptors, the thin nerve fibers which convey nociceptive input from the periphery to the spinal cord. Neuropathic pain is caused by malfunction or damage of the nervous system. Neuropathic pain is frequently difficult to treat being associated to sponta‐ neous pain, exaggerated responses to nociceptive stimuli (*hyperalgesia*) and nociceptive responses to stimuli which are usually non-nociceptive (*allodynia*).

The number of people affected by chronic pain is increasing due to multifactorial causes such as increasing aging of the population. In Europe, about 20% of people suffer from moderate to severe chronic pain [4]. In the United States the prevalence of chronic pain ranges from 2% to 40%, with a median of 15% [5], which cost the country 560 to 635 million dollars [6]. People suffering from chronic pain are less able to walk, sleep normally, perform social activities, exercise or have sexual relations. Chronic pain strongly affects the productivity. About 60% of chronic pain patients are unable or less able to work, 19% lost their jobs and 13% change jobs due to their pain [6]. Chronic pain is associated to several co-morbidities, namely depression and anxiety [6]. Besides all of these indirect costs, chronic pain is a burden due to direct costs of pain management. Despite major investments in basic and clinical pain research, the available analgesics remain considerably unchanged during the last decades. Opioids are useful to manage several pain types but they have a modest efficacy in several pain conditions (e.g. neuropathic pain). Furthermore, long term treatments with opioids frequently induce severe off-target effects, like nausea, constipation and addiction [7]. Intractable pain remains a clinical problem and a drama for the patients and their families [8]. During the last decade, pain clinicians and pain researchers were challenged to search for alternatives to conventional pain treatment, which should be more specific and sustained than conventional analgesics. Gene therapy outstands as a powerful technique to overcome some current problems of chronic pain treatments.

**Figure 1.** Schematic diagram of pain pathways involved in pain transmission and modulation. **Nociceptive informa‐ tion** is transmitted from the periphery to the spinal dorsal horn by primary sensory neurons. At the spinal level, these neurons transmit nociceptive information to second order neurons ("Ascending pathways") through the release of neurotransmitters like the excitatory amino acids (EAA) glutamate and aspartate, calcitonin gene-related peptide (CGRP), substance P (SP) galanin (Gal) and neuropeptide Y (NPY). In the brain, the nociceptive information is then per‐ ceived as a **pain sensation**. The transmission of nociceptive information at the spinal level is modulated by interneur‐ ons (mainly inhibitory) through the release of opioid pepides and GABA and also by supraspinal descending neurons ("Descending pathways") through the release of serotonin (5-HT) and noradrenaline (NA). Descending pathways may

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Gene therapy is an especially versatile tool for chronic pain management since it is based in a triad of controllable parameters: the vector, the transgene and the promoter. By knowing the neurobiological features of each chronic pain type, namely the neurotransmitters and receptors affected, it is possible to design gene therapy strategies based on the best combination of vectors, transgenes and promoters. As to **vectors**, gene therapy for pain uses mainly "vehicles" which have a "certified" experience in infecting neurons, namely replication-defective forms of viruses. Non-viral vectors have seldom been used in gene therapy studies for pain but their transduction efficiency and specificity are much lower than those of viral vectors. Some of these vectors have the ability to migrate retrogradely (i.e., contrary to the direction of nerve impulse) which is very useful to target neurons that are located in structures of difficult surgical

inhibit or enhance nociceptive transmission from the spinal cord.

Neurobiological research in the pain field provided solid information regarding the transmis‐ sion and modulation of nociceptive information from the periphery to the brain, where a pain sensation is produced (Fig. 1). Nociceptive signals are conveyed by primary afferent fibers from peripheral organs, like the bladder or muscles, to the spinal cord. This is the first relay station involved in the modulation of nociceptive information namely by local inhibitory interneurons that use opioid peptides or aminoacids (γ-amminobutiric acid-GABA- and glycine). Nociceptive information is then transmitted supraspinally, namely to the thalamus, and to several brainstem areas, where additional modulation of the nociceptive signal occurs. The thalamo-cortical pathway ensures that the nociceptive information reaches the somato‐ sensory and prefrontal cortices, where the nociceptive signal is finally perceived as a pain sensation [9, 10]. Some brain areas which directly or indirectly receive nociceptive information from the spinal cord are also involved in descending pain modulation. Both inhibition and facilitation may occur and chronic pain may derive from a reduction of the former and enhancement of the latter [9, 11]. This neurobiological knowledge has been used to design gene therapy studies for chronic pain, namely to choose the somatosensory system areas and neurotransmitters/receptors to be targeted in order to block nociceptive transmission.

Chronic pain may be divided into "nociceptive" and "neuropathic" [3]. Nociceptive pain is caused by activation of nociceptors, the thin nerve fibers which convey nociceptive input from the periphery to the spinal cord. Neuropathic pain is caused by malfunction or damage of the nervous system. Neuropathic pain is frequently difficult to treat being associated to sponta‐ neous pain, exaggerated responses to nociceptive stimuli (*hyperalgesia*) and nociceptive

The number of people affected by chronic pain is increasing due to multifactorial causes such as increasing aging of the population. In Europe, about 20% of people suffer from moderate to severe chronic pain [4]. In the United States the prevalence of chronic pain ranges from 2% to 40%, with a median of 15% [5], which cost the country 560 to 635 million dollars [6]. People suffering from chronic pain are less able to walk, sleep normally, perform social activities, exercise or have sexual relations. Chronic pain strongly affects the productivity. About 60% of chronic pain patients are unable or less able to work, 19% lost their jobs and 13% change jobs due to their pain [6]. Chronic pain is associated to several co-morbidities, namely depression and anxiety [6]. Besides all of these indirect costs, chronic pain is a burden due to direct costs of pain management. Despite major investments in basic and clinical pain research, the available analgesics remain considerably unchanged during the last decades. Opioids are useful to manage several pain types but they have a modest efficacy in several pain conditions (e.g. neuropathic pain). Furthermore, long term treatments with opioids frequently induce severe off-target effects, like nausea, constipation and addiction [7]. Intractable pain remains a clinical problem and a drama for the patients and their families [8]. During the last decade, pain clinicians and pain researchers were challenged to search for alternatives to conventional pain treatment, which should be more specific and sustained than conventional analgesics. Gene therapy outstands as a powerful technique to overcome some current problems of

Neurobiological research in the pain field provided solid information regarding the transmis‐ sion and modulation of nociceptive information from the periphery to the brain, where a pain sensation is produced (Fig. 1). Nociceptive signals are conveyed by primary afferent fibers from peripheral organs, like the bladder or muscles, to the spinal cord. This is the first relay station involved in the modulation of nociceptive information namely by local inhibitory interneurons that use opioid peptides or aminoacids (γ-amminobutiric acid-GABA- and glycine). Nociceptive information is then transmitted supraspinally, namely to the thalamus, and to several brainstem areas, where additional modulation of the nociceptive signal occurs. The thalamo-cortical pathway ensures that the nociceptive information reaches the somato‐ sensory and prefrontal cortices, where the nociceptive signal is finally perceived as a pain sensation [9, 10]. Some brain areas which directly or indirectly receive nociceptive information from the spinal cord are also involved in descending pain modulation. Both inhibition and facilitation may occur and chronic pain may derive from a reduction of the former and enhancement of the latter [9, 11]. This neurobiological knowledge has been used to design gene therapy studies for chronic pain, namely to choose the somatosensory system areas and

neurotransmitters/receptors to be targeted in order to block nociceptive transmission.

responses to stimuli which are usually non-nociceptive (*allodynia*).

chronic pain treatments.

686 Gene Therapy - Tools and Potential Applications

**Figure 1.** Schematic diagram of pain pathways involved in pain transmission and modulation. **Nociceptive informa‐ tion** is transmitted from the periphery to the spinal dorsal horn by primary sensory neurons. At the spinal level, these neurons transmit nociceptive information to second order neurons ("Ascending pathways") through the release of neurotransmitters like the excitatory amino acids (EAA) glutamate and aspartate, calcitonin gene-related peptide (CGRP), substance P (SP) galanin (Gal) and neuropeptide Y (NPY). In the brain, the nociceptive information is then per‐ ceived as a **pain sensation**. The transmission of nociceptive information at the spinal level is modulated by interneur‐ ons (mainly inhibitory) through the release of opioid pepides and GABA and also by supraspinal descending neurons ("Descending pathways") through the release of serotonin (5-HT) and noradrenaline (NA). Descending pathways may inhibit or enhance nociceptive transmission from the spinal cord.

Gene therapy is an especially versatile tool for chronic pain management since it is based in a triad of controllable parameters: the vector, the transgene and the promoter. By knowing the neurobiological features of each chronic pain type, namely the neurotransmitters and receptors affected, it is possible to design gene therapy strategies based on the best combination of vectors, transgenes and promoters. As to **vectors**, gene therapy for pain uses mainly "vehicles" which have a "certified" experience in infecting neurons, namely replication-defective forms of viruses. Non-viral vectors have seldom been used in gene therapy studies for pain but their transduction efficiency and specificity are much lower than those of viral vectors. Some of these vectors have the ability to migrate retrogradely (i.e., contrary to the direction of nerve impulse) which is very useful to target neurons that are located in structures of difficult surgical access. A good example is the application of replication-defective forms of Herpes Simplex Virus type 1 (HSV-1) at the periphery (e.g. the skin) to transduce neurons at the spinal ganglia (dorsal root ganglia-DRGs), which are difficult to access due to their bone protection. Regard‐ ing the **transgenes** to include in the vectors for gene therapy of pain, it is possible to increase the expression of neurotransmitters and receptors involved in nociceptive inhibition (e.g. opioids), neurotrophic factors or substances with anti-inflammatory properties. Finally, and in what concerns the **promoters**, it is possible to choose those that restrict transgene expression to a cell type, such as a neuron or a glial cell, or even target selective neurochemical neuronal populations. Examples of neuron-specific promoters are synapsin I, calcium/calmodulindependent protein kinase II, tubulin alpha I and neuron-specific enolase [12]. Some possibili‐ ties of controlling the vectors, transgenes and promoters will be discussed in the next two sections using gene therapy in animal models.

**Pain models Gene product Inoculation References Herpes Simplex type 1** Acute pain Pre-proenkephalin Subcutaneous [35] Inflammatory pain Pre-proenkephalin A Subcutaneous [14] Neuropathic pain Pre-proenkephalin A Subcutaneous [41] Cutaneous hyperalgesia Pre-preproenkephalin Subcutaneous [36] Bladder hyperactivity Pre-preproenkephalin Bladder wall [37] Inflammatory pain Endomorphin-2 Subcutaneous [15] Neuropathic pain Endomorphin-2 Subcutaneous [23] Neuropathic pain IL-4 Subcutaneous [24] Neuropathic pain sTNFRs Subcutaneous [25] Neuropathic pain GAD Subcutaneous [26, 47] Chronic pancreatitis Pre-proenkephalin Pancreas surface [22] Inflammatory pain Nav1.7 antisense Subcutaneous [16] Incision pain Pre-proenkephalin Subcutaneous [39] Cancer pain Pre-proenkephalin Subcutaneous [40] **Adenovirus**

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Inflammatory pain GAD Trigeminal ganglion [42] Neuropathic pain IL-10 Intrathecal [27] Inflammatory pain β-endorphin Intrathecal [17] Neuropathic pain IL-2 Intrathecal [28] **Adeno-associated vectors**

Neuropathic pain IL-10 Intrathecal [29] Neuropathic pain shGCH1 Intrathecal [30] Neuropathic pain Prepro- β-endorphin Intrathecal [31] Inflammatory pain µ-opioid receptors DRG [18]

**Lentivirus**

As to the transgenes included in the HSV-1 vectors, opioid peptides or their precursors largely prevail due to their well-known ability to block nociceptive transmission at the spinal cord. HSV-1-based delivery of opioids has additional advantages over classic opioids, namely by

Neuropathic pain GDNF Intraspinal [32] Neuropathic pain NFκB Repressor Intraspinal [33]

**Table 1.** Summary of experimental studies using viral vectors for gene transfer to the spinal cord.

#### **3. Gene therapy targeting the spinal cord in animal pain models**

One of the main advantages of experimental gene therapy studies is that they can be performed using several pain models. This is important since each pain type may induce specific changes in neuronal circuits devoted to the transmission and modulation of nociceptive transmission [13]. Studies of gene therapy for pain have used clinically relevant models of inflammatory [14-22] and neuropathic pain [23-34]. In a much lower incidence, models of acute [35-38], post-operative pain [39] and cancer [40] pain have been used in experimental gene therapy studies. The large majority of studies were performed in pain models affecting the limbs or the trunk, in the latter case being of visceral origin [22, 37]. Two studies used gene therapy to block nociceptive transmission coming from the head/ face in pain models that reproduces some types of craniofacial pain, like trigeminal neuralgia [41] or temporomandibular joint disorders [42].

Gene therapy studies for pain in animal models may be divided in studies targeting the spinal cord (Table 1) and studies directed to pain control centres located in the brain (Table 2). Studies directed to the spinal cord mainly aim to manipulate the expression of transgenes in order to block the transmission of nociceptive input at the spinal dorsal horn (Table 1). Most of the spinal cord studies using gene therapy for pain elected HSV-1 as the most suitable vector, due to its natural affinity to the neuron and its ability for retrograde transport [43]. HSV-1 has the additional advantage over other vectors of carrying multiple transgenes or large transgenes and not integrating in the host genome, which reduces the possibility of mutagenic events [44, 45]. After application of replication-defective forms of HSV-1 at the periphery in order to transduce DRG neurons (or trigeminal ganglion neurons), delivery of the transgene product by the spinal branch of transduced neurons at the spinal dorsal horn induced analgesia in several rodent models of pain (Table 1). Gene therapy in animal models of craniofacial pain [41, 42] aimed to release the transgene products at the level of the spinal trigeminal nucleus and this structure is homolog of the spinal cord, which prompted to include these studies in the section devoted to spinal cord studies.


access. A good example is the application of replication-defective forms of Herpes Simplex Virus type 1 (HSV-1) at the periphery (e.g. the skin) to transduce neurons at the spinal ganglia (dorsal root ganglia-DRGs), which are difficult to access due to their bone protection. Regard‐ ing the **transgenes** to include in the vectors for gene therapy of pain, it is possible to increase the expression of neurotransmitters and receptors involved in nociceptive inhibition (e.g. opioids), neurotrophic factors or substances with anti-inflammatory properties. Finally, and in what concerns the **promoters**, it is possible to choose those that restrict transgene expression to a cell type, such as a neuron or a glial cell, or even target selective neurochemical neuronal populations. Examples of neuron-specific promoters are synapsin I, calcium/calmodulindependent protein kinase II, tubulin alpha I and neuron-specific enolase [12]. Some possibili‐ ties of controlling the vectors, transgenes and promoters will be discussed in the next two

**3. Gene therapy targeting the spinal cord in animal pain models**

One of the main advantages of experimental gene therapy studies is that they can be performed using several pain models. This is important since each pain type may induce specific changes in neuronal circuits devoted to the transmission and modulation of nociceptive transmission [13]. Studies of gene therapy for pain have used clinically relevant models of inflammatory [14-22] and neuropathic pain [23-34]. In a much lower incidence, models of acute [35-38], post-operative pain [39] and cancer [40] pain have been used in experimental gene therapy studies. The large majority of studies were performed in pain models affecting the limbs or the trunk, in the latter case being of visceral origin [22, 37]. Two studies used gene therapy to block nociceptive transmission coming from the head/ face in pain models that reproduces some types of craniofacial pain, like trigeminal

Gene therapy studies for pain in animal models may be divided in studies targeting the spinal cord (Table 1) and studies directed to pain control centres located in the brain (Table 2). Studies directed to the spinal cord mainly aim to manipulate the expression of transgenes in order to block the transmission of nociceptive input at the spinal dorsal horn (Table 1). Most of the spinal cord studies using gene therapy for pain elected HSV-1 as the most suitable vector, due to its natural affinity to the neuron and its ability for retrograde transport [43]. HSV-1 has the additional advantage over other vectors of carrying multiple transgenes or large transgenes and not integrating in the host genome, which reduces the possibility of mutagenic events [44, 45]. After application of replication-defective forms of HSV-1 at the periphery in order to transduce DRG neurons (or trigeminal ganglion neurons), delivery of the transgene product by the spinal branch of transduced neurons at the spinal dorsal horn induced analgesia in several rodent models of pain (Table 1). Gene therapy in animal models of craniofacial pain [41, 42] aimed to release the transgene products at the level of the spinal trigeminal nucleus and this structure is homolog of the spinal cord, which prompted to include these studies in

sections using gene therapy in animal models.

688 Gene Therapy - Tools and Potential Applications

neuralgia [41] or temporomandibular joint disorders [42].

the section devoted to spinal cord studies.

**Table 1.** Summary of experimental studies using viral vectors for gene transfer to the spinal cord.

As to the transgenes included in the HSV-1 vectors, opioid peptides or their precursors largely prevail due to their well-known ability to block nociceptive transmission at the spinal cord. HSV-1-based delivery of opioids has additional advantages over classic opioids, namely by being deprived of major side-effects and preventing tolerance after repeated administrations of the vector [46]. Furthermore, opioid-based gene therapy can be very powerful in inducing analgesia if combined with administration of very low doses of classical opioids [46]. Besides opioid peptides, other transgenes were included in the HSV-1 vectors constructs. A transgene that increases the levels of the inhibitory neurotransmitter GABA, namely by overexpressing its synthetizing enzyme glutamate decarboxylase (GAD), induced analgesia in neuropathic pain models [26, 47]. HSV-1 based vectors have also been used to deliver transgenes that overexpress anti-inflammatory interleukins [24, 48] or the soluble receptor for tumor necrosis factor-α (TNF-α), which act as an antagonist of TNF-α in order to block its role as a proinflammatory mediator [25, 49]. A decrease in the levels of the α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) was also achieved using HSV-1 constructs but with the transgene inserted in antisense orientation [16].

manipulate the complex brain neuronal circuits involved in pain modulation. The spinal cord constitutes a less invasive delivery route when the aim is to manipulate descending modula‐ tory pathways (Fig. 1). This delivery route was recently explored by injecting intraspinally an adenovirus vector targeting the expression of a potassium channel into noradrenergic pontospinal neurons, which decreased the activity of those pontospinal neurons and induced hyperalgesia [51]. These experiments confirm the pain inhibiting role of the noradrenergic

**Pain models Gene product Delivery References**

Acute pain GAD Insular Cortex [38] Inflammatory pain Preproenkephalin Amygdala [21]

**Table 2.** Summary of animal studies using viral vectors for gene transfer to pain control centers in the brain.

Gene transfer in the brain used almost exclusively HSV-1 vectors to overexpress opioid peptides [19-21, 38] and, in a much more limited extent, GAD [38]. Our research group has a large experience in gene transfer to pain control centres at the medulla oblongata, namely the dorsal reticular nucleus (DRt), the caudal ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). These areas were elected based on the extensive neurobiological knowl‐ edge of their role in pain modulation [53, 54]. Overexpression of opioid precursors in the DRt and VLM induced analgesia in acute pain tests and models of sustained or chronic inflamma‐ tory pain [19, 20, 55]. Brain areas involved in pain control and which are of easier neurosurgical access are the amygdala and the rostral agranular insular cortex. Overexpression of opioid precursors in the central amygdalar nucleus [21] or GAD in the rostral agranular insular cortex induced analgesia in acute pain models [38]. Lentiviral vectors were delivered to the NTS, an area which is crucial in pain and cardiovascular integration, to decrease local expression of Nmethyl-D-aspartate (NMDA) receptor, a key receptor for the action of glutamate, and this approach was shown to decrease acute and inflammatory pain [56]. Since glutamate, is the most ubiquitous mediator of excitatory synaptic transmission in the central nervous system

nucleus (DRt) [55]

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nucleus (DRt) [34]

nucleus (VLM) [19]

nucleus (DRt) [20]

solitary tract [56]

neurons [51]

Medullary dorsal reticular

Acute pain Proenkephalin Medullary dorsal reticular

Inflammatory Proenkephalin Medullary ventral reticular

Inflammatory Proenkephalin Medullary dorsal reticular

antisense

Acute and Inflammatory pain NMDA antisense Medullary nucleus of the

neuropathic pain model Potassium channel (hKir2.1) Pontospinal noradrenergic

projections to the spinal cord [52].

Inflammatory and

Neuropathic Tyrosine Hydroxylase

Other viral vectors, namely adenoviruses, adeno-associated viruses and lentiviruses have been used to target the spinal cord, but unlike HSV-1 vectors which have been administered at the periphery following its natural route of retrograde transport to DRG neurons, these vectors were either directly injected into DRGs or trigeminal ganglion neurons, intrathecally or intraspinally (Table 1). The transgenes included in adenoviruses, adeno-associated viruses are similar to those used in HSV-1 vectors, namely opioids [17, 31], interleukins [27-29] and GAD [42]. Adeno-associated vectors have also carried transgenes that overexpress µ-opioid receptors [18] or block the expression of GTP cyclohydroxilase (GCH1) using small hairpin RNAs [30]. GCH1 is the rate-limiting enzyme of an essential co-factor for nitric oxide synthase (NOS), which modulates nociceptive transmission. Finally, lentiviral vectors have also been used in gene transfer studies directed to the spinal cord. Based on its ability to restrict transduction to the injection site, lentiviral vectors have been administered intraspinally in the dorsal horn to increase the levels of a neurotrophic factor (glial-derived neurotrofic factor, GDNF) [32] or decrease the expression of Nuclear Factor kB (NFκB), which regulates cellular inflammation responses [33]. In the latter study, microdelivery of an HIV pseudotyped lentiviral vector into the spinal dorsal horn led to a preferential transgene expression in glial cells. This shows that, besides the promoter, pseudotyping the vector is a way of directing transgene expression and glia is an important target in pain, inasmuch that chronic pain is associated to the activation of glial cells which produce algogenic mediators that exacerbate pain, namely NFκB. All of these approaches showed considerable analgesic efficacy and reduced side effects.

#### **4. Gene therapy targeting for pain: The challenge of targeting pain control circuits in the brain**

Abnormal descending pain modulation from the brain is a common feature of several chronic pain conditions, namely those characterized by widespread pain, like fibromyalgia, which derive from impairments in descending pain inhibition [50]. Studies with gene delivery into the brain (Table 2) are much scarcer than spinal cord deliveries. This is due both to the higher difficulty of surgical approaches to deliver the vectors into the brain and challenges to manipulate the complex brain neuronal circuits involved in pain modulation. The spinal cord constitutes a less invasive delivery route when the aim is to manipulate descending modula‐ tory pathways (Fig. 1). This delivery route was recently explored by injecting intraspinally an adenovirus vector targeting the expression of a potassium channel into noradrenergic pontospinal neurons, which decreased the activity of those pontospinal neurons and induced hyperalgesia [51]. These experiments confirm the pain inhibiting role of the noradrenergic projections to the spinal cord [52].

being deprived of major side-effects and preventing tolerance after repeated administrations of the vector [46]. Furthermore, opioid-based gene therapy can be very powerful in inducing analgesia if combined with administration of very low doses of classical opioids [46]. Besides opioid peptides, other transgenes were included in the HSV-1 vectors constructs. A transgene that increases the levels of the inhibitory neurotransmitter GABA, namely by overexpressing its synthetizing enzyme glutamate decarboxylase (GAD), induced analgesia in neuropathic pain models [26, 47]. HSV-1 based vectors have also been used to deliver transgenes that overexpress anti-inflammatory interleukins [24, 48] or the soluble receptor for tumor necrosis factor-α (TNF-α), which act as an antagonist of TNF-α in order to block its role as a proinflammatory mediator [25, 49]. A decrease in the levels of the α subunit of the voltage-gated sodium channel 1.7 (Nav 1.7) was also achieved using HSV-1 constructs but with the transgene

Other viral vectors, namely adenoviruses, adeno-associated viruses and lentiviruses have been used to target the spinal cord, but unlike HSV-1 vectors which have been administered at the periphery following its natural route of retrograde transport to DRG neurons, these vectors were either directly injected into DRGs or trigeminal ganglion neurons, intrathecally or intraspinally (Table 1). The transgenes included in adenoviruses, adeno-associated viruses are similar to those used in HSV-1 vectors, namely opioids [17, 31], interleukins [27-29] and GAD [42]. Adeno-associated vectors have also carried transgenes that overexpress µ-opioid receptors [18] or block the expression of GTP cyclohydroxilase (GCH1) using small hairpin RNAs [30]. GCH1 is the rate-limiting enzyme of an essential co-factor for nitric oxide synthase (NOS), which modulates nociceptive transmission. Finally, lentiviral vectors have also been used in gene transfer studies directed to the spinal cord. Based on its ability to restrict transduction to the injection site, lentiviral vectors have been administered intraspinally in the dorsal horn to increase the levels of a neurotrophic factor (glial-derived neurotrofic factor, GDNF) [32] or decrease the expression of Nuclear Factor kB (NFκB), which regulates cellular inflammation responses [33]. In the latter study, microdelivery of an HIV pseudotyped lentiviral vector into the spinal dorsal horn led to a preferential transgene expression in glial cells. This shows that, besides the promoter, pseudotyping the vector is a way of directing transgene expression and glia is an important target in pain, inasmuch that chronic pain is associated to the activation of glial cells which produce algogenic mediators that exacerbate pain, namely NFκB. All of these approaches showed considerable analgesic efficacy and

**4. Gene therapy targeting for pain: The challenge of targeting pain control**

Abnormal descending pain modulation from the brain is a common feature of several chronic pain conditions, namely those characterized by widespread pain, like fibromyalgia, which derive from impairments in descending pain inhibition [50]. Studies with gene delivery into the brain (Table 2) are much scarcer than spinal cord deliveries. This is due both to the higher difficulty of surgical approaches to deliver the vectors into the brain and challenges to

inserted in antisense orientation [16].

690 Gene Therapy - Tools and Potential Applications

reduced side effects.

**circuits in the brain**


**Table 2.** Summary of animal studies using viral vectors for gene transfer to pain control centers in the brain.

Gene transfer in the brain used almost exclusively HSV-1 vectors to overexpress opioid peptides [19-21, 38] and, in a much more limited extent, GAD [38]. Our research group has a large experience in gene transfer to pain control centres at the medulla oblongata, namely the dorsal reticular nucleus (DRt), the caudal ventrolateral medulla (VLM) and the nucleus of the solitary tract (NTS). These areas were elected based on the extensive neurobiological knowl‐ edge of their role in pain modulation [53, 54]. Overexpression of opioid precursors in the DRt and VLM induced analgesia in acute pain tests and models of sustained or chronic inflamma‐ tory pain [19, 20, 55]. Brain areas involved in pain control and which are of easier neurosurgical access are the amygdala and the rostral agranular insular cortex. Overexpression of opioid precursors in the central amygdalar nucleus [21] or GAD in the rostral agranular insular cortex induced analgesia in acute pain models [38]. Lentiviral vectors were delivered to the NTS, an area which is crucial in pain and cardiovascular integration, to decrease local expression of Nmethyl-D-aspartate (NMDA) receptor, a key receptor for the action of glutamate, and this approach was shown to decrease acute and inflammatory pain [56]. Since glutamate, is the most ubiquitous mediator of excitatory synaptic transmission in the central nervous system and NMDA receptors are also expressed by glial cells, the effects of gene therapy were restricted to NTS neurons by using the rat synapsin promoter.

Although it could be argued that this is due to lack of activity of the hCMV promoter in amygdalar and cortical neurons, other studies showed that hCMV is active in those neurons [21, 59]. These results rather point to a selective uptake of HSV-1 vectors injected in the brain parenchyma, probably due to interactions between neuronal receptors and glycoproteins of the HSV-1 envelope. By carefully mapping the brain areas exhibiting retrograde transport after HSV-1 injections in the brain using immunohistochemical detection of the gene reporter and in situ hybridization against the DNA of HSV-1, the problems of affecting brain afferents of

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The selective migration of HSV-1 in the brain can be a useful feature of the vector. After establishing the dynamics of the migration of HSV-1 in the brain after injection into the DRt (a facilitatory pain control centre of the brain), we used a tissue specific promoter (tyrosine hydroxylase-TH) to direct the expression of the vector to the noradrenergic afferents of the DRt (Fig. 3). Based on the analgesic effects of the administration of α1-adrenorecetor antago‐ nists into the DRt, the TH transgene was inserted in antisense orientation into the vector in order to decrease the levels of noradrenaline in the DRt [34]. A sustained analgesic effect was achieved in a model of neuropathic pain, which reproduces clinically relevant features of neuropathic pain. The fact that the analgesic effects were so long, lasting for 2 months with a single vector injection, and reversed several pain modalities, indicates that targeting pain control centres of the brain needs to be considered both in animal and pre-clinical studies.

**5. Gene therapy for chronic pain at the bedside: Human studies**

effects of reinoculation of the vector and assessment of the maximal dose [45, 62].

The progress of the clinical trials for cancer pain opened avenues to test gene therapy to block nociceptive transmission in the spinal cord in other pain conditions, such as painful diabetic neuropathy. This pain type, which is increasing to the pandemic occurrence of diabetes, is difficult to treat with conventional analgesics and only about one third of the patients achieve a 50% pain reduction beyond the placebo effect [63]. A clinical trial has recently been approved

The translational perspectives of the studies summarized in section 2, namely those using replication-defective HSV-1 vectors, favoured the approval of clinical trials for gene therapy for chronic pain. An important reinforcement of the proof-of-concept for the potential utility of HSV-based vector in rodent pain models was provided by equivalent studies performed in primates [36]. These studies were important since the translational perspectives of the rodent results were questioned for several reasons, such as the larger size of dermatomes of humans. The first clinical trial of gene therapy for pain was a safety and dose-escalation Phase I study in ten patients with mild to severe intractable pain due to terminal cancer [60]. The protocol consisted in the administration directly in the pain-reporting area of an HSV-1 replicationdefective vector containing the transgene of the precursor of enkephalin [61]. A dose-depend‐ ent analgesic effect was demonstrated with a reduction of pain scores lasting for at least 2 weeks and with no adverse effects. These encouraging results prompt to implement a Phase II trial in a larger cancer population and the study includes placebo controls, evaluation of the

the injected area can be circumvented.

There is a puzzling difference between gene therapy studies using HSV-1 vectors at the periphery or the brain. Whereas the ability for HSV-1 to migrate retrogradely is the main feature of studies at the periphery, the migration of HSV-1 in the brain is seldom evaluated. This can confound the effects of gene therapy on pain responses since the effect may derive from transduction of neurons that project to the injected area, and not at the targeted area itself. Our research group has pioneer work in studying the dynamics of HSV-1 migration in the brain after injections of the vector in pain control centres of the medulla oblongata, namely the caudal medulla oblongata (VLM) and the dorsal reticular nucleus (DRt). After injections of a HSV-1 vector expressing the lacZ reporter gene, under control of the human cytomegalovirus promoter (hCMV), in pain control centres of the medulla oblongata, migration in VLM and DRt afferents was detected [19, 55] (Fig. 2). However, not all the brain afferents of the VLM and DRt exhibited β-galactosidase (β-gal), the product of *lacZ* expression. For example, the amygdala and the cortex, which are important VLM and DRt afferents [57, 58] did not show neurons expressing β-gal.

**Figure 2.** Dynamics of HSV-1 migration in the brain after injection into the DRt. Photomicrographs of β-gal positive neurons in the cerebellum (A), the parabrachial complex (B), the locus *coeruleus* (C) and the VLM (D) at 7 days postinjection Scale bar D: 100 μm (photomicrographs A-C are at the same magnification).

Although it could be argued that this is due to lack of activity of the hCMV promoter in amygdalar and cortical neurons, other studies showed that hCMV is active in those neurons [21, 59]. These results rather point to a selective uptake of HSV-1 vectors injected in the brain parenchyma, probably due to interactions between neuronal receptors and glycoproteins of the HSV-1 envelope. By carefully mapping the brain areas exhibiting retrograde transport after HSV-1 injections in the brain using immunohistochemical detection of the gene reporter and in situ hybridization against the DNA of HSV-1, the problems of affecting brain afferents of the injected area can be circumvented.

and NMDA receptors are also expressed by glial cells, the effects of gene therapy were

There is a puzzling difference between gene therapy studies using HSV-1 vectors at the periphery or the brain. Whereas the ability for HSV-1 to migrate retrogradely is the main feature of studies at the periphery, the migration of HSV-1 in the brain is seldom evaluated. This can confound the effects of gene therapy on pain responses since the effect may derive from transduction of neurons that project to the injected area, and not at the targeted area itself. Our research group has pioneer work in studying the dynamics of HSV-1 migration in the brain after injections of the vector in pain control centres of the medulla oblongata, namely the caudal medulla oblongata (VLM) and the dorsal reticular nucleus (DRt). After injections of a HSV-1 vector expressing the lacZ reporter gene, under control of the human cytomegalovirus promoter (hCMV), in pain control centres of the medulla oblongata, migration in VLM and DRt afferents was detected [19, 55] (Fig. 2). However, not all the brain afferents of the VLM and DRt exhibited β-galactosidase (β-gal), the product of *lacZ* expression. For example, the amygdala and the cortex, which are important VLM and DRt afferents [57, 58] did not show

**Figure 2.** Dynamics of HSV-1 migration in the brain after injection into the DRt. Photomicrographs of β-gal positive neurons in the cerebellum (A), the parabrachial complex (B), the locus *coeruleus* (C) and the VLM (D) at 7 days post-

injection Scale bar D: 100 μm (photomicrographs A-C are at the same magnification).

restricted to NTS neurons by using the rat synapsin promoter.

neurons expressing β-gal.

692 Gene Therapy - Tools and Potential Applications

The selective migration of HSV-1 in the brain can be a useful feature of the vector. After establishing the dynamics of the migration of HSV-1 in the brain after injection into the DRt (a facilitatory pain control centre of the brain), we used a tissue specific promoter (tyrosine hydroxylase-TH) to direct the expression of the vector to the noradrenergic afferents of the DRt (Fig. 3). Based on the analgesic effects of the administration of α1-adrenorecetor antago‐ nists into the DRt, the TH transgene was inserted in antisense orientation into the vector in order to decrease the levels of noradrenaline in the DRt [34]. A sustained analgesic effect was achieved in a model of neuropathic pain, which reproduces clinically relevant features of neuropathic pain. The fact that the analgesic effects were so long, lasting for 2 months with a single vector injection, and reversed several pain modalities, indicates that targeting pain control centres of the brain needs to be considered both in animal and pre-clinical studies.

#### **5. Gene therapy for chronic pain at the bedside: Human studies**

The translational perspectives of the studies summarized in section 2, namely those using replication-defective HSV-1 vectors, favoured the approval of clinical trials for gene therapy for chronic pain. An important reinforcement of the proof-of-concept for the potential utility of HSV-based vector in rodent pain models was provided by equivalent studies performed in primates [36]. These studies were important since the translational perspectives of the rodent results were questioned for several reasons, such as the larger size of dermatomes of humans. The first clinical trial of gene therapy for pain was a safety and dose-escalation Phase I study in ten patients with mild to severe intractable pain due to terminal cancer [60]. The protocol consisted in the administration directly in the pain-reporting area of an HSV-1 replicationdefective vector containing the transgene of the precursor of enkephalin [61]. A dose-depend‐ ent analgesic effect was demonstrated with a reduction of pain scores lasting for at least 2 weeks and with no adverse effects. These encouraging results prompt to implement a Phase II trial in a larger cancer population and the study includes placebo controls, evaluation of the effects of reinoculation of the vector and assessment of the maximal dose [45, 62].

The progress of the clinical trials for cancer pain opened avenues to test gene therapy to block nociceptive transmission in the spinal cord in other pain conditions, such as painful diabetic neuropathy. This pain type, which is increasing to the pandemic occurrence of diabetes, is difficult to treat with conventional analgesics and only about one third of the patients achieve a 50% pain reduction beyond the placebo effect [63]. A clinical trial has recently been approved

circuits in the brain namely in what concerns the functional changes induced by the chronic pain condition in order to select the best brain areas to target to maximise the balance between

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The advances of gene therapy in other diseases of the nervous system rather than pain will be crucial to define the future of gene therapy for chronic pain, namely by the improve‐ ments in the delivery systems. Studies which improved the efficacy of non-viral vectors already inspired the construction of a non-viral, non plasmid immunologically defined gene expression (MIDGE) vector that overexpress β-endorphin and induced analgesia after injection into inflamed paws by increasing the concentration of β-endorphin in leukocytes [66]. Since chronic pain requires long-term transgene expression, the duration of the activity of promoters needs to be increased. It could be useful to design constructs that are activated only when pain lasts for longer periods and rises over a certain threshold. This would allow treating chronic pain but still preserve acute pain as an alert signal. An interesting possibility could be to control the activity of the promoter using inducible promoters, which have been used in gene therapy studies other than pain. The activity of these promoters can be induced exogenously, for example, by antibiotics. An ingenious idea was recently applied by using a ligand (glycine) which normally is not expressed in DRG neurons but can be administered to activate HSV-1 vectors to express glycine receptors in animal models of somatic and visceral pain [67]. Besides the vectors and the promoters, an election of effective transgenes for chronic pain will be important to define the future of gene therapy for chronic pain. Transgenes for opioid peptides have been overused in gene therapy studies in animal models but long-term treatments with classic opioids may induce pain, a phenomena known as opioid-induced hyperalgesia [68]. By achieving more sustained and strong transgene expression, it is possible that opioid-induced hyperalgesia could also be induced by gene therapy. New transgenes should be considered in future studies of gene therapy for chronic pain. Based on the role of the vanilloid receptor TRPV-1 (Transient Receptor Potential channel Vanilloid 1) as a pro-nociceptive cationic channel involved in pain signalling, and the clinical relevance of desensitization of TRPV1 receptors [69], this may be an important target molecule in the future. By decreasing the expression of protein kinase C-epsilon (PKC), which phosphorylates TRPV1 receptors, it was possible to induce analgesia in animal models [70]. Even more challenging is the possibility of targeting pain control centres in the brain using gene therapy. These studies will continue for large years to focus on animal pain models in order to determine neurobiological effects of chronic pain installation in pain control centres, using gene therapy as a method to prevent those changes. Finally, the emergent new field in pain research of genetics of pain has recently provided data which may explain the higher susceptibility of some persons to develop chronic pain [71]. Due to its versatility and the possibility of direct gene targeting, gene therapy can be the perfect tool to verify if the holy grail of a personalized pain treatment

efficacy and risk.

**6. Future challenges**

can be implemented.

**Figure 3.** HSV-1 injected at the DRt transduces noradrenergic afferents of the nucleus (**A**, **B**). Photomicrographs repre‐ senting double-labeled neurons for β-gal and TH (yellowish) in the locus *coeruleus* (**A**) and the A5 noradrenergic cell group (**B**). β-gal positive neurons are shown in red and TH positive neurons are shown in green (**A**). Scale bar in B: 40 µm (A is at the same magnification). The insertion of TH in antisense orientation into HSV-1 (THa vector) induced anal‐ gesia in the spared nerve injury (SNI) model of neuropathic pain (**C**, **D**). THa induced a sustained attenuation of me‐ chanical hyperalgesia evaluated by the pin-prick test (**C)** and cold allodynia evaluated by the acetone test (**D**). THa and the control vector were injected at time 0, i.e., 2 weeks after SNI induction. Data are presented as mean ± SEM (n=6 for each group); \*P<0.05, \*\*P<0.01, \*\*\*P<0.001 THa- *vs*. control- vector.

to use an HSV-1 vector that overexpress GAD to relief painful diabetic neuropathy [45]. Other therapeutic transgenes are being considered for future clinical trials of gene therapy, namely the overexpression of interleukins [45]. The future of gene therapy for chronic pain in humans will depend on the results of the clinical trials that are currently being performed but the promising results obtained so far indicate that gene therapy will add to the armamentarium of available pain treatments.

The application of gene therapy to block nociceptive transmission at supraspinal levels has been proposed by several pain specialists [64]. However, most experimental studies dealing with gene delivery at the brain were directed to pain control areas of the medulla oblongata, which are of difficult neurosurgical approach since they are in close vicinity to areas involved in the control of vital functions, such as cardiovascular and respiratory controls. Moving the focus of the gene delivery studies to areas that are more easy to approach may be useful namely in the context of widespread chronic pain, such as fibromyalgia and complex regional pain syndrome [65]. This can only be considered after a thorough characterization of the pain control circuits in the brain namely in what concerns the functional changes induced by the chronic pain condition in order to select the best brain areas to target to maximise the balance between efficacy and risk.

#### **6. Future challenges**

to use an HSV-1 vector that overexpress GAD to relief painful diabetic neuropathy [45]. Other therapeutic transgenes are being considered for future clinical trials of gene therapy, namely the overexpression of interleukins [45]. The future of gene therapy for chronic pain in humans will depend on the results of the clinical trials that are currently being performed but the promising results obtained so far indicate that gene therapy will add to the armamentarium

**Figure 3.** HSV-1 injected at the DRt transduces noradrenergic afferents of the nucleus (**A**, **B**). Photomicrographs repre‐ senting double-labeled neurons for β-gal and TH (yellowish) in the locus *coeruleus* (**A**) and the A5 noradrenergic cell group (**B**). β-gal positive neurons are shown in red and TH positive neurons are shown in green (**A**). Scale bar in B: 40 µm (A is at the same magnification). The insertion of TH in antisense orientation into HSV-1 (THa vector) induced anal‐ gesia in the spared nerve injury (SNI) model of neuropathic pain (**C**, **D**). THa induced a sustained attenuation of me‐ chanical hyperalgesia evaluated by the pin-prick test (**C)** and cold allodynia evaluated by the acetone test (**D**). THa and the control vector were injected at time 0, i.e., 2 weeks after SNI induction. Data are presented as mean ± SEM (n=6 for

The application of gene therapy to block nociceptive transmission at supraspinal levels has been proposed by several pain specialists [64]. However, most experimental studies dealing with gene delivery at the brain were directed to pain control areas of the medulla oblongata, which are of difficult neurosurgical approach since they are in close vicinity to areas involved in the control of vital functions, such as cardiovascular and respiratory controls. Moving the focus of the gene delivery studies to areas that are more easy to approach may be useful namely in the context of widespread chronic pain, such as fibromyalgia and complex regional pain syndrome [65]. This can only be considered after a thorough characterization of the pain control

of available pain treatments.

694 Gene Therapy - Tools and Potential Applications

each group); \*P<0.05, \*\*P<0.01, \*\*\*P<0.001 THa- *vs*. control- vector.

The advances of gene therapy in other diseases of the nervous system rather than pain will be crucial to define the future of gene therapy for chronic pain, namely by the improve‐ ments in the delivery systems. Studies which improved the efficacy of non-viral vectors already inspired the construction of a non-viral, non plasmid immunologically defined gene expression (MIDGE) vector that overexpress β-endorphin and induced analgesia after injection into inflamed paws by increasing the concentration of β-endorphin in leukocytes [66]. Since chronic pain requires long-term transgene expression, the duration of the activity of promoters needs to be increased. It could be useful to design constructs that are activated only when pain lasts for longer periods and rises over a certain threshold. This would allow treating chronic pain but still preserve acute pain as an alert signal. An interesting possibility could be to control the activity of the promoter using inducible promoters, which have been used in gene therapy studies other than pain. The activity of these promoters can be induced exogenously, for example, by antibiotics. An ingenious idea was recently applied by using a ligand (glycine) which normally is not expressed in DRG neurons but can be administered to activate HSV-1 vectors to express glycine receptors in animal models of somatic and visceral pain [67]. Besides the vectors and the promoters, an election of effective transgenes for chronic pain will be important to define the future of gene therapy for chronic pain. Transgenes for opioid peptides have been overused in gene therapy studies in animal models but long-term treatments with classic opioids may induce pain, a phenomena known as opioid-induced hyperalgesia [68]. By achieving more sustained and strong transgene expression, it is possible that opioid-induced hyperalgesia could also be induced by gene therapy. New transgenes should be considered in future studies of gene therapy for chronic pain. Based on the role of the vanilloid receptor TRPV-1 (Transient Receptor Potential channel Vanilloid 1) as a pro-nociceptive cationic channel involved in pain signalling, and the clinical relevance of desensitization of TRPV1 receptors [69], this may be an important target molecule in the future. By decreasing the expression of protein kinase C-epsilon (PKC), which phosphorylates TRPV1 receptors, it was possible to induce analgesia in animal models [70]. Even more challenging is the possibility of targeting pain control centres in the brain using gene therapy. These studies will continue for large years to focus on animal pain models in order to determine neurobiological effects of chronic pain installation in pain control centres, using gene therapy as a method to prevent those changes. Finally, the emergent new field in pain research of genetics of pain has recently provided data which may explain the higher susceptibility of some persons to develop chronic pain [71]. Due to its versatility and the possibility of direct gene targeting, gene therapy can be the perfect tool to verify if the holy grail of a personalized pain treatment can be implemented.

#### **Financial support**

This work was supported by FCT and COMPTE project PTDC/SAU-NSC/110954/2009.

**Author details**

Portugal

**References**

Isaura Tavares1,2 and Isabel Martins1,2

\*Address all correspondence to: isatav@med.up.pt

can Academy of Pain Management.

lation. Neuron, 2007. 55(3): p. 377-91.

2012. 13(8): p. 715-24.

Suppl): p. S105-20.

p. 267-84.

and treatment. Eur J Pain, 2006. 10(4): p. 287-333.

view of the literature. Pain, 1998. 77(3): p. 231-9.

1 Department of Experimental Biology, Faculty of Medicine of Porto, University of Porto,

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697

[1] Merskey, H., et al., Pain terms: a list with definitions and notes on usage. Recom‐

[2] Classification of chronic pain. Descriptions of chronic pain syndromes and defini‐ tions of pain terms. Prepared by the International Association for the Study of Pain,

[3] Thienhaus, O. and B.E. Cole, Classification of pain. Weiner RS, 6th ed. 2002: Ameri‐

[4] Breivik, H., et al., Survey of chronic pain in Europe: prevalence, impact on daily life,

[5] Verhaak, P.F., et al., Prevalence of chronic benign pain disorder among adults: a re‐

[6] Gaskin, D.J. and P. Richard, The economic costs of pain in the United States. J Pain,

[7] Benyamin, R., et al., Opioid complications and side effects. Pain Physician, 2008. 11(2

[8] Mao, J., M.S. Gold, and M.M. Backonja, Combination drug therapy for chronic pain:

[9] Tracey, I. and P.W. Mantyh, The cerebral signature for pain perception and its modu‐

[10] Basbaum, A.I., et al., Cellular and molecular mechanisms of pain. Cell, 2009. 139(2):

[11] Heinricher, M.M., et al., Descending control of nociception: Specificity, recruitment

mended by the IASP Subcommittee on Taxonomy. Pain, 1979. 6(3): p. 249.

Subcommittee on Taxonomy. Pain Suppl, 1986. 3: p. S1-226.

a call for more clinical studies. J Pain, 2011. 12(2): p. 157-66.

and plasticity. Brain Res Rev, 2009. 60(1): p. 214-25.

2 IBMC - Instituto de Biologia Molecular e Celular, University of Porto, Portugal

#### **List of abbreviations**


#### **Author details**

**Financial support**

696 Gene Therapy - Tools and Potential Applications

**List of abbreviations**

DRG- Dorsal root ganglion

DRt- Dorsal reticular nucleus

GABA- γ-amminobutiric acid

GAD- glutamate decarboxylase

hCMV- Human cytomegalovirus

IL2- Interleukin 2

IL4- Interleukin 4

IL10- Interleukin 10

NFκB- Nuclear Factor kB

NMDA- N-methyl-D-aspartate

PKC- Protein kinase C-epsilon

sTNFRs- tumor necrosis factor-α

VLM- Caudal ventrolateral medulla

TH- Tyrosine hydroxylase

NTS- Nucleus of the solitary tract

HSV-1- Herpes Simplex Virus type 1

Nav 1.7- Voltage-gated sodium channel 1.7

IASP- International Association for the Study of Pain

MIDGE- Non plasmid immunologically defined gene expression

shGCH1- small hairpin RNAs for GTP cyclohydroxilase

TRPV-1- Transient Receptor Potential channel Vanilloid 1

GDNF- Glial-derived neurotrofic factor

β-gal- β-galactosidase

This work was supported by FCT and COMPTE project PTDC/SAU-NSC/110954/2009.

Isaura Tavares1,2 and Isabel Martins1,2

\*Address all correspondence to: isatav@med.up.pt

1 Department of Experimental Biology, Faculty of Medicine of Porto, University of Porto, Portugal

2 IBMC - Instituto de Biologia Molecular e Celular, University of Porto, Portugal

#### **References**


[12] Hioki, H., et al., Efficient gene transduction of neurons by lentivirus with enhanced neuron-specific promoters. Gene Ther, 2007. 14(11): p. 872-82.

[27] Milligan, E.D., et al., Controlling pathological pain by adenovirally driven spinal pro‐ duction of the anti-inflammatory cytokine, interleukin-10. Eur J Neurosci, 2005. 21(8):

Gene Therapy for Chronic Pain Management

http://dx.doi.org/10.5772/55050

699

[28] Yao, M.Z., et al., Adenovirus-mediated interleukin-2 gene therapy of nociception.

[29] Milligan, E.D., et al., Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10. Mol Pain, 2005. 1: p. 9.

[30] Kim, S.J., et al., Effective relief of neuropathic pain by adeno-associated virus-mediat‐ ed expression of a small hairpin RNA against GTP cyclohydrolase 1. Mol Pain, 2009.

[31] Storek, B., et al., Sensory neuron targeting by self-complementary AAV8 via lumbar puncture for chronic pain. Proc Natl Acad Sci U S A, 2008. 105(3): p. 1055-60.

[32] Pezet, S., et al., Reversal of neurochemical changes and pain-related behavior in a model of neuropathic pain using modified lentiviral vectors expressing GDNF. Mol

[33] Meunier, A., et al., Lentiviral-mediated targeted NF-kappaB blockade in dorsal spi‐ nal cord glia attenuates sciatic nerve injury-induced neuropathic pain in the rat. Mol

[34] Martins, I., et al., Reversal of neuropathic pain by HSV-1-mediated decrease of nora‐ drenaline in a pain facilitatory area of the brain. Pain, 2010. 151(1): p. 137-45.

[35] Wilson, S.P., et al., Antihyperalgesic effects of infection with a preproenkephalin-en‐

[36] Yeomans, D.C., et al., Recombinant herpes vector-mediated analgesia in a primate

[37] Yokoyama, H., et al., Gene therapy for bladder overactivity and nociception with herpes simplex virus vectors expressing preproenkephalin. Hum Gene Ther, 2009.

[38] Jasmin, L., et al., Analgesia and hyperalgesia from GABA-mediated modulation of

[39] Cabanero, D., et al., The pro-nociceptive effects of remifentanil or surgical injury in mice are associated with a decrease in delta-opioid receptor mRNA levels: Preven‐ tion of the nociceptive response by on-site delivery of enkephalins. Pain, 2009.

[40] Goss, J.R., et al., Herpes vector-mediated expression of proenkephalin reduces bone

coding herpes virus. Proc Natl Acad Sci U S A, 1999. 96(6): p. 3211-6.

model of hyperalgesia. Mol Ther, 2006. 13(3): p. 589-97.

the cerebral cortex. Nature, 2003. 424(6946): p. 316-20.

cancer pain. Ann Neurol, 2002. 52(5): p. 662-5.

p. 2136-48.

5: p. 67.

Gene Ther, 2003. 10(16): p. 1392-9.

Ther, 2006. 13(6): p. 1101-9.

Ther, 2007. 15(4): p. 687-97.

20(1): p. 63-71.

141(1-2): p. 88-96.


[27] Milligan, E.D., et al., Controlling pathological pain by adenovirally driven spinal pro‐ duction of the anti-inflammatory cytokine, interleukin-10. Eur J Neurosci, 2005. 21(8): p. 2136-48.

[12] Hioki, H., et al., Efficient gene transduction of neurons by lentivirus with enhanced

[13] Porreca, F., M.H. Ossipov, and G.F. Gebhart, Chronic pain and medullary descend‐

[14] Braz, J., et al., Therapeutic efficacy in experimental polyarthritis of viral-driven enke‐ phalin overproduction in sensory neurons. J Neurosci, 2001. 21(20): p. 7881-8.

[15] Hao, S., et al., Effects of transgene-mediated endomorphin-2 in inflammatory pain.

[16] Yeomans, D.C., et al., Decrease in inflammatory hyperalgesia by herpes vector-medi‐ ated knockdown of Nav1.7 sodium channels in primary afferents. Hum Gene Ther,

[17] Finegold, A.A., A.J. Mannes, and M.J. Iadarola, A paracrine paradigm for in vivo gene therapy in the central nervous system: treatment of chronic pain. Hum Gene

[18] Xu, Y., et al., Adeno-associated viral transfer of opioid receptor gene to primary sen‐ sory neurons: a strategy to increase opioid antinociception. Proc Natl Acad Sci U S A,

[19] Martins, I., et al., Reversal of inflammatory pain by HSV-1-mediated overexpression of enkephalin in the caudal ventrolateral medulla. Eur J Pain, 2011. 15(10): p. 1008-14.

[20] Pinto, M., et al., Opioids modulate pain facilitation from the dorsal reticular nucleus.

[21] Kang, W., et al., Herpes virus-mediated preproenkephalin gene transfer to the amyg‐

[22] Lu, Y., et al., Treatment of inflamed pancreas with enkephalin encoding HSV-1 re‐ combinant vector reduces inflammatory damage and behavioral sequelae. Mol Ther,

[23] Wolfe, D., et al., Engineering an endomorphin-2 gene for use in neuropathic pain

[24] Hao, S., et al., HSV-mediated expression of interleukin-4 in dorsal root ganglion neu‐

[25] Peng, X.M., et al., Tumor necrosis factor-alpha contributes to below-level neuropathic

[26] Hao, S., et al., Gene transfer of glutamic acid decarboxylase reduces neuropathic

neuron-specific promoters. Gene Ther, 2007. 14(11): p. 872-82.

ing facilitation. Trends Neurosci, 2002. 25(6): p. 319-25.

Eur J Pain, 2009. 13(4): p. 380-6.

2005. 16(2): p. 271-7.

698 Gene Therapy - Tools and Potential Applications

Ther, 1999. 10(7): p. 1251-7.

2003. 100(10): p. 6204-9.

2007. 15(10): p. 1812-9.

Mol Cell Neurosci, 2008. 39(4): p. 508-18.

therapy. Pain, 2007. 133(1-3): p. 29-38.

pain. Ann Neurol, 2005. 57(6): p. 914-8.

dala is antinociceptive. Brain Res, 1998. 792(1): p. 133-5.

rons reduces neuropathic pain. Mol Pain, 2006. 2: p. 6.

pain after spinal cord injury. Ann Neurol, 2006. 59(5): p. 843-51.


[41] Meunier, A., et al., Attenuation of pain-related behavior in a rat model of trigeminal neuropathic pain by viral-driven enkephalin overproduction in trigeminal ganglion neurons. Mol Ther, 2005. 11(4): p. 608-16.

[56] Marques-Lopes, J., et al., Decrease in the expression of N-methyl-D-aspartate recep‐ tors in the nucleus tractus solitarii induces antinociception and increases blood pres‐

Gene Therapy for Chronic Pain Management

http://dx.doi.org/10.5772/55050

701

[57] Almeida, A., et al., Brain afferents to the medullary dorsal reticular nucleus: a retro‐ grade and anterograde tracing study in the rat. Eur J Neurosci, 2002. 16(1): p. 81-95.

[58] Cobos, A., et al., Brain afferents to the lateral caudal ventrolateral medulla: a retro‐ grade and anterograde tracing study in the rat. Neuroscience, 2003. 120(2): p. 485-98.

[59] Chiocca, E.A., et al., Transfer and expression of the lacZ gene in rat brain neurons mediated by herpes simplex virus mutants. New Biol, 1990. 2(8): p. 739-46.

[60] Fink, D.J., et al., Gene therapy for pain: results of a phase I clinical trial. Ann Neurol,

[61] Akil, H., et al., Endogenous opioids: biology and function. Annu Rev Neurosci, 1984.

[62] Fink, D.J. and D. Wolfe, Gene Therapy for Pain: A Perspective. Pain Manag, 2011.

[63] Turk, D.C., Clinical effectiveness and cost-effectiveness of treatments for patients

[64] Wu, C.L., et al., Gene therapy for the management of pain: Part I: Methods and strat‐

[65] Weiss, K. and N.M. Boulis, Herpes Simplex Virus-Based Gene Therapies for Chronic

[66] Machelska, H., et al., Peripheral non-viral MIDGE vector-driven delivery of beta-en‐

[67] Goss, J.R., et al., HSV delivery of a ligand-regulated endogenous ion channel gene to sensory neurons results in pain control following channel activation. Mol Ther, 2011.

[68] Lee, M., et al., A comprehensive review of opioid-induced hyperalgesia. Pain Physi‐

[69] Kissin, I. and A. Szallasi, Therapeutic targeting of TRPV1 by resiniferatoxin, from preclinical studies to clinical trials. Curr Top Med Chem, 2011. 11(17): p. 2159-70.

[70] Srinivasan, R., et al., Protein kinase C epsilon contributes to basal and sensitizing re‐ sponses of TRPV1 to capsaicin in rat dorsal root ganglion neurons. Eur J Neurosci,

[71] Doehring, A., G. Geisslinger, and J. Lotsch, Epigenetics in pain and analgesia: an im‐

with chronic pain. Clin J Pain, 2002. 18(6): p. 355-65.

Pain. J Pain Palliat Care Pharmacother, 2012. 26(3): p. 291-3.

dorphin in inflammatory pain. Mol Pain, 2009. 5: p. 72.

minent research field. Eur J Pain, 2011. 15(1): p. 11-6.

egies. Anesthesiology, 2001. 94(6): p. 1119-32.

sure. J Neurosci Res, 2012. 90(2): p. 356-66.

2011. 70(2): p. 207-12.

7: p. 223-55.

1(5): p. 379-381.

19(3): p. 500-6.

cian, 2011. 14(2): p. 145-61.

2008. 28(7): p. 1241-54.


[56] Marques-Lopes, J., et al., Decrease in the expression of N-methyl-D-aspartate recep‐ tors in the nucleus tractus solitarii induces antinociception and increases blood pres‐ sure. J Neurosci Res, 2012. 90(2): p. 356-66.

[41] Meunier, A., et al., Attenuation of pain-related behavior in a rat model of trigeminal neuropathic pain by viral-driven enkephalin overproduction in trigeminal ganglion

[42] Vit, J.P., et al., Adenovector GAD65 gene delivery into the rat trigeminal ganglion

[43] Glorioso, J.C. and D.J. Fink, Herpes vector-mediated gene transfer in the treatment of

[44] Goss, J.R., M.S. Gold, and J.C. Glorioso, HSV vector-mediated modification of pri‐ mary nociceptor afferents: an approach to inhibit chronic pain. Gene Ther, 2009.

[45] Goins, W.F., J.B. Cohen, and J.C. Glorioso, Gene therapy for the treatment of chronic

[46] Hao, S., et al., Transgene-mediated enkephalin release enhances the effect of mor‐ phine and evades tolerance to produce a sustained antiallodynic effect in neuropath‐

[47] Liu, J., et al., Peripherally delivered glutamic acid decarboxylase gene therapy for

[48] Zhou, Z., et al., HSV-mediated transfer of interleukin-10 reduces inflammatory pain through modulation of membrane tumor necrosis factor alpha in spinal cord micro‐

[49] Hao, S., et al., Gene transfer to interfere with TNFalpha signaling in neuropathic

[50] Staud, R., Evidence for shared pain mechanisms in osteoarthritis, low back pain, and

[51] Howorth, P.W., et al., Retrograde viral vector-mediated inhibition of pontospinal noradrenergic neurons causes hyperalgesia in rats. J Neurosci, 2009. 29(41): p.

[52] Pertovaara, A., Noradrenergic pain modulation. Prog Neurobiol, 2006. 80(2): p. 53-83.

[53] Tavares, I. and D. Lima, The caudal ventrolateral medulla as an important inhibitory modulator of pain transmission in the spinal cord. J Pain, 2002. 3(5): p. 337-46.

[54] Lima, D. and A. Almeida, The medullary dorsal reticular nucleus as a pronociceptive

[55] Martins, I., et al., Dynamic of migration of HSV-1 from a medullary pronociceptive centre: antinociception by overexpression of the preproenkephalin transgene. Eur J

centre of the pain control system. Prog Neurobiol, 2002. 66(2): p. 81-108.

peripheral nervous system pain. Neurobiol Dis, 2012. 48(2): p. 255-70.

neurons. Mol Ther, 2005. 11(4): p. 608-16.

chronic pain. Mol Ther, 2009. 17(1): p. 13-8.

ic pain. Pain, 2003. 102(1-2): p. 135-42.

glia. Gene Ther, 2008. 15(3): p. 183-90.

pain. Gene Ther, 2007. 14(13): p. 1010-6.

Neurosci, 2008. 28(10): p. 2075-83.

16(4): p. 493-501.

700 Gene Therapy - Tools and Potential Applications

12855-64.

produces orofacial analgesia. Mol Pain, 2009. 5: p. 42.

spinal cord injury pain. Mol Ther, 2004. 10(1): p. 57-66.

fibromyalgia. Curr Rheumatol Rep, 2011. 13(6): p. 513-20.


**Chapter 29**

**Insulin Trafficking in a Glucose Responsive Engineered**

Type I diabetes is caused by the autoimmune destruction of pancreatic beta (β) cells [1]. Cur‐ rent treatment requires multiple daily injections of insulin to control blood glucose levels. Tight glucose control lowers, but does not eliminate, the onset of diabetic complications, which greatly reduce the quality and longevity of life for patients. Transplantation of pan‐ creatic tissue as a treatment is restricted by the scarcity of donors and the requirement for lifelong immunosuppression to preserve the graft, which carries adverse side-effects. This is of particular concern as Type 1 diabetes predominantly affects children. Lack of glucose con‐ trol could be overcome by genetically engineering "an artificial β-cell" that is capable of syn‐ thesising, storing and secreting insulin in response to metabolic signals. The donor cell type must be readily accessible and capable of being engineered to synthesise, process, store and

The cell type of choice for the gene therapy of diabetes is not the β-cell. β-cells are greatly reduced or absent in people with Type I diabetes because of their autoimmune destruction. This fact will actively work against gene therapists trying to derive surrogate β-cells from

> © 2013 Simpson et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Human Liver Cell Line is Regulated by the Interaction**

**of ATP-Sensitive Potassium Channels and Voltage-**

**Gated Calcium Channels**

Ann M. Simpson, M. Anne Swan, Guo Jun Liu, Chang Tao, Bronwyn A O'Brien, Edwin Ch'ng,

Bernard E. Tuch and Graham M. Nicholson

Additional information is available at the end of the chapter

secrete insulin under physiological conditions.

http://dx.doi.org/10.5772/52839

**1. Introduction**

Leticia M. Castro, Julia Ting, Zehra Elgundi, Tony An, Mark Lutherborrow, Fraser Torpy, Donald K. Martin,

**Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-Sensitive Potassium Channels and Voltage-Gated Calcium Channels**

Ann M. Simpson, M. Anne Swan, Guo Jun Liu, Chang Tao, Bronwyn A O'Brien, Edwin Ch'ng, Leticia M. Castro, Julia Ting, Zehra Elgundi, Tony An, Mark Lutherborrow, Fraser Torpy, Donald K. Martin, Bernard E. Tuch and Graham M. Nicholson

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52839

**1. Introduction**

Type I diabetes is caused by the autoimmune destruction of pancreatic beta (β) cells [1]. Cur‐ rent treatment requires multiple daily injections of insulin to control blood glucose levels. Tight glucose control lowers, but does not eliminate, the onset of diabetic complications, which greatly reduce the quality and longevity of life for patients. Transplantation of pan‐ creatic tissue as a treatment is restricted by the scarcity of donors and the requirement for lifelong immunosuppression to preserve the graft, which carries adverse side-effects. This is of particular concern as Type 1 diabetes predominantly affects children. Lack of glucose con‐ trol could be overcome by genetically engineering "an artificial β-cell" that is capable of syn‐ thesising, storing and secreting insulin in response to metabolic signals. The donor cell type must be readily accessible and capable of being engineered to synthesise, process, store and secrete insulin under physiological conditions.

The cell type of choice for the gene therapy of diabetes is not the β-cell. β-cells are greatly reduced or absent in people with Type I diabetes because of their autoimmune destruction. This fact will actively work against gene therapists trying to derive surrogate β-cells from

© 2013 Simpson et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. stem cells. There are innumerable theories describing putative mechanisms for preventing a patient's immune system from re-attacking transplanted β-cells, but the fact that the basic processes of islet cell attack have not been fully elucidated makes the search for relevant genes problematic. Thus, the engineering of non-pancreatic β-cells to synthesise, process, store and secrete insulin has several advantages, the most important of which is the ready availability of donor cells. If non β-cells from a diabetic individual can be engineered to pro‐ duce insulin, then cellular rejection is less likely to occur since donor and recipient are autol‐ ogous. In pursuit of this goal, hepatocytes have been shown to be suitable target cells for the generation of artificial β-cells [2-9]. Moreover, liver cells that produce insulin may not be prone to autoimmune attack [10]. The suitability of hepatocytes as a β-cell replacement is at‐ tributable, in part, to their inherent glucose responsiveness and their embryonic origin from the same endodermal precursor cells as the β-cell. Most importantly, liver cells express the high capacity glucose transporter, GLUT 2 [11], and the high capacity phosphorylation en‐ zyme, glucokinase [12], which constitute the key elements of the "glucose sensing system" that regulates insulin release from pancreatic β-cells in response to small extracellular nu‐ trient changes.

For an insulin-producing liver cell to be of maximum benefit *in vivo* it must be capable of rapid responsive secretion of biologically active insulin. This characteristic demands that ar‐ tificial β-cells process proinsulin to insulin and store it in granules. Our previous studies have shown that the insertion of genes encoding for insulin and the glucose transporter, GLUT2, into the HEPG2 human hepatoma cell line, resulted in synthesis and storage of (pro)insulin in structures resembling the secretory granules of pancreatic β-cells (HEPG2/ins/g), and the near physiological secretion of (pro)insulin in response to glucose [2, 3]. Similar to pancreatic β-cells, HEPG2ins/g cells responded to glucose via signalling path‐ ways dependent upon KATP channels [27]. Therefore, expression of both insulin and GLUT2 in HEPG2 liver cells appeared to be sufficient for the generation of functional KATP channels, unlike the parental cell line that required pharmacological stimulation to activate the KATP channels [28]. It has previously been shown that stable transfection of the insulin gene into the human liver cell line, Huh7 (which endogenously expresses GLUT2), resulted in synthe‐ sis, storage, and regulated release of insulin to the physiological stimulus glucose (Huh7ins cells) [7]. Huh7ins cells are more akin to pancreatic β-cells than HEPG2/ins/g cells. They ex‐ press a range of β-cell transcription factors [7, 29] and possess storage granules that cleave proinsulin to biologically active diarginyl insulin, due to the expression of the proconvertas‐ es PC1 and PC2 [7]. As Huh7ins cells also rapidly secrete insulin in a tightly regulated man‐ ner in response to glucose, the Huh7ins cells were able to reverse chemically induced diabetes when transplanted into an animal model [7], which HEPG2ins/g cells [3] failed to

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

705

This chapter will detail the use of electrophysiological and biochemical techniques to show that Huh7ins cells respond to a glucose stimulus by closure of KATP channels and activation of CaV channels, which is an analogous mechanism to pancreatic β-cells. Patch-clamp elec‐ trophysiology of Huh7ins cells yielded current-voltage (*I-V*) curves that indicated the pres‐ ence of potassium-selective currents; in contrast, currents recorded from Huh7 cells were non-selective. The presence of functional ATP-sensitive potassium (KATP) channels and volt‐ age-gated calcium (CaV) channels was further validated by measurement of acute insulin se‐ cretion by Huh7ins cells in response to pharmacological channel inhibitors and activators and by calcium imaging and patch-clamp electrophysiology experiments. Molecular analy‐ ses were used to confirm that the Huh7ins cells express CaV and all the subunits of KATP channels. The secretion of insulin from granules in live Huh7ins cells was revealed by confo‐ cal microscopy which allowed visualization of secretion of insulin to a zinquin probe or an insulin-enhanced green fluorescent protein (EGFP) fusion protein (EGFP-ins). The glucose responsive mechanism that we observed in the Huh7ins cells was the same as that reported for the pancreatic β-cell line, MIN6 [30]. Prior to this study, the physiological interaction of KATP channels and CaV channels had never been shown in liver cells engineered to secrete insulin. As the biochemical properties of Huh7ins cells are akin to those of pancreatic β-cells, engineering hepatocytes in this way opens a promising avenue for the ultimate replacement of the endogenous β-cell function that is lost in Type I diabetes, by modifying a patient's own liver cells to become artificial β-cells. This is the first study that clearly delineates the control of insulin trafficking in a functioning artificial β-cell line that was derived from a hu‐

achieve [Tuch, unpublished results].

man liver cell.

In pancreatic β-cells, a small increase in plasma glucose concentration stimulates significant insulin secretion. Therefore, glucose is the major modulator of β-cell function and this behav‐ iour must be mimicked in insulin-secreting liver cells. In pancreatic β-cells, KATP channels, which are composed of four sulphonylurea receptor (SUR) subunits and four inwardly-recti‐ fying potassium channel (KIR6.2) subunits [13-18], maintain resting membrane potentials and link plasma glucose concentrations to the insulin secretory machinery. The triggering path‐ way for insulin release begins with the uptake of glucose via the glucose carrier, GLUT2, and an acceleration of metabolism, such that glucose is used to generate ATP. An increase in the absolute intracellular concentration of ATP, with respect to ADP, stimulates the closure of KATP channels [19, 20]. Potassium conductance of the plasma membrane decreases, allowing a background current to shift the membrane potential away from the equilibrium potential for K+ , thus depolarising the membrane. Consequently, the pancreatic β-cell is able to translate metabolic signals to electrical signals, the latter regulating insulin secretion. Lack of function‐ al KATP channels in insulin-secreting NES2Y cells resulted in the unregulated release of insu‐ lin, which was restored by expression of both KIR6.2 and SUR1 [21].

When depolarisation of the pancreatic β-cell reaches the threshold for activation of L-type (CaV1.3), and to a lesser extent P/Q (CaV1.2) and T-type (CaV3.x) voltage-gated calcium chan‐ nels, these open allowing Ca2+ influx down their electrochemical gradient [22]. The opening of CaV channels is intermittent, fluctuating with the membrane potential, therefore generat‐ ing oscillations in the intracellular (cytosolic) calcium concentration ([Ca2+]i ), which, in turn, triggers pulsatile insulin secretion. In β-cells, elevation of [Ca2+]i occurs via the release of Ca2+ from intracellular stores (endoplasmic reticulum, mitochondria and secretory granules) and/or influx of extracellular Ca2+ through CaV channels [23, 24]. No functional CaV channels have been previously described in liver cells, however the presence of an α1-subunit lacking the voltage sensor has been reported in the rat liver cell line H4IIE [25] and an L-type α1 subunit has been detected at low levels in rat liver by RT-PCR [26].

#### Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-Sensitive Potassium Channels and Voltage-Gated Calcium Channels http://dx.doi.org/10.5772/52839 705

stem cells. There are innumerable theories describing putative mechanisms for preventing a patient's immune system from re-attacking transplanted β-cells, but the fact that the basic processes of islet cell attack have not been fully elucidated makes the search for relevant genes problematic. Thus, the engineering of non-pancreatic β-cells to synthesise, process, store and secrete insulin has several advantages, the most important of which is the ready availability of donor cells. If non β-cells from a diabetic individual can be engineered to pro‐ duce insulin, then cellular rejection is less likely to occur since donor and recipient are autol‐ ogous. In pursuit of this goal, hepatocytes have been shown to be suitable target cells for the generation of artificial β-cells [2-9]. Moreover, liver cells that produce insulin may not be prone to autoimmune attack [10]. The suitability of hepatocytes as a β-cell replacement is at‐ tributable, in part, to their inherent glucose responsiveness and their embryonic origin from the same endodermal precursor cells as the β-cell. Most importantly, liver cells express the high capacity glucose transporter, GLUT 2 [11], and the high capacity phosphorylation en‐ zyme, glucokinase [12], which constitute the key elements of the "glucose sensing system" that regulates insulin release from pancreatic β-cells in response to small extracellular nu‐

In pancreatic β-cells, a small increase in plasma glucose concentration stimulates significant insulin secretion. Therefore, glucose is the major modulator of β-cell function and this behav‐ iour must be mimicked in insulin-secreting liver cells. In pancreatic β-cells, KATP channels, which are composed of four sulphonylurea receptor (SUR) subunits and four inwardly-recti‐ fying potassium channel (KIR6.2) subunits [13-18], maintain resting membrane potentials and link plasma glucose concentrations to the insulin secretory machinery. The triggering path‐ way for insulin release begins with the uptake of glucose via the glucose carrier, GLUT2, and an acceleration of metabolism, such that glucose is used to generate ATP. An increase in the absolute intracellular concentration of ATP, with respect to ADP, stimulates the closure of KATP channels [19, 20]. Potassium conductance of the plasma membrane decreases, allowing a background current to shift the membrane potential away from the equilibrium potential for

, thus depolarising the membrane. Consequently, the pancreatic β-cell is able to translate metabolic signals to electrical signals, the latter regulating insulin secretion. Lack of function‐ al KATP channels in insulin-secreting NES2Y cells resulted in the unregulated release of insu‐

When depolarisation of the pancreatic β-cell reaches the threshold for activation of L-type (CaV1.3), and to a lesser extent P/Q (CaV1.2) and T-type (CaV3.x) voltage-gated calcium chan‐ nels, these open allowing Ca2+ influx down their electrochemical gradient [22]. The opening of CaV channels is intermittent, fluctuating with the membrane potential, therefore generat‐

from intracellular stores (endoplasmic reticulum, mitochondria and secretory granules) and/or influx of extracellular Ca2+ through CaV channels [23, 24]. No functional CaV channels have been previously described in liver cells, however the presence of an α1-subunit lacking the voltage sensor has been reported in the rat liver cell line H4IIE [25] and an L-type α1-

), which, in turn,

occurs via the release of Ca2+

lin, which was restored by expression of both KIR6.2 and SUR1 [21].

triggers pulsatile insulin secretion. In β-cells, elevation of [Ca2+]i

subunit has been detected at low levels in rat liver by RT-PCR [26].

ing oscillations in the intracellular (cytosolic) calcium concentration ([Ca2+]i

trient changes.

704 Gene Therapy - Tools and Potential Applications

K+

For an insulin-producing liver cell to be of maximum benefit *in vivo* it must be capable of rapid responsive secretion of biologically active insulin. This characteristic demands that ar‐ tificial β-cells process proinsulin to insulin and store it in granules. Our previous studies have shown that the insertion of genes encoding for insulin and the glucose transporter, GLUT2, into the HEPG2 human hepatoma cell line, resulted in synthesis and storage of (pro)insulin in structures resembling the secretory granules of pancreatic β-cells (HEPG2/ins/g), and the near physiological secretion of (pro)insulin in response to glucose [2, 3]. Similar to pancreatic β-cells, HEPG2ins/g cells responded to glucose via signalling path‐ ways dependent upon KATP channels [27]. Therefore, expression of both insulin and GLUT2 in HEPG2 liver cells appeared to be sufficient for the generation of functional KATP channels, unlike the parental cell line that required pharmacological stimulation to activate the KATP channels [28]. It has previously been shown that stable transfection of the insulin gene into the human liver cell line, Huh7 (which endogenously expresses GLUT2), resulted in synthe‐ sis, storage, and regulated release of insulin to the physiological stimulus glucose (Huh7ins cells) [7]. Huh7ins cells are more akin to pancreatic β-cells than HEPG2/ins/g cells. They ex‐ press a range of β-cell transcription factors [7, 29] and possess storage granules that cleave proinsulin to biologically active diarginyl insulin, due to the expression of the proconvertas‐ es PC1 and PC2 [7]. As Huh7ins cells also rapidly secrete insulin in a tightly regulated man‐ ner in response to glucose, the Huh7ins cells were able to reverse chemically induced diabetes when transplanted into an animal model [7], which HEPG2ins/g cells [3] failed to achieve [Tuch, unpublished results].

This chapter will detail the use of electrophysiological and biochemical techniques to show that Huh7ins cells respond to a glucose stimulus by closure of KATP channels and activation of CaV channels, which is an analogous mechanism to pancreatic β-cells. Patch-clamp elec‐ trophysiology of Huh7ins cells yielded current-voltage (*I-V*) curves that indicated the pres‐ ence of potassium-selective currents; in contrast, currents recorded from Huh7 cells were non-selective. The presence of functional ATP-sensitive potassium (KATP) channels and volt‐ age-gated calcium (CaV) channels was further validated by measurement of acute insulin se‐ cretion by Huh7ins cells in response to pharmacological channel inhibitors and activators and by calcium imaging and patch-clamp electrophysiology experiments. Molecular analy‐ ses were used to confirm that the Huh7ins cells express CaV and all the subunits of KATP channels. The secretion of insulin from granules in live Huh7ins cells was revealed by confo‐ cal microscopy which allowed visualization of secretion of insulin to a zinquin probe or an insulin-enhanced green fluorescent protein (EGFP) fusion protein (EGFP-ins). The glucose responsive mechanism that we observed in the Huh7ins cells was the same as that reported for the pancreatic β-cell line, MIN6 [30]. Prior to this study, the physiological interaction of KATP channels and CaV channels had never been shown in liver cells engineered to secrete insulin. As the biochemical properties of Huh7ins cells are akin to those of pancreatic β-cells, engineering hepatocytes in this way opens a promising avenue for the ultimate replacement of the endogenous β-cell function that is lost in Type I diabetes, by modifying a patient's own liver cells to become artificial β-cells. This is the first study that clearly delineates the control of insulin trafficking in a functioning artificial β-cell line that was derived from a hu‐ man liver cell.

#### **2. Understanding the mechanism by which liver-derived artificial beta cells respond to glucose and pharmacological stimulators and inhibitors of insulin secretion:**

(DMEM) supplemented with 10% v/v fetal calf serum (FCS) (Trace Biosciences, Australia) in 5% CO2 at 37°C. Although of murine origin, MIN6 cells are one of the few β-cell lines that are responsive to glucose in the physiological range, and, accordingly provide an establish‐ ed β-cell-like cell line for comparative purposes [30]. MIN6 cells were grown in DMEM sup‐ plemented with 15% v/v FCS (37°C, 5% CO2). For the Huh7ins cell line, the selective

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

707

**Figure 1.** Sensitivity of potassium channels to glucose and diazoxide in Huh7ins cells. The upper three sets of current traces in panels A and B show superimposed families of whole-cell K+ currents elicited by 450 ms test pulses from –80 to +80 mV in 10-mV steps. Lower graphs show the I-V relationship of the late current measured at the end of the test pulse and shows the mean ± SEM at each potential. (A) Glucose (20 mM) reversibly inhibited the potassium currents of MIN6 and Huh7ins cells (left and centre columns; n = 4), however glucose did not affect the non-selective currents of Huh7 cells (right column; n = 3). (B) The channel opener diazoxide (100 μM) reversibly increased the potassium cur‐ rents of MIN6 and Huh7ins cells (left and centre columns; n = 9), but did not effect the non-selective currents of Huh7

To determine if functional KATP channels were present in Huh7ins or Huh7 cells, KATP chan‐ nel currents were recorded using whole-cell patch-clamp electrophysiology, with MIN6 cells

cells (right column; n = 3).

antibiotic G418 (0.55 mg/ml) was added to maintain stable transfectants.

The mechanisms by which liver-derived artificial β-cells respond to glucose are poorly un‐ derstood. Indeed, the majority of engineered insulin-secreting liver cells lack a truly regulat‐ ed pathway of insulin release [31]. As pancreas and liver are derived from the same endodermal origin, the capacity of liver cells to differentiate into cells bearing pancreatic characteristics is well documented. A number of studies have shown that the expression of β-cell transcription factors in liver cells leads to pancreatic transdifferentiation, glucoseregulated insulin secretion and reversal of diabetes [4-7, 9, 32, 33]. Spontaneous pancreatic transdifferentiation and glucose-regulated insulin secretion have also been shown in dedif‐ ferentiated liver cells that express β-cell transcription factors such as the HEPG2ins/g and Huh7ins liver cell lines [3, 7, 9], as well as liver cells that have experienced a metabolic insult such as hepatic oval cells cultured in high glucose [34]. Consistent with this, our laboratory has shown spontaneous pancreatic transdifferentiation in hyperglycaemic rat livers and re‐ versal of diabetes following the delivery of the insulin gene using a lentiviral vector [8]. Oth‐ er recent studies in our laboratory have employed the H4IIE liver cell line, which does not express β-cell transcription factors and lacks a regulated pathway of insulin release [31]. When engineered to express the β-cell transcription factor *Neurod1* and rat insulin (H4IIEins/ ND), H4IIE cells underwent pancreatic transdifferentiation and glucose-regulated insulin se‐ cretion from secretory granules. However, when *Neurod1* alone was expressed, an array of β-cell transcription factors and pancreatic hormones were expressed, but glucose-regulated insulin-secretion was not observed [9]. The Huh7 parent cell line, from which the insulinsecreting Huh7ins cells were derived, represents an ideal candidate for the engineering of an artificial β-cell. These cells possess several characteristics inherent to β-cells but not intrinsic to primary hepatocytes, such as the expression of β-cell transcription factors *Neurod1* [7], *Pdx1*, *Nkx2-2, Nkx6-1, Neurog3* and *Pax 6* [29]. Importantly, however, the process of transfec‐ tion with insulin resulted in the formation of insulin secretory granules and the develop‐ ment of a regulated insulin secretory pathway [7] as was observed in the rodent H4IIEins/ND cells. Results of a mechanistic microarray analysis comparing Huh and Huh7ins cells following insulin transfection indicated that the formation of secretory gran‐ ules and the development of a regulated secretory pathway was likely related to a protein interaction or posttranslational effect in combination with increased gene expression of se‐ cretory granule proteins such as chromgranin A [29]..

#### **2.1. Huh7ins cells possess potassium-selective plasma membrane channels**

Huh7 (parental human liver cell line), Huh7ins (parental human liver cell line transfected with human insulin cDNA) [7] were maintained in Dulbecco's Modified Eagle's Medium Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-Sensitive Potassium Channels and Voltage-Gated Calcium Channels http://dx.doi.org/10.5772/52839 707

(DMEM) supplemented with 10% v/v fetal calf serum (FCS) (Trace Biosciences, Australia) in 5% CO2 at 37°C. Although of murine origin, MIN6 cells are one of the few β-cell lines that are responsive to glucose in the physiological range, and, accordingly provide an establish‐ ed β-cell-like cell line for comparative purposes [30]. MIN6 cells were grown in DMEM sup‐ plemented with 15% v/v FCS (37°C, 5% CO2). For the Huh7ins cell line, the selective antibiotic G418 (0.55 mg/ml) was added to maintain stable transfectants.

**2. Understanding the mechanism by which liver-derived artificial beta cells respond to glucose and pharmacological stimulators and inhibitors**

The mechanisms by which liver-derived artificial β-cells respond to glucose are poorly un‐ derstood. Indeed, the majority of engineered insulin-secreting liver cells lack a truly regulat‐ ed pathway of insulin release [31]. As pancreas and liver are derived from the same endodermal origin, the capacity of liver cells to differentiate into cells bearing pancreatic characteristics is well documented. A number of studies have shown that the expression of β-cell transcription factors in liver cells leads to pancreatic transdifferentiation, glucoseregulated insulin secretion and reversal of diabetes [4-7, 9, 32, 33]. Spontaneous pancreatic transdifferentiation and glucose-regulated insulin secretion have also been shown in dedif‐ ferentiated liver cells that express β-cell transcription factors such as the HEPG2ins/g and Huh7ins liver cell lines [3, 7, 9], as well as liver cells that have experienced a metabolic insult such as hepatic oval cells cultured in high glucose [34]. Consistent with this, our laboratory has shown spontaneous pancreatic transdifferentiation in hyperglycaemic rat livers and re‐ versal of diabetes following the delivery of the insulin gene using a lentiviral vector [8]. Oth‐ er recent studies in our laboratory have employed the H4IIE liver cell line, which does not express β-cell transcription factors and lacks a regulated pathway of insulin release [31]. When engineered to express the β-cell transcription factor *Neurod1* and rat insulin (H4IIEins/ ND), H4IIE cells underwent pancreatic transdifferentiation and glucose-regulated insulin se‐ cretion from secretory granules. However, when *Neurod1* alone was expressed, an array of β-cell transcription factors and pancreatic hormones were expressed, but glucose-regulated insulin-secretion was not observed [9]. The Huh7 parent cell line, from which the insulinsecreting Huh7ins cells were derived, represents an ideal candidate for the engineering of an artificial β-cell. These cells possess several characteristics inherent to β-cells but not intrinsic to primary hepatocytes, such as the expression of β-cell transcription factors *Neurod1* [7], *Pdx1*, *Nkx2-2, Nkx6-1, Neurog3* and *Pax 6* [29]. Importantly, however, the process of transfec‐ tion with insulin resulted in the formation of insulin secretory granules and the develop‐ ment of a regulated insulin secretory pathway [7] as was observed in the rodent H4IIEins/ND cells. Results of a mechanistic microarray analysis comparing Huh and Huh7ins cells following insulin transfection indicated that the formation of secretory gran‐ ules and the development of a regulated secretory pathway was likely related to a protein interaction or posttranslational effect in combination with increased gene expression of se‐

**of insulin secretion:**

706 Gene Therapy - Tools and Potential Applications

cretory granule proteins such as chromgranin A [29]..

**channels**

**2.1. Huh7ins cells possess potassium-selective plasma membrane**

Huh7 (parental human liver cell line), Huh7ins (parental human liver cell line transfected with human insulin cDNA) [7] were maintained in Dulbecco's Modified Eagle's Medium

**Figure 1.** Sensitivity of potassium channels to glucose and diazoxide in Huh7ins cells. The upper three sets of current traces in panels A and B show superimposed families of whole-cell K+ currents elicited by 450 ms test pulses from –80 to +80 mV in 10-mV steps. Lower graphs show the I-V relationship of the late current measured at the end of the test pulse and shows the mean ± SEM at each potential. (A) Glucose (20 mM) reversibly inhibited the potassium currents of MIN6 and Huh7ins cells (left and centre columns; n = 4), however glucose did not affect the non-selective currents of Huh7 cells (right column; n = 3). (B) The channel opener diazoxide (100 μM) reversibly increased the potassium cur‐ rents of MIN6 and Huh7ins cells (left and centre columns; n = 9), but did not effect the non-selective currents of Huh7 cells (right column; n = 3).

To determine if functional KATP channels were present in Huh7ins or Huh7 cells, KATP chan‐ nel currents were recorded using whole-cell patch-clamp electrophysiology, with MIN6 cells being included as the positive control. Whole-cell patch-clamp recordings from potassium channels were made as previously described [27]. Cells grown on coverslips were transfer‐ red to a recording chamber and were perfused with a bath solution of the following compo‐ sition (in mM): 140 Na acetate, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4). Patch pipettes were filled with an internal solution containing (in mM): 136 K acetate, 5 CsF, 5 KCl, 1 EGTA, 10 HEPES (pH 7.3). For inside-out patch-clamp recordings, the patch pipette was filled with (in mM): 135 NaCl, 5 KCl, 5 CaCl2, 2 MgSO4, 5 HEPES or a high K+ extracellular solution in which KCl replaced NaCl. The bath solution contained (in mM): 107 KCl, 11 EGTA, 2 MgSO4, 1 CaCl2, 11 HEPES (pH 7.2). For CaV channel analyses the bath solution contained (in mM): 115 NaCl, 5 KCl, 10 CaCl2, 10 HEPES, 2 D-glucose and 100 µM tetrodotoxin (pH 7.4) and the internal solution contained (in mM): 10 CsCl, 115 Cs aspartate, 2.5 EGTA, 10 HEPES (pH 7.2). Chan‐ nel currents were amplified and filtered using a MultiClamp amplifier (Molecular Devices, MDS Analytical Technologies, Toronto, Canada) and sampled on-line using a Digidata 1322 (A/D converter) and pClamp 8.2 software program (Molecular Devices).

for a K+


Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

versed close to 0 mV, with a mean slope conductance of 48.5 pS (–80 to –10 mV). In compari‐

for KATP channels in Huh7 cells indicated that currents from these cells were non-selective as the reversal potential was closer to 0 mV (Figure 2B). As secretory granules require KATP channels for the appropriate release of insulin [35, 36], it is likely that Huh7ins cells also con‐

**2.2. Secretion of insulin observed in real time in response to glucose and**

In order to observe, in real time, the secretion of insulin from granules in response to stimu‐ lators and inhibitors of insulin secretion by confocal microscopy, Huh7 and MIN 6 cells were engineered to express insulin fused to EGFP. To accomplish this, human insulin cDNA pC2 (a gift from Dr. M. Walker, Weizmann Institute, Israel) [7] was cloned into the multicloning site of the pEGFP-N1 vector (Clontech, CA, USA). As there were no intervening stop codons, EGFP/insulin (EGFPins) was expressed as a fusion protein, which allowed visuali‐ zation and localization of the fusion protein in cells. The construct (20 µg) or vector alone was introduced into Huh7 and MIN6 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the instructions of the manufacturer. To obtain stable transfectants, contain‐ ing the construct (EGFPins) or empty vector (EGFP), G418 antibiotic (0.55 mg/ml) (Gibco Laboratories, Grand Island, NY) was added to the culture medium after 48 h. Media and G418 were changed every 2–3 days. After 3–4 weeks of selection, 25 colonies were chosen and screened for production of insulin by radioimmunoassay (RIA) [7] and EGFP by fluo‐ rescence microscopy. Human c-peptide was measured as previously described [8]. Clones were expanded into mass cultures and maintained in G418 selection media (37°C, 5% CO2). Huh7-EGFP (parental human liver cell line expressing EGFP) and Huh7-EGFPins (parental human liver cell line expressing EGFPins) cells were maintained in DMEM supplemented with 10% v/v fetal calf serum (FCS) (Trace Biosciences, Australia) in 5% CO2 at 37°C. MIN6- EGFP (EGFP-expressing MIN6 cells) and MIN6-EGFPins (EGFPins-expressing MIN6 cells) cells were grown in DMEM supplemented with 15% v/v FCS (37°C, 5% CO2). For these

son, the slope conductance was reduced to 12.4 pS (0 to +60 mV) when the [K+

tained KATP channels located intracellularly at the secretory granule membrane.

transfected cell lines, the selective antibiotic G418 (0.55 mg/ml) was added.

To compare the function of Huh7-EGFPins and Huh7ins cells, chronic insulin secretion, in‐ sulin storage, and glucose-responsiveness were assessed. Acute insulin secretion was meas‐ ured by static stimulation in basal medium consisting of PBS supplemented with (in mM): 1 CaCl2, 20 HEPES, 2 mg/ml BSA, 1.0 D-glucose; pH 7.4, as previously described [7]. Insulin was measured by RIA using human or rodent standards as previously described [7]. To as‐ say insulin content, insulin was extracted from cells using 0.18 N HCl in 70% ethanol for 18 h at 4°C, as previously described [7]. To assess the quantity of human as compared to rodent insulin secreted by MIN6-EGFPins cells, a commercial RIA for human insulin (Linco Re‐

to 5 mM, indicating that the channel was K+

**KATP channel blockers**

] re‐

709

]o was reduced

http://dx.doi.org/10.5772/52839


Sensitive Potassium Channels and Voltage-Gated Calcium Channels

The electrophysiological properties of the KATP channel in the Huh7ins cells closely resemble those reported for normal pancreatic β-cells [19]. The outward potassium currents of MIN6 and Huh7ins cells were sensitive to glucose and inhibited by perfusing 20 mM glucose for 5 min, with partial recovery of current amplitude after the washout of glucose for 10 min. In contrast, the non-selective outward and inward currents of Huh7 cells were not altered by the addition of 20 mM glucose (Figure 1A). The outward potassium currents of MIN6 and Huh7ins cells were also reversibly increased by perfusing with the KATP channel opener, di‐ azoxide, 100 µM (Figure 1B), whereas the non-selective currents of Huh7 cells were unaffect‐ ed by diazoxide.

**Figure 2.** I-V curves of Huh7ins and Huh7 cells. (A) Mean current-voltage relations for inside-out patches of Huh7ins cells exposed to an external K+ concentration of either 140 mM or 5 mM K+ (n = 6). (B) Using an internal and external K <sup>+</sup> concentration of 5 mM and 140 mM respectively, the reversal potential (Erev) of Huh7 whole-cell currents (n = 6), was approximately 0 mV, indicating a non-selective current. Values represent means ± SEM.

Further support for the presence of functional KATP channels in Huh7ins cells was obtained by analysis of current-voltage (*I*-*V*) relationships of single channel currents, which had simi‐ lar kinetics to that of pancreatic β-cells. Recordings were made from inside-out patches ex‐ posed to 140 mM [K+ ]i and either 140 mM K+ [K+ ]o or 5 mM K+ [K+ ]o. As would be expected for a K+ -selective channel, the single channel currents recorded with symmetrical [K+ ] re‐ versed close to 0 mV, with a mean slope conductance of 48.5 pS (–80 to –10 mV). In compari‐ son, the slope conductance was reduced to 12.4 pS (0 to +60 mV) when the [K+ ]o was reduced to 5 mM, indicating that the channel was K+ -selective (Figure 2A). In contrast the *I-V* curve for KATP channels in Huh7 cells indicated that currents from these cells were non-selective as the reversal potential was closer to 0 mV (Figure 2B). As secretory granules require KATP channels for the appropriate release of insulin [35, 36], it is likely that Huh7ins cells also con‐ tained KATP channels located intracellularly at the secretory granule membrane.

being included as the positive control. Whole-cell patch-clamp recordings from potassium channels were made as previously described [27]. Cells grown on coverslips were transfer‐ red to a recording chamber and were perfused with a bath solution of the following compo‐ sition (in mM): 140 Na acetate, 1 CaCl2, 1 MgCl2, 10 HEPES (pH 7.4). Patch pipettes were filled with an internal solution containing (in mM): 136 K acetate, 5 CsF, 5 KCl, 1 EGTA, 10 HEPES (pH 7.3). For inside-out patch-clamp recordings, the patch pipette was filled with (in mM):

replaced NaCl. The bath solution contained (in mM): 107 KCl, 11 EGTA, 2 MgSO4, 1 CaCl2, 11 HEPES (pH 7.2). For CaV channel analyses the bath solution contained (in mM): 115 NaCl, 5 KCl, 10 CaCl2, 10 HEPES, 2 D-glucose and 100 µM tetrodotoxin (pH 7.4) and the internal solution contained (in mM): 10 CsCl, 115 Cs aspartate, 2.5 EGTA, 10 HEPES (pH 7.2). Chan‐ nel currents were amplified and filtered using a MultiClamp amplifier (Molecular Devices, MDS Analytical Technologies, Toronto, Canada) and sampled on-line using a Digidata 1322

The electrophysiological properties of the KATP channel in the Huh7ins cells closely resemble those reported for normal pancreatic β-cells [19]. The outward potassium currents of MIN6 and Huh7ins cells were sensitive to glucose and inhibited by perfusing 20 mM glucose for 5 min, with partial recovery of current amplitude after the washout of glucose for 10 min. In contrast, the non-selective outward and inward currents of Huh7 cells were not altered by the addition of 20 mM glucose (Figure 1A). The outward potassium currents of MIN6 and Huh7ins cells were also reversibly increased by perfusing with the KATP channel opener, di‐ azoxide, 100 µM (Figure 1B), whereas the non-selective currents of Huh7 cells were unaffect‐

**Figure 2.** I-V curves of Huh7ins and Huh7 cells. (A) Mean current-voltage relations for inside-out patches of Huh7ins cells exposed to an external K+ concentration of either 140 mM or 5 mM K+ (n = 6). (B) Using an internal and external K <sup>+</sup> concentration of 5 mM and 140 mM respectively, the reversal potential (Erev) of Huh7 whole-cell currents (n = 6), was

Further support for the presence of functional KATP channels in Huh7ins cells was obtained by analysis of current-voltage (*I*-*V*) relationships of single channel currents, which had simi‐ lar kinetics to that of pancreatic β-cells. Recordings were made from inside-out patches ex‐

]o or 5 mM K+ [K+

]o. As would be expected

approximately 0 mV, indicating a non-selective current. Values represent means ± SEM.

and either 140 mM K+ [K+

extracellular solution in which KCl

135 NaCl, 5 KCl, 5 CaCl2, 2 MgSO4, 5 HEPES or a high K+

708 Gene Therapy - Tools and Potential Applications

ed by diazoxide.

posed to 140 mM [K+

]i

(A/D converter) and pClamp 8.2 software program (Molecular Devices).

### **2.2. Secretion of insulin observed in real time in response to glucose and KATP channel blockers**

In order to observe, in real time, the secretion of insulin from granules in response to stimu‐ lators and inhibitors of insulin secretion by confocal microscopy, Huh7 and MIN 6 cells were engineered to express insulin fused to EGFP. To accomplish this, human insulin cDNA pC2 (a gift from Dr. M. Walker, Weizmann Institute, Israel) [7] was cloned into the multicloning site of the pEGFP-N1 vector (Clontech, CA, USA). As there were no intervening stop codons, EGFP/insulin (EGFPins) was expressed as a fusion protein, which allowed visuali‐ zation and localization of the fusion protein in cells. The construct (20 µg) or vector alone was introduced into Huh7 and MIN6 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA), following the instructions of the manufacturer. To obtain stable transfectants, contain‐ ing the construct (EGFPins) or empty vector (EGFP), G418 antibiotic (0.55 mg/ml) (Gibco Laboratories, Grand Island, NY) was added to the culture medium after 48 h. Media and G418 were changed every 2–3 days. After 3–4 weeks of selection, 25 colonies were chosen and screened for production of insulin by radioimmunoassay (RIA) [7] and EGFP by fluo‐ rescence microscopy. Human c-peptide was measured as previously described [8]. Clones were expanded into mass cultures and maintained in G418 selection media (37°C, 5% CO2). Huh7-EGFP (parental human liver cell line expressing EGFP) and Huh7-EGFPins (parental human liver cell line expressing EGFPins) cells were maintained in DMEM supplemented with 10% v/v fetal calf serum (FCS) (Trace Biosciences, Australia) in 5% CO2 at 37°C. MIN6- EGFP (EGFP-expressing MIN6 cells) and MIN6-EGFPins (EGFPins-expressing MIN6 cells) cells were grown in DMEM supplemented with 15% v/v FCS (37°C, 5% CO2). For these transfected cell lines, the selective antibiotic G418 (0.55 mg/ml) was added.

To compare the function of Huh7-EGFPins and Huh7ins cells, chronic insulin secretion, in‐ sulin storage, and glucose-responsiveness were assessed. Acute insulin secretion was meas‐ ured by static stimulation in basal medium consisting of PBS supplemented with (in mM): 1 CaCl2, 20 HEPES, 2 mg/ml BSA, 1.0 D-glucose; pH 7.4, as previously described [7]. Insulin was measured by RIA using human or rodent standards as previously described [7]. To as‐ say insulin content, insulin was extracted from cells using 0.18 N HCl in 70% ethanol for 18 h at 4°C, as previously described [7]. To assess the quantity of human as compared to rodent insulin secreted by MIN6-EGFPins cells, a commercial RIA for human insulin (Linco Re‐ search, MO, USA), was used. This has less than 1% and 6% cross-reactivity with rodent insu‐ lin and human proinsulin, respectively.

initial time point. Confocal microscopy detected intracellular EGFP-ins or Zinquin-E as punctuate fluorescence, which was indicative of insulin stored within secretion granules.

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

711

**Figure 3.** Confocal microscopic visualization of HuH7-EGFPins cells after exposure to glucose and diazoxide. (A) Huh7- EGFPins and (B) MIN6-EGFPins cells were incubated in DMEM containing 5 mM glucose (CLSM medium), then stimu‐ lated with glucose (20 mM, G) and diazoxide (150 μM, D). Images were recorded in CLSM medium at 0, 10 and 20 min after glucose addition. At 20 min, cells were placed in CLSM medium containing diazoxide and images were recorded at 10 and 20 min after diazoxide exposure (bars = 10 μm). Normalized EGFP density indicated that (C) Huh7-EGFPins (n = 60) and (D) MIN6-EGFPins cells (n = 42) showed decreasing EGFP density after addition of glucose, whereas (E) Huh7-EGFP (n = 19) and (F) MIN6-EGFP cells (n = 12) showed increasing EGFP density after addition of glucose. Values

For statistical analysis of all the confocal measurements described below SPSS version 11.5 (SPSS Inc) was used to determine a one-way analysis of variance after testing for homogene‐ ity of variance using the Levene statistic. Huh7-EGFPins and MIN6-EGFPins cells respond‐ ed in the same way to 20 mM glucose after 10 and 15 min, with loss of fluorescence from the ROI indicative of insulin secretion (Figure 3A-D). There was no significant difference be‐ tween the response of Huh7-EGFPins and MIN6-EGFPins cells at 10 min (*p*> 0.5, *n* = 60) and 15 min (*p* ≥ 0.7, *n* = 42). The MIN6-EGFPins cells responded more rapidly to the glucose stimulus than the Huh7ins-EGFP cells, with close to maximum loss of fluorescence achieved at 5 min, but after 10 min the two cell lines had achieved the same response level (Figure 3C-D). When diazoxide (150 µM) was added to cells that had been stimulated by 20 mM glu‐ cose for 20 min, cytoplasmic fluorescence accumulated, as the release of insulin from

represent the mean ± SEM.

Of the 25 clones of Huh7-EGFPins isolated for analysis, insulin secretion differed 3-fold (0.11 ± 0.2 *vs*. 0.32 ± 0.2 pmol insulin/106 cells/24 h; *n* = 6) and insulin storage varied 2-fold (3.4 ± 1.2 *vs*. 7.1 ± 0.3 pmol insulin/106 cells; *n* = 6). Subsequently, six clones which secreted and stored the highest levels of insulin and exhibited consistently bright EGFP fluorescence, were examined for glucose responsiveness. Whilst all clones were glucose responsive, one clone (clone 16) was most comparable to Huh7ins cells [7] as it secreted equal amounts of insulin over a 24 h period (0.32 ± 0.2 vs. 0.30 ± 0.1 pmol insulin/106 cells for Huh7ins cells; *n* = 6). Insulin storage was also comparable between the two cell lines with Huh7-EGFPins (clone 16) and Huh7ins cells storing 7.1 ± 0.3 and 7.0 ± 0.2 pmol/106 cells (*n* = 6), respectively. Glucose concentration-response curves for the Huh7-EGFPins (clone 16) and Huh7ins cell lines were also determined and revealed that there was no significant difference to previous‐ ly published values [7] (data not shown). Levels of human proinsulin (not insulin) were 11.4 ± 1.2% of total insulin (*n* = 6). Human c-peptide levels were 1.0 ± 0.4% of total insulin activity (*n* = 6). Therefore clone 16 was used for all subsequent analyses, and is referred to as Huh7- EGFPins hereafter. As expected, Huh7-EGFP cells did not synthesize, store nor secrete insu‐ lin. Examination of the insulin secreted chronically by MIN6-EGFPins cells revealed that 20.5 ± 2.3% (*n* = 6) was of human origin, the remainder being rodent insulin. Of the insulin stored by MIN6-EGFPins cells, 17.9 ± 2.4% (*n* = 6) was human insulin. As expected, all of the insulin stored and secreted by MIN6-EGFP cells was of rodent origin. These data suggest that MIN6 cells handled EGFPins in a similar fashion to native rodent insulin.

In order to perform confocal microscopy, cells were plated on coverslips (Marienfeld superi‐ or 22 mm diameter) and grown for 2–4 days. Each coverslip was inserted into a Perspex cell chamber, sealed with silicone grease, and overlaid with 1 ml DMEM containing 5 mM glu‐ cose (confocal scanning laser microscope [CSLM] medium). For Zinquin-E (zinquin ester, ethyl[2-methyl-8-*p*-toluenesulphonamido-6-quinolyloxy]acetate) staining, cells were incu‐ bated at 37°C for 30 min with CSLM medium containing 25 µM zinquin E (Luminis Pty Ltd, Australia), as previously described [27]. After incubation, cells were rinsed with CSLM me‐ dium before recording confocal images with a Leica TCSNT (Wetzlar, Germany) with an in‐ verted microscope (Leica DMRBE). Cells were imaged with a UV laser, oil 100x (N.A.1.4 UV-corrected Planapo) or oil 63x (N.A.1.32 UV-corrected Planapo). Emissions were collected with a BP490/440 filter. For analyses of stable transfectants expressing EGFP, incubation with a fluorescent probe was not required. These cells were imaged with an Ar/Kr laser and DP488/568 dichroic and emissions were collected with a BP525/550 filter.

CSLM medium or test solutions containing glibenclamide (20 µM), or diazoxide (150 µM) in CSLM medium, or DMEM containing 20 mM glucose, were exchanged at 37°C. Density measurements on images were performed using the public domain NIH Image program [37].

Defined regions of interest (ROI) for individual cells (10–30 cells per experiment) were fol‐ lowed through a time series before, and after, addition of test solutions. All values were nor‐ malized by subtracting the initial density, before addition of the test solution, from all the measurements in the series for each individual ROI to give a value of zero density for the


search, MO, USA), was used. This has less than 1% and 6% cross-reactivity with rodent insu‐

Of the 25 clones of Huh7-EGFPins isolated for analysis, insulin secretion differed 3-fold (0.11

stored the highest levels of insulin and exhibited consistently bright EGFP fluorescence, were examined for glucose responsiveness. Whilst all clones were glucose responsive, one clone (clone 16) was most comparable to Huh7ins cells [7] as it secreted equal amounts of

6). Insulin storage was also comparable between the two cell lines with Huh7-EGFPins

Glucose concentration-response curves for the Huh7-EGFPins (clone 16) and Huh7ins cell lines were also determined and revealed that there was no significant difference to previous‐ ly published values [7] (data not shown). Levels of human proinsulin (not insulin) were 11.4 ± 1.2% of total insulin (*n* = 6). Human c-peptide levels were 1.0 ± 0.4% of total insulin activity (*n* = 6). Therefore clone 16 was used for all subsequent analyses, and is referred to as Huh7- EGFPins hereafter. As expected, Huh7-EGFP cells did not synthesize, store nor secrete insu‐ lin. Examination of the insulin secreted chronically by MIN6-EGFPins cells revealed that 20.5 ± 2.3% (*n* = 6) was of human origin, the remainder being rodent insulin. Of the insulin stored by MIN6-EGFPins cells, 17.9 ± 2.4% (*n* = 6) was human insulin. As expected, all of the insulin stored and secreted by MIN6-EGFP cells was of rodent origin. These data suggest

In order to perform confocal microscopy, cells were plated on coverslips (Marienfeld superi‐ or 22 mm diameter) and grown for 2–4 days. Each coverslip was inserted into a Perspex cell chamber, sealed with silicone grease, and overlaid with 1 ml DMEM containing 5 mM glu‐ cose (confocal scanning laser microscope [CSLM] medium). For Zinquin-E (zinquin ester, ethyl[2-methyl-8-*p*-toluenesulphonamido-6-quinolyloxy]acetate) staining, cells were incu‐ bated at 37°C for 30 min with CSLM medium containing 25 µM zinquin E (Luminis Pty Ltd, Australia), as previously described [27]. After incubation, cells were rinsed with CSLM me‐ dium before recording confocal images with a Leica TCSNT (Wetzlar, Germany) with an in‐ verted microscope (Leica DMRBE). Cells were imaged with a UV laser, oil 100x (N.A.1.4 UV-corrected Planapo) or oil 63x (N.A.1.32 UV-corrected Planapo). Emissions were collected with a BP490/440 filter. For analyses of stable transfectants expressing EGFP, incubation with a fluorescent probe was not required. These cells were imaged with an Ar/Kr laser and

CSLM medium or test solutions containing glibenclamide (20 µM), or diazoxide (150 µM) in CSLM medium, or DMEM containing 20 mM glucose, were exchanged at 37°C. Density measurements on images were performed using the public domain NIH Image program [37]. Defined regions of interest (ROI) for individual cells (10–30 cells per experiment) were fol‐ lowed through a time series before, and after, addition of test solutions. All values were nor‐ malized by subtracting the initial density, before addition of the test solution, from all the measurements in the series for each individual ROI to give a value of zero density for the

insulin over a 24 h period (0.32 ± 0.2 vs. 0.30 ± 0.1 pmol insulin/106

(clone 16) and Huh7ins cells storing 7.1 ± 0.3 and 7.0 ± 0.2 pmol/106

that MIN6 cells handled EGFPins in a similar fashion to native rodent insulin.

DP488/568 dichroic and emissions were collected with a BP525/550 filter.

cells/24 h; *n* = 6) and insulin storage varied 2-fold (3.4 ±

cells for Huh7ins cells; *n* =

cells (*n* = 6), respectively.

cells; *n* = 6). Subsequently, six clones which secreted and

lin and human proinsulin, respectively.

± 0.2 *vs*. 0.32 ± 0.2 pmol insulin/106

1.2 *vs*. 7.1 ± 0.3 pmol insulin/106

710 Gene Therapy - Tools and Potential Applications

**Figure 3.** Confocal microscopic visualization of HuH7-EGFPins cells after exposure to glucose and diazoxide. (A) Huh7- EGFPins and (B) MIN6-EGFPins cells were incubated in DMEM containing 5 mM glucose (CLSM medium), then stimu‐ lated with glucose (20 mM, G) and diazoxide (150 μM, D). Images were recorded in CLSM medium at 0, 10 and 20 min after glucose addition. At 20 min, cells were placed in CLSM medium containing diazoxide and images were recorded at 10 and 20 min after diazoxide exposure (bars = 10 μm). Normalized EGFP density indicated that (C) Huh7-EGFPins (n = 60) and (D) MIN6-EGFPins cells (n = 42) showed decreasing EGFP density after addition of glucose, whereas (E) Huh7-EGFP (n = 19) and (F) MIN6-EGFP cells (n = 12) showed increasing EGFP density after addition of glucose. Values represent the mean ± SEM.

For statistical analysis of all the confocal measurements described below SPSS version 11.5 (SPSS Inc) was used to determine a one-way analysis of variance after testing for homogene‐ ity of variance using the Levene statistic. Huh7-EGFPins and MIN6-EGFPins cells respond‐ ed in the same way to 20 mM glucose after 10 and 15 min, with loss of fluorescence from the ROI indicative of insulin secretion (Figure 3A-D). There was no significant difference be‐ tween the response of Huh7-EGFPins and MIN6-EGFPins cells at 10 min (*p*> 0.5, *n* = 60) and 15 min (*p* ≥ 0.7, *n* = 42). The MIN6-EGFPins cells responded more rapidly to the glucose stimulus than the Huh7ins-EGFP cells, with close to maximum loss of fluorescence achieved at 5 min, but after 10 min the two cell lines had achieved the same response level (Figure 3C-D). When diazoxide (150 µM) was added to cells that had been stimulated by 20 mM glu‐ cose for 20 min, cytoplasmic fluorescence accumulated, as the release of insulin from secretory granules was blocked (Figure 3A-B). The response of the control cell lines, Huh7- EGFP and Min6-EGFP to 20 mM glucose was significantly different (*p*< 0.00001) at all time points from that of the engineered cell lines. In the control cell lines fluorescence increased over time (Figure 3E-F) since cells accumulated considerable amounts of EGFP within their cytoplasm and they were unresponsive to 20 mM glucose. Presumably this phenomenon is attributable to an inability of the parental cell lines to direct EGFP to secretory granules, whereas the cells engineered to synthesize insulin responded by releasing insulin from se‐ cretory granules so that their fluorescence decreased (Figure 3C-D).

GGCAGGTGGGAAATCATCGTTA, R: TCCCCACCTTCAGTGACAA') and SUR2B (F: GATGCGGTTGTCACTGAA, R: ACTCCTTCACATGTCTGC). Primers were also designed to amplify the α1-subunit of the CaV1.3 channel of pancreatic β-cells (F: TGGCAGGAGAT‐ CATGCTGG, R: CTAATCTCTTGCTCGCTACC). RT-PCR analyses were performed using the cDNA synthesised from RNA isolated from Huh7 and Huh7ins cells using TRIzol® Re‐ agent (Invitrogen). Positive controls were HEPG2 cells that express the human KIR6.2 and

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

713

Immunoblot analyses were performed using protein extracted from Huh7 and Huh7ins cells and human pancreatic islets to detect the human KATP subunits, SUR1, SUR2A and SUR2B, and the α1-subunit of the CaV1.3 channel. Detection of the KIR6.2 subunit was determined as previously described [27]. For detection of KIR6.2, SUR1, SUR2A, SUR2B and the α1-subunit of the CaV1.3 channel, cell supernatants were suspended in buffer I containing (in mM): 10 Tris, 20 NaH2PO4, 1 EDTA, 0.1 PMSF, 10 µg/ml pepstatin, 10 µl/ml leupeptin (pH 7.8), sub‐ jected to three freeze-thaw cycles, and then incubated for 20 min at 4°C. The protein concen‐ tration of the supernatant was determined using a Micro Bicinchoninic Protein Assay Reagent Kit (PIERCE, Thermo Fisher Scientific, Rockford, Il, USA). Protein samples (15 µg) were electrophoresed in 10% polyacrylamide gels (100 V) and then transferred to nitrocellu‐ lose membranes (Millipore Corporation, USA) for immunoblot analyses. Nitrocellulose membranes were blocked in PBS with 5% w/v skim milk overnight at 4°C. Immunoblotting was performed using a 1:1000 dilution of goat anti-human KIR6.2, SUR1, SUR2A, SUR2B and the α1-subunit of the CaV1.3 channel polyclonal IgGs (Santa Cruz Biotech. USA) and detec‐ tion was achieved using monoclonal (mouse) anti-goat/sheep horseradish peroxidase IgG

RT-PCR analysis revealed that the Huh7 and Huh7ins cells expressed the human KATP chan‐ nel subunit, KIR6.2, and the β-cell sulfonylurea receptor subunits, SUR2A and SUR2B (Figure 4A-C), together with the human α1-subunit of the CaV1.3 channel (Figure 4E). SUR1 was on‐ ly detected in the Huh7ins liver cell line (Figure 4D). Immunoblot analysis for the presence of KIR6.2, SUR1, SUR2A and SUR2B, revealed strong expression in Huh7ins cells and human pancreatic islets, with no detectable expression in Huh7 cells (Figure 4F-I). The presence of protein product for the α1-subunit of the CaV1.3 channel was confirmed by immunoblot analysis of protein extracted from Huh7ins cells, with only low expression in Huh7 cells

Thus, unlike the glucose-responsive insulin-secreting cell line, HEPG2ins/g [26], the Huh7ins cells expressed the SUR1 receptor as do pancreatic β-cells. The functional recording of KATP activity in Huh7ins cells are supported by the immunoblot blot analyses, which sug‐ gests that KIR6.2, SUR1, SUR2A and SUR2B are strongly expressed in Huh7ins cells. There was no detectable expression of KIR6.2, SUR1, SUR2A and SUR2B in Huh7 cells, which is supported by the absence of KATP currents in the patch-clamp recordings (Figure 2B). Ex‐ pression of KIR6.2 and SUR1, the two relevant subunits of the pancreatic β-cell KATP channel, is commonly seen in primary hepatocytes, although dedifferentiated cell lines such as HEPG2 [28] and Huh7 cells appear to have lost expression of SUR1 at the mRNA level. It is apparent that the process of pancreatic transdifferentiation, which has caused the formation

SUR2A subunits [27], or human pancreatic islets.

conjugate (1:800 dilution) (Sigma).

(Figure 4J).

Huh7ins and Min6 cells stained with the zinquin-E probe responded to 20 mM glucose with decreasing fluorescence. There was no significant difference between Huh7ins cells labelled with zinquin-E and Huh7-EGFPins cells in their response to 20 mM glucose after 5 min (*p*> 0.8, *n* = 41) and 15 min (*P*> 0.1, *n* = 59). After incubation with the KATP channel blocking sul‐ phonylurea, glibenclamide (20 µM), the two cell lines responded as they did in the presence of glucose (i.e. decreasing fluorescence was observed). Conversely, treatment with 150 µM diazoxide, which inhibits glucose-activated β-cell depolarisation by suppressing closure of KATP channels, caused increased fluorescence, showing that secretion of insulin was blocked in Huh7ins-EGFP cells (Figure 3A) and Huh7ins cells (with zinquin-E probe).

Huh7ins-EGFP and MIN6ins-EGFP cells responded to either glucose or glibenclamide with a decrease in fluorescence (indicative of insulin secretion). The same secretory response to glucose or glibenclamide was seen in MIN6 and Huh7ins cells using the zinquin probe. Through its high affinity for the sulphonylurea subunit of the KATP channel, glibenclamide renders the KATP channel inactive and calcium influx through CaV channels ensues due to depolarisation of the cell membrane. The release of insulin from intracellular storage gran‐ ules is the net result of these processes. As this response to glibenclamide was observed for Huh7ins, Huh7-EGFPins, MIN6 and MIN6-EGFPins cells, insulin secretion likely occurred via the classic insulin triggering pathway utilized by pancreatic β-cells. In contrast, the nega‐ tive controls (Huh7-EGFP and MIN6-EGFP cells) or Huh7 cells in the case of Zinquin-E la‐ belling, were unresponsive to glucose or glibenclamide. The increased fluorescence of the negative control cells MIN6-EGFP and Huh7-EGFP after glucose stimulation showed that there was no trafficking of EGFP to secretory granules. In an earlier publication, Arvan and Halban [38] questioned the specificity of the trans Golgi network sorting process, but the fact that in our cell lines the secretion of EGFP-ins was regulated while EGFP was not, shows that the sorting of EGFP was specific, with only EGFP-insulin being trafficked to se‐ cretory granules.

#### **2.3. Huh7ins cells express the KATP channel subunits, KIR6.2, SUR2A and SUR2B, and the α1-subunit of the CaV1.3 channel**

Primers were designed to the cDNA sequences encoding the human KATP subunits, KIR6.2 (F: AGCCCAAGTTCAGCATCTCTCC, R:CCAGAAATAGCATAGTGACAAGTGCC), SUR1 (F: TCAGGGTTGTGAACCGCA, R: GTTTCTGCGAAGCATAGGC), SUR2A (F: GGCAGGTGGGAAATCATCGTTA, R: TCCCCACCTTCAGTGACAA') and SUR2B (F: GATGCGGTTGTCACTGAA, R: ACTCCTTCACATGTCTGC). Primers were also designed to amplify the α1-subunit of the CaV1.3 channel of pancreatic β-cells (F: TGGCAGGAGAT‐ CATGCTGG, R: CTAATCTCTTGCTCGCTACC). RT-PCR analyses were performed using the cDNA synthesised from RNA isolated from Huh7 and Huh7ins cells using TRIzol® Re‐ agent (Invitrogen). Positive controls were HEPG2 cells that express the human KIR6.2 and SUR2A subunits [27], or human pancreatic islets.

secretory granules was blocked (Figure 3A-B). The response of the control cell lines, Huh7- EGFP and Min6-EGFP to 20 mM glucose was significantly different (*p*< 0.00001) at all time points from that of the engineered cell lines. In the control cell lines fluorescence increased over time (Figure 3E-F) since cells accumulated considerable amounts of EGFP within their cytoplasm and they were unresponsive to 20 mM glucose. Presumably this phenomenon is attributable to an inability of the parental cell lines to direct EGFP to secretory granules, whereas the cells engineered to synthesize insulin responded by releasing insulin from se‐

Huh7ins and Min6 cells stained with the zinquin-E probe responded to 20 mM glucose with decreasing fluorescence. There was no significant difference between Huh7ins cells labelled with zinquin-E and Huh7-EGFPins cells in their response to 20 mM glucose after 5 min (*p*> 0.8, *n* = 41) and 15 min (*P*> 0.1, *n* = 59). After incubation with the KATP channel blocking sul‐ phonylurea, glibenclamide (20 µM), the two cell lines responded as they did in the presence of glucose (i.e. decreasing fluorescence was observed). Conversely, treatment with 150 µM diazoxide, which inhibits glucose-activated β-cell depolarisation by suppressing closure of KATP channels, caused increased fluorescence, showing that secretion of insulin was blocked

Huh7ins-EGFP and MIN6ins-EGFP cells responded to either glucose or glibenclamide with a decrease in fluorescence (indicative of insulin secretion). The same secretory response to glucose or glibenclamide was seen in MIN6 and Huh7ins cells using the zinquin probe. Through its high affinity for the sulphonylurea subunit of the KATP channel, glibenclamide renders the KATP channel inactive and calcium influx through CaV channels ensues due to depolarisation of the cell membrane. The release of insulin from intracellular storage gran‐ ules is the net result of these processes. As this response to glibenclamide was observed for Huh7ins, Huh7-EGFPins, MIN6 and MIN6-EGFPins cells, insulin secretion likely occurred via the classic insulin triggering pathway utilized by pancreatic β-cells. In contrast, the nega‐ tive controls (Huh7-EGFP and MIN6-EGFP cells) or Huh7 cells in the case of Zinquin-E la‐ belling, were unresponsive to glucose or glibenclamide. The increased fluorescence of the negative control cells MIN6-EGFP and Huh7-EGFP after glucose stimulation showed that there was no trafficking of EGFP to secretory granules. In an earlier publication, Arvan and Halban [38] questioned the specificity of the trans Golgi network sorting process, but the fact that in our cell lines the secretion of EGFP-ins was regulated while EGFP was not, shows that the sorting of EGFP was specific, with only EGFP-insulin being trafficked to se‐

**2.3. Huh7ins cells express the KATP channel subunits, KIR6.2, SUR2A and**

Primers were designed to the cDNA sequences encoding the human KATP subunits, KIR6.2 (F: AGCCCAAGTTCAGCATCTCTCC, R:CCAGAAATAGCATAGTGACAAGTGCC), SUR1 (F: TCAGGGTTGTGAACCGCA, R: GTTTCTGCGAAGCATAGGC), SUR2A (F:

**SUR2B, and the α1-subunit of the CaV1.3 channel**

cretory granules so that their fluorescence decreased (Figure 3C-D).

712 Gene Therapy - Tools and Potential Applications

in Huh7ins-EGFP cells (Figure 3A) and Huh7ins cells (with zinquin-E probe).

cretory granules.

Immunoblot analyses were performed using protein extracted from Huh7 and Huh7ins cells and human pancreatic islets to detect the human KATP subunits, SUR1, SUR2A and SUR2B, and the α1-subunit of the CaV1.3 channel. Detection of the KIR6.2 subunit was determined as previously described [27]. For detection of KIR6.2, SUR1, SUR2A, SUR2B and the α1-subunit of the CaV1.3 channel, cell supernatants were suspended in buffer I containing (in mM): 10 Tris, 20 NaH2PO4, 1 EDTA, 0.1 PMSF, 10 µg/ml pepstatin, 10 µl/ml leupeptin (pH 7.8), sub‐ jected to three freeze-thaw cycles, and then incubated for 20 min at 4°C. The protein concen‐ tration of the supernatant was determined using a Micro Bicinchoninic Protein Assay Reagent Kit (PIERCE, Thermo Fisher Scientific, Rockford, Il, USA). Protein samples (15 µg) were electrophoresed in 10% polyacrylamide gels (100 V) and then transferred to nitrocellu‐ lose membranes (Millipore Corporation, USA) for immunoblot analyses. Nitrocellulose membranes were blocked in PBS with 5% w/v skim milk overnight at 4°C. Immunoblotting was performed using a 1:1000 dilution of goat anti-human KIR6.2, SUR1, SUR2A, SUR2B and the α1-subunit of the CaV1.3 channel polyclonal IgGs (Santa Cruz Biotech. USA) and detec‐ tion was achieved using monoclonal (mouse) anti-goat/sheep horseradish peroxidase IgG conjugate (1:800 dilution) (Sigma).

RT-PCR analysis revealed that the Huh7 and Huh7ins cells expressed the human KATP chan‐ nel subunit, KIR6.2, and the β-cell sulfonylurea receptor subunits, SUR2A and SUR2B (Figure 4A-C), together with the human α1-subunit of the CaV1.3 channel (Figure 4E). SUR1 was on‐ ly detected in the Huh7ins liver cell line (Figure 4D). Immunoblot analysis for the presence of KIR6.2, SUR1, SUR2A and SUR2B, revealed strong expression in Huh7ins cells and human pancreatic islets, with no detectable expression in Huh7 cells (Figure 4F-I). The presence of protein product for the α1-subunit of the CaV1.3 channel was confirmed by immunoblot analysis of protein extracted from Huh7ins cells, with only low expression in Huh7 cells (Figure 4J).

Thus, unlike the glucose-responsive insulin-secreting cell line, HEPG2ins/g [26], the Huh7ins cells expressed the SUR1 receptor as do pancreatic β-cells. The functional recording of KATP activity in Huh7ins cells are supported by the immunoblot blot analyses, which sug‐ gests that KIR6.2, SUR1, SUR2A and SUR2B are strongly expressed in Huh7ins cells. There was no detectable expression of KIR6.2, SUR1, SUR2A and SUR2B in Huh7 cells, which is supported by the absence of KATP currents in the patch-clamp recordings (Figure 2B). Ex‐ pression of KIR6.2 and SUR1, the two relevant subunits of the pancreatic β-cell KATP channel, is commonly seen in primary hepatocytes, although dedifferentiated cell lines such as HEPG2 [28] and Huh7 cells appear to have lost expression of SUR1 at the mRNA level. It is apparent that the process of pancreatic transdifferentiation, which has caused the formation of secretory granules, has resulted in expression of KIR6.2 protein and SUR1 at the mRNA level and protein expression in Huh7ins cells.

**2.4. Huh7ins cells possess CaV channels**

immediately stimulated an elevated level of free [Ca2+]i

cantly increase the level of free [Ca2+]i

corded in Huh7ins cells (Figure 5A).

produced a more delayed increase in the [Ca2+]i

and diazoxide-induced increase in free [Ca2+]i

The level of intracellular free Ca2+ was measured using Fluo4-AM and pluronic F-127 with a Zeiss microscope (Axiovert 200M; Zeiss, Germany). Cells were grown on coverslips until 50– 70% confluent and were then incubated in culture medium containing 8 µM Fluo4-AM (Invi‐ trogen, Carlsbad, CA) and 0.1% pluronic F-127 (Invitrogen) at 37°C for 60 min. To remove excess Fluo4-AM and F-127, the cells were incubated with HEPES buffer containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 D-glucose, 10 HEPES (pH 7.4), for 30 min images were captured. The coverslips were then placed in a chamber containing HEPES buffer. After control images were taken (before addition of glucose or glibenclamide), the cells were exposed to 20 mM glucose or 20 µM glibenclamide until the completion of experiments. For the experi‐ ments in the presence of CaV channel blocker, the cells were incubated with 10 µM verapa‐ mil for 30 min before the addition of glucose or glibenclamide. Fluorescence intensity was observed under a Zeiss microscope and images were captured with a digital camera (Axio‐ Cam, Zeiss) and the Axiovision program (Zeiss). Images were taken every 20 s and ana‐ lyzed using ImageJ software [39]. Results were presented as relative fluorescence values (*F*/*F* 0), where *F*0 represents the fluorescence of controls (before addition of glucose or glibenclamide). While the expression of CaV channels in pancreatic β-cells has been well documented [23, 40], their precise role in hepatocytes is yet to be elucidated. It has been reported that CaV1 channels are found in endocrine (pancreatic), cardiac and neural cells [41], but no physiolog‐ ically-active CaV1 channels have been identified in hepatocytes prior to this study. Calcium imaging revealed that an increase in the extracellular glucose concentration from 5 to 20 mM

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

in 2 min and then gradually recovered to the level observed prior to application of 20 mM glucose (Figure 5A). The *F*/*F* 0 value at 2 min after the application of 20 mM glucose in Huh7ins cells was 1.14 ± 0.038 (*n* = 33, Figure 5B). However, 20 mM glucose did not signifi‐

significantly lower than that of Huh7ins cells (Fig 5A-B). To examine if blockade of KATP channels mimicked the effect of 20 mM glucose, glibenclamide (20 µM) was applied in the bath solution containing 5 mM glucose. Glibenclamide dramatically increased the level of intracellular free Ca2+ (*F*/*F* o = 1.87 ± 0.24, *n* = 25), which had a similar time course to that observed in the presence of 20 mM glucose, but with a greater peak amplitude. Similar to the effects of 20 mM glucose on Huh7 cells, glibenclamide did not alter calcium flux in Huh7 cells (Figure 5A-B). It should be noted that both 20 mM glucose and 20 µM glibenclamide

Verapamil (10 µM), a phenylalkylamine CaV1.x channel blocker, inhibited the increase in [Ca2+]I in Huh7ins cells produced by 20 mM glucose (1.04 ± 0.02, *n* = 31) and glibenclamide (0.99 ± 0.02, *n* = 31; Figure 5A and C). This indicated that the observed glucose-induced block

idate this interpretation, we used the whole-cell patch-clamp technique to measure the effect of increased glucose on membrane currents in Huh7ins and Huh7 cells. The resultant *I*-*V* curve indicated that increasing the concentration of glucose from 2 to 20 mM resulted in ac‐

in Huh7ins cells, which peaked with‐

in Huh7 cells (*F*/*F* o = 1.02 ± 0.01, *n* = 19), which was

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

715

in Huh7 cells in comparison with data re‐

was mediated by CaV channels. To further val‐

**Figure 4.** RT-PCR and immunoblot analysis for KATP and CaV channel subunits. RT-PCR analysis of liver cell lines for (A) human KIR6.2: HuH7 (lane 1), Huh7ins (lane 2), Huh7-EGFPins (lane 3), HEPG2 (lane 4, positive control), and no cDNA control (lane 5); (B) human SUR2A: Huh7 (lane 1), Huh7ins (lane 2), Huh7-EGFPins (lane 3), HEPG2 (lane 4, positive control), and no cDNA control (lane 5); (C) human SUR2B: no cDNA (lane 1), human pancreas (lane 2, positive control), Huh7 (lane 3), Huh7ins (lane 4), Huh7-EGFPins (lane 5), (D) Human SUR1: no cDNA (lane 1), Huh7 (lane 2), Huh7ins (lane 3), Huh7-EGFPins (lane 4), human pancreas (lane 5, positive control), (E) human α1-subunit of the CaV1.3 chan‐ nel: no cDNA (lane 1), human pancreas (lane 2, positive control), Huh7 (lane 3), Huh7ins (lane 4), Huh7-EGFPins (lane 5). Immunoblot analysis for (F) human KIR6.2, (G) human SUR2A, (H) SUR2B, (I) SUR1 and (J) the α1-subunit of the CaV1.3 channel in Huh7 (lane 1), Huh7ins (lane 2) and human islet (lane 3).

### **2.4. Huh7ins cells possess CaV channels**

of secretory granules, has resulted in expression of KIR6.2 protein and SUR1 at the mRNA

**Figure 4.** RT-PCR and immunoblot analysis for KATP and CaV channel subunits. RT-PCR analysis of liver cell lines for (A) human KIR6.2: HuH7 (lane 1), Huh7ins (lane 2), Huh7-EGFPins (lane 3), HEPG2 (lane 4, positive control), and no cDNA control (lane 5); (B) human SUR2A: Huh7 (lane 1), Huh7ins (lane 2), Huh7-EGFPins (lane 3), HEPG2 (lane 4, positive control), and no cDNA control (lane 5); (C) human SUR2B: no cDNA (lane 1), human pancreas (lane 2, positive control), Huh7 (lane 3), Huh7ins (lane 4), Huh7-EGFPins (lane 5), (D) Human SUR1: no cDNA (lane 1), Huh7 (lane 2), Huh7ins (lane 3), Huh7-EGFPins (lane 4), human pancreas (lane 5, positive control), (E) human α1-subunit of the CaV1.3 chan‐ nel: no cDNA (lane 1), human pancreas (lane 2, positive control), Huh7 (lane 3), Huh7ins (lane 4), Huh7-EGFPins (lane 5). Immunoblot analysis for (F) human KIR6.2, (G) human SUR2A, (H) SUR2B, (I) SUR1 and (J) the α1-subunit of the

CaV1.3 channel in Huh7 (lane 1), Huh7ins (lane 2) and human islet (lane 3).

level and protein expression in Huh7ins cells.

714 Gene Therapy - Tools and Potential Applications

The level of intracellular free Ca2+ was measured using Fluo4-AM and pluronic F-127 with a Zeiss microscope (Axiovert 200M; Zeiss, Germany). Cells were grown on coverslips until 50– 70% confluent and were then incubated in culture medium containing 8 µM Fluo4-AM (Invi‐ trogen, Carlsbad, CA) and 0.1% pluronic F-127 (Invitrogen) at 37°C for 60 min. To remove excess Fluo4-AM and F-127, the cells were incubated with HEPES buffer containing (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 5 D-glucose, 10 HEPES (pH 7.4), for 30 min images were captured. The coverslips were then placed in a chamber containing HEPES buffer. After control images were taken (before addition of glucose or glibenclamide), the cells were exposed to 20 mM glucose or 20 µM glibenclamide until the completion of experiments. For the experi‐ ments in the presence of CaV channel blocker, the cells were incubated with 10 µM verapa‐ mil for 30 min before the addition of glucose or glibenclamide. Fluorescence intensity was observed under a Zeiss microscope and images were captured with a digital camera (Axio‐ Cam, Zeiss) and the Axiovision program (Zeiss). Images were taken every 20 s and ana‐ lyzed using ImageJ software [39]. Results were presented as relative fluorescence values (*F*/*F* 0), where *F*0 represents the fluorescence of controls (before addition of glucose or glibenclamide).

While the expression of CaV channels in pancreatic β-cells has been well documented [23, 40], their precise role in hepatocytes is yet to be elucidated. It has been reported that CaV1 channels are found in endocrine (pancreatic), cardiac and neural cells [41], but no physiolog‐ ically-active CaV1 channels have been identified in hepatocytes prior to this study. Calcium imaging revealed that an increase in the extracellular glucose concentration from 5 to 20 mM immediately stimulated an elevated level of free [Ca2+]i in Huh7ins cells, which peaked with‐ in 2 min and then gradually recovered to the level observed prior to application of 20 mM glucose (Figure 5A). The *F*/*F* 0 value at 2 min after the application of 20 mM glucose in Huh7ins cells was 1.14 ± 0.038 (*n* = 33, Figure 5B). However, 20 mM glucose did not signifi‐ cantly increase the level of free [Ca2+]i in Huh7 cells (*F*/*F* o = 1.02 ± 0.01, *n* = 19), which was significantly lower than that of Huh7ins cells (Fig 5A-B). To examine if blockade of KATP channels mimicked the effect of 20 mM glucose, glibenclamide (20 µM) was applied in the bath solution containing 5 mM glucose. Glibenclamide dramatically increased the level of intracellular free Ca2+ (*F*/*F* o = 1.87 ± 0.24, *n* = 25), which had a similar time course to that observed in the presence of 20 mM glucose, but with a greater peak amplitude. Similar to the effects of 20 mM glucose on Huh7 cells, glibenclamide did not alter calcium flux in Huh7 cells (Figure 5A-B). It should be noted that both 20 mM glucose and 20 µM glibenclamide produced a more delayed increase in the [Ca2+]i in Huh7 cells in comparison with data re‐ corded in Huh7ins cells (Figure 5A).

Verapamil (10 µM), a phenylalkylamine CaV1.x channel blocker, inhibited the increase in [Ca2+]I in Huh7ins cells produced by 20 mM glucose (1.04 ± 0.02, *n* = 31) and glibenclamide (0.99 ± 0.02, *n* = 31; Figure 5A and C). This indicated that the observed glucose-induced block and diazoxide-induced increase in free [Ca2+]i was mediated by CaV channels. To further val‐ idate this interpretation, we used the whole-cell patch-clamp technique to measure the effect of increased glucose on membrane currents in Huh7ins and Huh7 cells. The resultant *I*-*V* curve indicated that increasing the concentration of glucose from 2 to 20 mM resulted in ac‐ tivation of an inwardly-rectifying current in Huh7ins cells (Figure 5D). This current was blocked by the addition of CsCl thereby lending further support to the premise that it was mediated via K+ channels. No activation was seen when Huh7 cells were used in these ex‐ periments (results not shown). CaV channel currents recorded from Huh7ins cells were in‐ hibited by verapamil (10 µM), indicating that CaV1.x channels were involved in the response (Figure 5E). This further corroborates the calcium imaging data described above.

ing that Huh7ins and Huh7 cells possess CaV1.3 channels that are similar to those found in pancreatic β-cells. Ca2+ imaging and patch-clamp electrophysiology experiments further de‐ tected a CaV channel current in Huh7ins cells, which was stimulated by glucose and inhibit‐ ed by verapamil. The expression of functional CaV channels in Huh7ins cells may explain, in part, the acute secretion of insulin in response to glucose stimulation. The mechanism of in‐ sulin secretion depends upon the activities of ion channels in the plasma membrane, and, more critically, upon the activation of CaV channels, caused indirectly by increased glucose metabolism. Influx of Ca2+, through open CaV channels, is responsible for the exocytosis of insulin storage granules, emphasising the importance of CaV channels in glucose-stimulated insulin secretion [41]. The lack of functional CaV channels in Huh7 cells is likely related to the low level of expression of the CaV1.3 α1-subunit. Once it was determined that Huh7ins cells possessed functional CaV channels, static stimulation experiments using the inhibitor verapamil, and the activator BayK8644, established that CaV channels in Huh7ins cells func‐

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

717

**2.5. Huh7ins cells appear to be glucose-responsive through the presence**

To measure insulin secretion, monolayers of cells were incubated with KATP channel modu‐ lators, using concentrations determined from concentration-response curves in the corre‐ sponding cell lines. These included the KATP channel activators tolbutamide (100 µM) or diazoxide (150 µM) and the KATP channel blocker glibenclamide (20 µM) with or without 20 mM glucose for 1 h. The effects of the CaV channel blocker verapamil (10 µM), the CaV chan‐ nel activator Bay K8644 (1 µM), the sarcoplasmic and endoplasmic reticulum family of Ca2+- ATPases (SERCA) blocker ryanodine (20 µM), the SERCA stimulator thapsigargin (1 µM), and the hemi-channel blocker oleic acid (20 µM) were also assessed. Inhibitors and activa‐ tors were purchased from Sigma, Sydney, Australia. Results were expressed as means ± standard error of the mean (SEM). The statistical analysis of insulin RIA results was by uni‐ variate repeated measures analysis of variance using Systat™ version 9. Post-hoc compari‐

Stimulation with 20 mM glucose resulted in a 3.6- and 5.2-fold increase in insulin secretion by Huh7ins and MIN6 cells, respectively (Figures 6 and 7). Incubation of Huh7-EGFPins cells with the KATP channel blocker, glibenclamide, significantly increased insulin secretion by Huh7- EGFPins from 0.06 ± 0.01 to 0.26 ± 0.03 pmol/106 cells (*p*< 0.001, *n* = 6). The KATP activator, diazoxide, completely inhibited glucose-stimulated insulin release from Huh7-EGFPins (0.05

diazoxide treatment prevented glucose-induced insulin secretion in Huh7ins and Huh7-EGF‐ Pins cells. Diazoxide causes sustained opening of KATP channels causing hyperpolarisation of the cell membrane, thereby preventing the voltage-dependant calcium response and inhibit‐ ing insulin exocytosis [43]. Static glucose stimulation experiments demonstrated that the insulin secretory response of Huh7ins and Huh7-EGFPins cells functioned via the channel-depend‐ ant pathway of insulin secretion. The responses of Huh7ins and MIN6 cells to diazoxide and

cells, *n* = 6) and MIN6-EGFPins cells (data not shown). It was also noted that,

sons were made using Tukey's HSD test (Minitab™ version 13, Minitab Inc).

tion in a similar manner to CaV channels in pancreatic β-cells.

**of functional KATP channels and CaV channels**

± 0.02 pmol/106

**Figure 5.** Calcium imaging and patch-clamp electrophysiology of Huh7ins and Huh7 cells. High glucose and blockade of KATP channels elevated levels of intracellular free Ca2+ in Huh7ins cells. (A) Averaged time courses of relative fluores‐ cence intensity (F/F0) induced by 20 mM glucose (gluc) and 20 μM glibenclamide (gliben) in the presence, and ab‐ sence, of 10 μM verapamil (verap, CaV1.x channel blocker) in Huh7ins and Huh7 cells. The black bar at the base of panel A represents the time of application of glucose or glibenclamide. Each trace represents an average F/F0 value of the cells investigated. (B) Glucose and glibenclamide increased the level of free [Ca2+]i in Huh7ins cells, but not in Huh7 cells. (C) Glucose- and glibenclamide-induced increases in intracellular free Ca2+ in Huh7ins cells were significantly in‐ hibited by 10 μM verapamil. The values shown in B and C were taken 2 min after application of glucose or glibencla‐ mide. \* p < 0.05 and \*\*\* p < 0.001. (D) Mean I-V relationship in Huh7ins cells under low (2 mM) and high (20 mM) glucose conditions (n = 4). (E) I-V curves for CaV channel currents in Huh7ins cells in the presence of 20 mM glucose and following the addition of 10 μM verapamil (n = 6). Values are expressed as means ± SEM

The CaV1.3 α1 subunit (Figure 4J), expressed in pancreatic β-cells [42], was detected in both Huh7ins cells and the parental Huh7 cells, at both the mRNA and the protein level, suggest‐ Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-Sensitive Potassium Channels and Voltage-Gated Calcium Channels http://dx.doi.org/10.5772/52839 717

ing that Huh7ins and Huh7 cells possess CaV1.3 channels that are similar to those found in pancreatic β-cells. Ca2+ imaging and patch-clamp electrophysiology experiments further de‐ tected a CaV channel current in Huh7ins cells, which was stimulated by glucose and inhibit‐ ed by verapamil. The expression of functional CaV channels in Huh7ins cells may explain, in part, the acute secretion of insulin in response to glucose stimulation. The mechanism of in‐ sulin secretion depends upon the activities of ion channels in the plasma membrane, and, more critically, upon the activation of CaV channels, caused indirectly by increased glucose metabolism. Influx of Ca2+, through open CaV channels, is responsible for the exocytosis of insulin storage granules, emphasising the importance of CaV channels in glucose-stimulated insulin secretion [41]. The lack of functional CaV channels in Huh7 cells is likely related to the low level of expression of the CaV1.3 α1-subunit. Once it was determined that Huh7ins cells possessed functional CaV channels, static stimulation experiments using the inhibitor verapamil, and the activator BayK8644, established that CaV channels in Huh7ins cells func‐ tion in a similar manner to CaV channels in pancreatic β-cells.

tivation of an inwardly-rectifying current in Huh7ins cells (Figure 5D). This current was blocked by the addition of CsCl thereby lending further support to the premise that it was

periments (results not shown). CaV channel currents recorded from Huh7ins cells were in‐ hibited by verapamil (10 µM), indicating that CaV1.x channels were involved in the response

**Figure 5.** Calcium imaging and patch-clamp electrophysiology of Huh7ins and Huh7 cells. High glucose and blockade of KATP channels elevated levels of intracellular free Ca2+ in Huh7ins cells. (A) Averaged time courses of relative fluores‐ cence intensity (F/F0) induced by 20 mM glucose (gluc) and 20 μM glibenclamide (gliben) in the presence, and ab‐ sence, of 10 μM verapamil (verap, CaV1.x channel blocker) in Huh7ins and Huh7 cells. The black bar at the base of panel A represents the time of application of glucose or glibenclamide. Each trace represents an average F/F0 value of the cells investigated. (B) Glucose and glibenclamide increased the level of free [Ca2+]i in Huh7ins cells, but not in Huh7 cells. (C) Glucose- and glibenclamide-induced increases in intracellular free Ca2+ in Huh7ins cells were significantly in‐ hibited by 10 μM verapamil. The values shown in B and C were taken 2 min after application of glucose or glibencla‐ mide. \* p < 0.05 and \*\*\* p < 0.001. (D) Mean I-V relationship in Huh7ins cells under low (2 mM) and high (20 mM) glucose conditions (n = 4). (E) I-V curves for CaV channel currents in Huh7ins cells in the presence of 20 mM glucose

The CaV1.3 α1 subunit (Figure 4J), expressed in pancreatic β-cells [42], was detected in both Huh7ins cells and the parental Huh7 cells, at both the mRNA and the protein level, suggest‐

and following the addition of 10 μM verapamil (n = 6). Values are expressed as means ± SEM

(Figure 5E). This further corroborates the calcium imaging data described above.

channels. No activation was seen when Huh7 cells were used in these ex‐

mediated via K+

716 Gene Therapy - Tools and Potential Applications

#### **2.5. Huh7ins cells appear to be glucose-responsive through the presence of functional KATP channels and CaV channels**

To measure insulin secretion, monolayers of cells were incubated with KATP channel modu‐ lators, using concentrations determined from concentration-response curves in the corre‐ sponding cell lines. These included the KATP channel activators tolbutamide (100 µM) or diazoxide (150 µM) and the KATP channel blocker glibenclamide (20 µM) with or without 20 mM glucose for 1 h. The effects of the CaV channel blocker verapamil (10 µM), the CaV chan‐ nel activator Bay K8644 (1 µM), the sarcoplasmic and endoplasmic reticulum family of Ca2+- ATPases (SERCA) blocker ryanodine (20 µM), the SERCA stimulator thapsigargin (1 µM), and the hemi-channel blocker oleic acid (20 µM) were also assessed. Inhibitors and activa‐ tors were purchased from Sigma, Sydney, Australia. Results were expressed as means ± standard error of the mean (SEM). The statistical analysis of insulin RIA results was by uni‐ variate repeated measures analysis of variance using Systat™ version 9. Post-hoc compari‐ sons were made using Tukey's HSD test (Minitab™ version 13, Minitab Inc).

Stimulation with 20 mM glucose resulted in a 3.6- and 5.2-fold increase in insulin secretion by Huh7ins and MIN6 cells, respectively (Figures 6 and 7). Incubation of Huh7-EGFPins cells with the KATP channel blocker, glibenclamide, significantly increased insulin secretion by Huh7- EGFPins from 0.06 ± 0.01 to 0.26 ± 0.03 pmol/106 cells (*p*< 0.001, *n* = 6). The KATP activator, diazoxide, completely inhibited glucose-stimulated insulin release from Huh7-EGFPins (0.05 ± 0.02 pmol/106 cells, *n* = 6) and MIN6-EGFPins cells (data not shown). It was also noted that, diazoxide treatment prevented glucose-induced insulin secretion in Huh7ins and Huh7-EGF‐ Pins cells. Diazoxide causes sustained opening of KATP channels causing hyperpolarisation of the cell membrane, thereby preventing the voltage-dependant calcium response and inhibit‐ ing insulin exocytosis [43]. Static glucose stimulation experiments demonstrated that the insulin secretory response of Huh7ins and Huh7-EGFPins cells functioned via the channel-depend‐ ant pathway of insulin secretion. The responses of Huh7ins and MIN6 cells to diazoxide and glibenclamide treatment were identical to that observed in each of the cell lines in which insulin was fused to EGFP (data not shown). Therefore, fusion of EGFP to insulin did not alter the physiological mechanism of insulin secretion.

potential of resting β-cells [43]. Rather, it acts on the Ca<sup>V</sup> channel in the open state, failing to affect basal insulin secretion at non-stimulatory glucose concentrations [43], but exaggerat‐ ing glucose-stimulated insulin secretion [44, 45]. The addition of BayK8644 increased insulin secretion by both the Huh7ins and MIN6 cells. However, the amount of insulin secreted in the presence of BayK8644 was lower than that released in response to 20 mM glucose alone. Putatively, this concentration of glucose may have stimulated the influx of extracellular Ca2+,

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

ways to such an extent that the total increase of Ca2+ in the cell was higher in the presence of

Consistent with reports that BayK8644 is known to stimulate the opening of CaV channels in pancreatic β-cells without altering the membrane potential [44], static stimulation of Huh7ins cells with 1 µM BayK8644 plus 20 mM glucose amplified glucose-stimulated insu‐ lin release. However, BayK8644 failed to amplify glucose-stimulated insulin secretion in MIN6 cells. This finding may be attributable to the ability of 20 mM glucose alone to cause the maximum threshold in the activation of insulin release in MIN6 cells, such that the addi‐ tion of BayK8644 was unable to exert any additional stimulatory effects. Nevertheless, these results demonstrate that the insulin secretory response of the Huh7ins cells is dependent

Static stimulation of Huh7ins cells with the highly specific SERCA blocker thapsigargin, which induces the release of Ca2+ from intracellular stores resulted in a significant increase (two-fold increase over basal levels), in insulin secretion in the absence of extracellular Ca2+ (*p*< 0.05, *n* = 6; Figure 6C). Consistent with results from Tuch *et al*. [7], the response of the Huh7ins cells to glucose was abolished when Ca2+ was removed from the basal medium be‐ fore 20 mM glucose was added (*p*> 0.05 *vs*. control, *n* = 6; Figure 6C). MIN6 cells showed a similar response; namely, in the absence of extracellular Ca2+ the glucose-responsiveness was abolished (*p*> 0.05 *vs*. control, *n* = 6; Figure 6C), and the presence of 1 µM thapsigargin significantly increased insulin secretion 1.8-fold over basal levels (*P*<0.05, *n* = 6; Figure 6C). The connexon (hemi-channel blocker), oleic acid, significantly reduced acute insulin secre‐ tion by 1.4-fold (*p*< 0.05, *n* = 6; Figure 7A), while verapamil (10 µM) resulted in a significant decrease in insulin secretion to glucose in both cell lines (*p*< 0.05, *n* = 6; Figure 7B). However, the combination of verapamil and ryanodine did not exert an additive effect on insulin se‐ cretion, compared to treatment with verapamil alone (*p*< 0.05, *n* = 6; Fig. 7B). Nevertheless, a greater decrease in insulin secretion was observed after the addition of verapamil, ryano‐ dine and oleic acid in both Huh7ins (*p*< 0.05, *n* = 6) and MIN6 cells (*p*< 0.05, *n* = 6; Figure 7C). SERCA operate to restore diminished intracellular endoplasmic and sarcoplasmic reticulum Ca2+ stores, thereby decreasing cytoplasmic Ca2+ levels [46-50]. Thapsigargin is a highly se‐ lective inhibitor of SERCA. Stimulation of β-cells with glucose causes an initial, thapsigar‐

Ca2+ into the endoplasmic reticulum [51, 52]. Blocking of SERCA by thapsigargin augments

which then facilitates the opening of CaV channels [51, 53, 54]. Consistent with the results reported by Tuch et al, [7], in the absence of extracellular Ca2+, the glucose responsiveness of

upon the activation of CaV channels, as is the case for pancreatic β-cells.

from intracellular stores and increased Ca2+ via other Ca2+-related path‐

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

719

that precedes the increase in [Ca2+]i due to the pumping of

increase by activating a depolarising store-operated current,

the release of [Ca2+]i

gin-inhibitable, drop in [Ca2+]i

the glucose-induced [Ca2+]i

20 mM glucose as compared to BayK8644 alone.

**Figure 6.** Secretion of insulin from Huh7ins cells and MIN6 cells. Insulin secretion was activated in response to 20 mM glucose alone or (A) 1 µM BayK8644 ± 20 mM glucose. (B) 20 µM ryanodine ± 20 mM glucose; and (C) 1 µM thapsigar‐ gin in the absence of extracellular calcium. Cells were incubated in basal medium for two consecutive 1 h periods be‐ fore being exposed to the stimulus for 1 h, followed by a third period of basal incubation. Cells in the control group were treated throughout with basal medium. Values are expressed as means ± SEM (n = 6).

The application of the CaV1.x channel activator, BayK8644 to Huh7ins and MIN6 cells signif‐ icantly increased insulin secretion above basal levels (*p*< 0.01, *n* = 6; Figure 6A). In the pres‐ ence of 20 mM glucose, BayK8644 further amplified glucose-stimulated insulin secretion in Huh7ins cells (*p*< 0.05, *n* = 6, Fig. 6A). Application of the SERCA blocker, ryanodine, which prevents increases in [Ca2+]i , caused a decrease in glucose-stimulated insulin secretion from Huh7ins and MIN6 cells (*p*< 0.05, *n* = 6; Figure 6B).

The dihydropyridine, BayK8644, functions as a CaV1 channel agonist, which interacts with the α1 subunit of CaV channels to stabilise the channel in the open state, thereby enhancing Ca2+ influx to cause the exocytosis of insulin [41]. BayK8644 does not change the membrane Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-Sensitive Potassium Channels and Voltage-Gated Calcium Channels http://dx.doi.org/10.5772/52839 719

potential of resting β-cells [43]. Rather, it acts on the Ca<sup>V</sup> channel in the open state, failing to affect basal insulin secretion at non-stimulatory glucose concentrations [43], but exaggerat‐ ing glucose-stimulated insulin secretion [44, 45]. The addition of BayK8644 increased insulin secretion by both the Huh7ins and MIN6 cells. However, the amount of insulin secreted in the presence of BayK8644 was lower than that released in response to 20 mM glucose alone. Putatively, this concentration of glucose may have stimulated the influx of extracellular Ca2+, the release of [Ca2+]i from intracellular stores and increased Ca2+ via other Ca2+-related path‐ ways to such an extent that the total increase of Ca2+ in the cell was higher in the presence of 20 mM glucose as compared to BayK8644 alone.

glibenclamide treatment were identical to that observed in each of the cell lines in which insulin was fused to EGFP (data not shown). Therefore, fusion of EGFP to insulin did not alter the

**Figure 6.** Secretion of insulin from Huh7ins cells and MIN6 cells. Insulin secretion was activated in response to 20 mM glucose alone or (A) 1 µM BayK8644 ± 20 mM glucose. (B) 20 µM ryanodine ± 20 mM glucose; and (C) 1 µM thapsigar‐ gin in the absence of extracellular calcium. Cells were incubated in basal medium for two consecutive 1 h periods be‐ fore being exposed to the stimulus for 1 h, followed by a third period of basal incubation. Cells in the control group

The application of the CaV1.x channel activator, BayK8644 to Huh7ins and MIN6 cells signif‐ icantly increased insulin secretion above basal levels (*p*< 0.01, *n* = 6; Figure 6A). In the pres‐ ence of 20 mM glucose, BayK8644 further amplified glucose-stimulated insulin secretion in Huh7ins cells (*p*< 0.05, *n* = 6, Fig. 6A). Application of the SERCA blocker, ryanodine, which

The dihydropyridine, BayK8644, functions as a CaV1 channel agonist, which interacts with the α1 subunit of CaV channels to stabilise the channel in the open state, thereby enhancing Ca2+ influx to cause the exocytosis of insulin [41]. BayK8644 does not change the membrane

, caused a decrease in glucose-stimulated insulin secretion from

were treated throughout with basal medium. Values are expressed as means ± SEM (n = 6).

prevents increases in [Ca2+]i

Huh7ins and MIN6 cells (*p*< 0.05, *n* = 6; Figure 6B).

physiological mechanism of insulin secretion.

718 Gene Therapy - Tools and Potential Applications

Consistent with reports that BayK8644 is known to stimulate the opening of CaV channels in pancreatic β-cells without altering the membrane potential [44], static stimulation of Huh7ins cells with 1 µM BayK8644 plus 20 mM glucose amplified glucose-stimulated insu‐ lin release. However, BayK8644 failed to amplify glucose-stimulated insulin secretion in MIN6 cells. This finding may be attributable to the ability of 20 mM glucose alone to cause the maximum threshold in the activation of insulin release in MIN6 cells, such that the addi‐ tion of BayK8644 was unable to exert any additional stimulatory effects. Nevertheless, these results demonstrate that the insulin secretory response of the Huh7ins cells is dependent upon the activation of CaV channels, as is the case for pancreatic β-cells.

Static stimulation of Huh7ins cells with the highly specific SERCA blocker thapsigargin, which induces the release of Ca2+ from intracellular stores resulted in a significant increase (two-fold increase over basal levels), in insulin secretion in the absence of extracellular Ca2+ (*p*< 0.05, *n* = 6; Figure 6C). Consistent with results from Tuch *et al*. [7], the response of the Huh7ins cells to glucose was abolished when Ca2+ was removed from the basal medium be‐ fore 20 mM glucose was added (*p*> 0.05 *vs*. control, *n* = 6; Figure 6C). MIN6 cells showed a similar response; namely, in the absence of extracellular Ca2+ the glucose-responsiveness was abolished (*p*> 0.05 *vs*. control, *n* = 6; Figure 6C), and the presence of 1 µM thapsigargin significantly increased insulin secretion 1.8-fold over basal levels (*P*<0.05, *n* = 6; Figure 6C).

The connexon (hemi-channel blocker), oleic acid, significantly reduced acute insulin secre‐ tion by 1.4-fold (*p*< 0.05, *n* = 6; Figure 7A), while verapamil (10 µM) resulted in a significant decrease in insulin secretion to glucose in both cell lines (*p*< 0.05, *n* = 6; Figure 7B). However, the combination of verapamil and ryanodine did not exert an additive effect on insulin se‐ cretion, compared to treatment with verapamil alone (*p*< 0.05, *n* = 6; Fig. 7B). Nevertheless, a greater decrease in insulin secretion was observed after the addition of verapamil, ryano‐ dine and oleic acid in both Huh7ins (*p*< 0.05, *n* = 6) and MIN6 cells (*p*< 0.05, *n* = 6; Figure 7C).

SERCA operate to restore diminished intracellular endoplasmic and sarcoplasmic reticulum Ca2+ stores, thereby decreasing cytoplasmic Ca2+ levels [46-50]. Thapsigargin is a highly se‐ lective inhibitor of SERCA. Stimulation of β-cells with glucose causes an initial, thapsigar‐ gin-inhibitable, drop in [Ca2+]i that precedes the increase in [Ca2+]i due to the pumping of Ca2+ into the endoplasmic reticulum [51, 52]. Blocking of SERCA by thapsigargin augments the glucose-induced [Ca2+]i increase by activating a depolarising store-operated current, which then facilitates the opening of CaV channels [51, 53, 54]. Consistent with the results reported by Tuch et al, [7], in the absence of extracellular Ca2+, the glucose responsiveness of both Huh7ins and MIN6 cells in the absence of extracellular Ca2+, was lost, while normal glucose responsiveness was seen when Ca2+ was present in the medium. However, thapsi‐ gargin, which raises cytosolic Ca2+, stimulated insulin secretion by both Huh7ins and MIN6 cells in the absence of extracellular Ca2+. This finding further supports the role of intracellu‐ lar Ca2+ storage in insulin secretion in both pancreatic β-cells and in the insulin-secreting liv‐ er cell line, Huh7ins.

complete abrogation of glucose-responsiveness upon extracellular Ca2+ levels has been pre‐ viously reported for pancreatic β-cells [40, 43, 56]. As expected the addition of oleic acid to Huh7ins and MIN6 cells resulted in reduced glucose responsiveness, due to the blockage of

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

721

The results described in this chapter indicate that insulin secretion in engineered hepato‐ cytes (Huh7ins cells) was controlled, as precisely as in the pancreatic β-cell, by a fully func‐ tional KATP and CaV channel system. The results clearly document that Huh7ins cells respond to glucose via insulin secretion from secretory granules by the same mechanism ob‐ served in pancreatic β-cells. This is the first study to demonstrate a clear physiological and biochemical interaction of KATP channels and CaV channels in liver cells, and as such reveals that hepatocytes are ideal candidates for the engineering of artificial β-cells. Testament to this, we have successfully engineered a liver cell line to synthesize, store and secrete insulin. Regardless of whether this hepatoma cell line will be a viable β-cell alternative for trans‐ plantation into patients, the present study provides valuable information with regards to the future engineering of glucose-responsive insulin-secreting liver cells. Elucidation of the min‐ imal molecular modifications required for the creation of an artificial β-cell from a hepato‐ cyte may one day provide therapeutic avenues to engineer a patient's own liver cells to

This work was supported by grants from Diabetes Australia Research Trust, Rebecca L. Cooper Medical Research Foundation and the University of Technology Sydney. We would like to thank Wayne Hawthorne and Philip O'Connell from the Westmead Millennium Insti‐

, Guo Jun Liu3

, Donald K. Martin1

1 School of Medical & Molecular Biosciences, University of Technology Sydney, Sydney,

, Julia Ting1

, Chang Tao1

, Zehra Elgundi1

, Bronwyn A O'Brien1

and

,

, Tony An2

, Bernard E. Tuch6

,

hemi-channels, similar to what has been reported in pancreatic β-cells [57].

synthesize, store and secrete insulin in response to metabolic stimuli.

tute for human pancreatic islets and Richard Limburg for IT support.

**3. Conclusion**

**Acknowledgements**

**Author details**

Mark Lutherborrow4

Graham M. Nicholson1

Edwin Ch'ng1

Australia

Ann M. Simpson1\*, M. Anne Swan2

, Leticia M. Castro1

, Fraser Torpy5

\*Address all correspondence to: Ann.Simpson@uts.edu.au

**Figure 7.** Secretion of insulin from Huh7ins cells and MIN6 cells. Insulin secretion was activated in response to 20 mM glucose alone or in the presence of (A) 20 µM oleic acid; (B) 10 µM verapamil ± 10 µM ryanodine and (C) 10 µM vera‐ pamil, 20 µM ryanodine and 20 µM oleic acid. Cells were incubated in basal medium for two consecutive 1 h periods before being exposed to the stimulus for 1 h, followed by a third period of basal incubation. Cells in the control group were treated throughout with basal medium. Values are expressed as means ± SEM (n = 6).

The presence of 20 µM ryanodine, which blocks CaV channels at concentrations ≥ 10 µM [55] and prevents the release of Ca2+ from the endoplasmic reticulum, reduced the glucose-re‐ sponsiveness of both Huh7ins cells and MIN6 cells, although to a lesser extent than was ob‐ served in the presence of 10 µM verapamil. This finding is consistent with previous reports that intracellular Ca2+ stores (and therefore SERCA) contribute to the intracellular Ca2+ re‐ sponse during insulin secretion. The application of verapamil to Huh7ins cells caused a complete abrogation of glucose-responsiveness upon extracellular Ca2+ levels has been pre‐ viously reported for pancreatic β-cells [40, 43, 56]. As expected the addition of oleic acid to Huh7ins and MIN6 cells resulted in reduced glucose responsiveness, due to the blockage of hemi-channels, similar to what has been reported in pancreatic β-cells [57].

#### **3. Conclusion**

both Huh7ins and MIN6 cells in the absence of extracellular Ca2+, was lost, while normal glucose responsiveness was seen when Ca2+ was present in the medium. However, thapsi‐ gargin, which raises cytosolic Ca2+, stimulated insulin secretion by both Huh7ins and MIN6 cells in the absence of extracellular Ca2+. This finding further supports the role of intracellu‐ lar Ca2+ storage in insulin secretion in both pancreatic β-cells and in the insulin-secreting liv‐

**Figure 7.** Secretion of insulin from Huh7ins cells and MIN6 cells. Insulin secretion was activated in response to 20 mM glucose alone or in the presence of (A) 20 µM oleic acid; (B) 10 µM verapamil ± 10 µM ryanodine and (C) 10 µM vera‐ pamil, 20 µM ryanodine and 20 µM oleic acid. Cells were incubated in basal medium for two consecutive 1 h periods before being exposed to the stimulus for 1 h, followed by a third period of basal incubation. Cells in the control group

The presence of 20 µM ryanodine, which blocks CaV channels at concentrations ≥ 10 µM [55] and prevents the release of Ca2+ from the endoplasmic reticulum, reduced the glucose-re‐ sponsiveness of both Huh7ins cells and MIN6 cells, although to a lesser extent than was ob‐ served in the presence of 10 µM verapamil. This finding is consistent with previous reports that intracellular Ca2+ stores (and therefore SERCA) contribute to the intracellular Ca2+ re‐ sponse during insulin secretion. The application of verapamil to Huh7ins cells caused a

were treated throughout with basal medium. Values are expressed as means ± SEM (n = 6).

er cell line, Huh7ins.

720 Gene Therapy - Tools and Potential Applications

The results described in this chapter indicate that insulin secretion in engineered hepato‐ cytes (Huh7ins cells) was controlled, as precisely as in the pancreatic β-cell, by a fully func‐ tional KATP and CaV channel system. The results clearly document that Huh7ins cells respond to glucose via insulin secretion from secretory granules by the same mechanism ob‐ served in pancreatic β-cells. This is the first study to demonstrate a clear physiological and biochemical interaction of KATP channels and CaV channels in liver cells, and as such reveals that hepatocytes are ideal candidates for the engineering of artificial β-cells. Testament to this, we have successfully engineered a liver cell line to synthesize, store and secrete insulin. Regardless of whether this hepatoma cell line will be a viable β-cell alternative for trans‐ plantation into patients, the present study provides valuable information with regards to the future engineering of glucose-responsive insulin-secreting liver cells. Elucidation of the min‐ imal molecular modifications required for the creation of an artificial β-cell from a hepato‐ cyte may one day provide therapeutic avenues to engineer a patient's own liver cells to synthesize, store and secrete insulin in response to metabolic stimuli.

#### **Acknowledgements**

This work was supported by grants from Diabetes Australia Research Trust, Rebecca L. Cooper Medical Research Foundation and the University of Technology Sydney. We would like to thank Wayne Hawthorne and Philip O'Connell from the Westmead Millennium Insti‐ tute for human pancreatic islets and Richard Limburg for IT support.

#### **Author details**

Ann M. Simpson1\*, M. Anne Swan2 , Guo Jun Liu3 , Chang Tao1 , Bronwyn A O'Brien1 , Edwin Ch'ng1 , Leticia M. Castro1 , Julia Ting1 , Zehra Elgundi1 , Tony An2 , Mark Lutherborrow4 , Fraser Torpy5 , Donald K. Martin1 , Bernard E. Tuch6 and Graham M. Nicholson1

\*Address all correspondence to: Ann.Simpson@uts.edu.au

1 School of Medical & Molecular Biosciences, University of Technology Sydney, Sydney, Australia

2 School of Medical Sciences (Anatomy & Histology) and Bosch Institute, University of Syd‐ ney, Australia

[8] Ren, B. H., O'Brien, B. A., Swan, M. A., Kiona, M. E., Nassif, N., Wei, M. Q., & Simp‐ son, A. M. (2007). Long-term correction of diabetes in rats following lentiviral hepatic

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

http://dx.doi.org/10.5772/52839

723

[9] Simpson, A. M., Tao, C., Swan, M. A., Ren, B., & O'Brien, B. A. (2008). Glucose regu‐ lated production of human insulin in H4IIE rat liver cells. *Diabetes*, 56(1), A120. [10] Tabiin, M. T., Tuch, B. E., Bai, L., Han-G, X., & Simpson, A. M. (2001). Susceptibility of insulin-secreting hepatocytes to the toxicity of pro-inflammatory cytokines. *J Auto‐*

[11] Permutt, MA, Koranyi, L., Keller, K., Lacy, P. E., & Scharp, D. W. (1989). Cloning and functional expression of a human pancreatic islet glucose-transporter cDNA. *Proc*

[12] Weinhouse, S. (1976). *In: Current topics in Cellular regulation. BL Horecker & ER Stadt‐*

[13] Aguilar-Bryan, L., Nichols, C. G., Wechsler, S. W., Clement, J. P., Boyd, A. E., Gonzá‐ lez, G., Herrera-Sosa, H., Nguy, K., Bryan, J., & Nelson, D. A. (1995). Cloning of the beta cell high-affinity sulfonylurea receptor: a regulator of insulin secretion. *Science*,

[14] Inagaki, N., Gonoi, T., Clement, J. P., Namba, N., Inazawa, J., Gonzalez, G., Aguilar-Bryan, L., Seino, S., & Bryan, J. (1995). Reconstitution of IKATP: an inward rectifier

[15] Inagaki, N., Gonoi, T., & Seino, S. (1997). Subunit stoichiometry of the pancreatic be‐

 channel. *FEBS Lett*, 409, 232-236. [16] Clement, J. P., Kunjilwar, K., Gonzalez, G., Schwanstecher, M., Panten, U., Aguilar-Bryan, L., & Bryan, J. (1997). Association and stoichiometry of KATP channel subunits.

[17] Shyng, S., & Nichols, C. G. (1997). Octameric stoichiometry of the KATP channel com‐

[18] Aguilar-Bryan, L., Clement, J. P., Gonzalez, G., Kunjilwar, K., Babenko, A., & Bryan, J. (1998). Toward understanding the assembly and structure of KATP channels. *Physiol*

[19] Ashcroft, F. M., & Rorsman, P. (1989). Electrophysiology of the pancreatic beta-cell.

[20] Lang, J. (1999). Molecular mechanisms and regulation of insulin exocytosis as a para‐

[21] Macfarlane, W. M., O'Brien, R. E., Barnes, P. D., Shepherd, R. M., Cosgrove, K. E., Lindley, K. J., Aynsley-Green, A., James, R. F., Docherty, K., & Dunne, MJ. (2000). Sulfonylurea receptor 1 and Kir6.2 expression in the novel human insulin-secreting

subunit plus the sulfonylurea receptor. *Science*, 270-1166.

insulin gene therapy. *Diabetologia*, 50, 1910-1920.

*Natl Acad Sci USA*, 86(22), 8688-8692.

*man., editors. Academic Press*.

ta-cell ATP-sensitive K+

*Neuron*, 18, 827-838.

*Rev*, 78-227.

plex. *J Gen Physiol*, 110, 655-664.

*Prog Biophys Mol Biol*, 54-87.

cell line NES2Y. *Diabetes*, 49-953.

digm of endocrine secretion. *Eur J Biochem*, 259-3.

*immunity*, 17-229.

268-423.

3 Brain and Mind Research Institute, Faculty of Health Sciences, University of Sydney and Life Sciences, Australian Nuclear Science and Technology Organization, Sydney, Australia

4 Australian Foundation for Diabetes Research & Diabetes Transplant Unit, Sydney, Aus‐ tralia

5 School of the Environment, University of Technology Sydney, Sydney, Australia

6 Australian Foundation for Diabetes Research & Diabetes Transplant Unit, Prince of Wales Hospital, and CSIRO, Division of Materials Science and Engineering, Sydney, Australia

#### **References**


[8] Ren, B. H., O'Brien, B. A., Swan, M. A., Kiona, M. E., Nassif, N., Wei, M. Q., & Simp‐ son, A. M. (2007). Long-term correction of diabetes in rats following lentiviral hepatic insulin gene therapy. *Diabetologia*, 50, 1910-1920.

2 School of Medical Sciences (Anatomy & Histology) and Bosch Institute, University of Syd‐

3 Brain and Mind Research Institute, Faculty of Health Sciences, University of Sydney and Life Sciences, Australian Nuclear Science and Technology Organization, Sydney, Australia

4 Australian Foundation for Diabetes Research & Diabetes Transplant Unit, Sydney, Aus‐

6 Australian Foundation for Diabetes Research & Diabetes Transplant Unit, Prince of Wales Hospital, and CSIRO, Division of Materials Science and Engineering, Sydney, Australia

[1] Eisenbarth, G. S. (1986). Type I diabetes mellitus: a chronic autoimmune disease. *N*

[2] Simpson, A. M., Tuch, B. E., Swan, Tu. J., & Marshall, G. M. (1995). Functional ex‐ pression of the human insulin gene in a human hepatoma cell line (HEP G2). *Gene*

[3] Simpson, A. M., Marshall, G. M., Tuch, B. E., Maxwell, L., Swan, MA, Tu, J., Beynon, S., Szymanska, B., & Camacho, M. (1997). Gene therapy of diabetes: glucose-stimulat‐

[4] Ber, I., Shternhall, K., Perl, S., Ohanuna, Z., Goldberg, I., Barshack, I., Benvenisti-Za‐ rum, L., Meivar-Levy, I., & Ferber, S. (2003). Functional, persistent, and extended liv‐

[5] Ferber, S., Halkin, A., Cohen, H., Ber, I., Einav, Y., Goldberg, I., Barshack, I., Seijffers, R., Kopolovic, J., Kaiser, N., & Karasik, A. (2000). Pancreatic and duodenal homeobox gene 1 induces expression of insulin genes in liver and ameliorates streptozotocin-in‐

[6] Kojima, H., Fujimiya, M., Matsumara, K., Yunan, P., Imaeda, H., Maeda, M., & Chan, L. (2003). NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and

[7] Tuch, B. E., Szymanska, B., Yao, M., Tabiin, M., Gross, D., Holman, S., Swan, MA, Humphrey, R., Marshall, G. M., & Simpson, A. M. (2003). Function of a genetically modified human liver cell line that stores, processes and secretes insulin. *Gene Thera‐*

ed insulin secretion in a human hepatoma cell line. *Gene Therapy*, 4-1202.

er to pancreas transdifferentiation. *J Biol Chem*, 278-31950.

duced hyperglycaemia. *Nature Med*, 6-568.

reverses diabetes in mice. *Nature Med*, 9-596.

5 School of the Environment, University of Technology Sydney, Sydney, Australia

ney, Australia

722 Gene Therapy - Tools and Potential Applications

**References**

*Engl J Med*, 4-1360.

*Therapy*, 2-231.

*py*, 10-490.

tralia


[22] Braun, M., Ramracheya, R., Zhang, Q., Karanauskaite, J., Partridge, C., Johnson, P. R., & Rorsman, P. (2008). Voltage-gated ion channels in human pancreatic β-cells: Elec‐ trophysiological characterization and role in insulin secretion. *Diabetes*, 57-1618.

[34] Yang, L., Li, S., Hatch, H., Ahrens, K., Cornelius, J. G., Petersen, B. E., & Peck, A. B. (2002). In vitro trans-differentiation of adult hepatic stem cells into pancreatic endo‐

Insulin Trafficking in a Glucose Responsive Engineered Human Liver Cell Line is Regulated by the Interaction of ATP-

[36] Quesada, I., Chin, W. C., Steed, J., Campos-Bedolla, P., & Verdugo, P. (2001). Mouse mast cell secretory granules can function as intracellular ionic oscillators. *Biophys J*,

[37] National Institutes of Health. (2008). NIH Image. http://rsb.info.hih.gov/nih-image/

[38] Arvan, P., & Halban, P. A. (2004). Sorting ourselves out: seeking consensus on traf‐

[39] National Institutes of Health. (2009). Image J. http://rsb.info.nih.gov/ij/Accessed 20

[40] Yoon, N., Nataliya, S., Jeong-J, M., Lee, T., Lee-S, M., Kim-L, H., Chin, H., Suh-G, P., Kim, S., & Shin-S, H. (2003). Requirement for the L-type Ca2+ channel α1D subunit in

[41] Catterall, W. A., & Striessnig, J. (1992). Receptor sites for Ca2+ channel antagonists.

[42] Henquin, J. C. (2000). Triggering and amplifying pathways of regulation of insulin

[43] Ammälä, C., Moorhouse, A., & Ashcroft, F. M. (1996). The sulphonylurea receptor

[44] Larsson-Nyren, G., & Sehlin, J. (1996). Comparison of the effects of perchlorate and Bay K 8644 on the dynamics of cytoplasmic Ca2+ concentration and insulin secretion

[45] Malaisse-Lagae, F., Matthias, P. C. F., & Malaisse, W. J. (1984). Gating and blocking of calcium channels by dihydropyridines in the pancreatic β-cell. *Biochem Biophys Res*

[46] Lytton, J., Westlin, M., & Hanley, M. R. (1991). Thapsigargin inhibits the sarcoplas‐ mic or endoplasmic reticulum Ca-ATPase family of calcium pumps. *J Biol Chem*,

[47] Kirby, M. S., Sagara, Y., Gaa, S., Inesi, G., Lederer, W. J., & Rogers, T. B. (1992). Thap‐ sigargin inhibits contraction and Ca2+ transient in cardiac cells by specific inhibition

of the sarcoplasmic reticulum Ca2+ pump. *J Biol Chem*, 267, 12545-12551.

postnatal pancreatic β-cell generation. *J Clin Inves*, 108, 1015-1022.

confers diazoxide sensitivity on the inwardly rectifying K+

in human embryonic kidney cells. *J Physiol*, 494, 709-714.

ion exchange in intra‐

http://dx.doi.org/10.5772/52839

725

Sensitive Potassium Channels and Voltage-Gated Calcium Channels

channel Kir6.1 expressed

crine hormone-producing cells. *Proc Natl Acad Sci USA*, 99-8078.

[35] Nguyen, T., Chin, W. C., & Verdugo, P. (1998). Role of Ca2+/K+

cellular storage and release of Ca2+. *Nature*, 395-908.

ficking in the beta-cell. *Traffic* , 5, 53-61.

secretion by glucose. *Diabetes*, 49, 1751-1760.

in mouse β-cells. *Biochem J*, 314-167.

*Comm*, 123-1062.

266-17067.

80, 2133-2139.

September).

Trends Pharm Sci; , 13-256.

Accessed 1 July,).


[34] Yang, L., Li, S., Hatch, H., Ahrens, K., Cornelius, J. G., Petersen, B. E., & Peck, A. B. (2002). In vitro trans-differentiation of adult hepatic stem cells into pancreatic endo‐ crine hormone-producing cells. *Proc Natl Acad Sci USA*, 99-8078.

[22] Braun, M., Ramracheya, R., Zhang, Q., Karanauskaite, J., Partridge, C., Johnson, P. R., & Rorsman, P. (2008). Voltage-gated ion channels in human pancreatic β-cells: Elec‐ trophysiological characterization and role in insulin secretion. *Diabetes*, 57-1618. [23] Wollheim, C. B., & Sharp, G. W. (1981). Regulation of insulin release by calcium.

[24] Gilon, P., & Henquin, J. C. (2001). Mechanisms and physiological significance of the

[25] Bereton, H. M., Harland, M. L., Froscio, M., Petronijevic, T., & Barrit, G. J. (1997). Novel variants of voltage-operated calcium channel alpha 1-subunit transcripts in a rat liver-derived cell line: deletion in the IVS4 voltage sensing region. *Cell Calcium*,

[26] Snutch, T. P., Tomlinson, W. J., Leonard, J. P., & Gilbert, M. M. (1991). Distinct calci‐ um channels are generated by alternative splicing and are differentially expressed in

[27] Liu, G. J., Simpson, A. M., Swan, Tao. C., Tuch, B. E., Crawford, R. M., Jovanovic, A., & Martin, D. K. (2003). ATP-sensitive potassium channels induced in liver cells after transfection with insulin cDNA and the GLUT 2 transporter regulate glucose-stimu‐

[28] Malhi, H., Irani, A. N., Rajvanshi, P., Suadicani, S. O., Spray, D. C., Mc Donald, T. V., & Gupta, S. (2000). KATP channels regulate mitogenically induced proliferation in pri‐

[29] Lutherborrow, M. A., Appavoo, M., Simpson, A. M., & Tuch, B. E. (2009). Gene ex‐ pression profiling of Huh7ins lack of a granulogenic function for chromagranin A.

[30] Miyazaki-I, J., Araki, K., Yamato, E., Ikegami, H., Asano, T., Shibasaki, Y., Oka, Y., & Yamamura, K. (1990). Establishment of a pancreatic beta cell line that retains glucoseinducible insulin secretion: Special reference to expression of glucose transporter. *En‐*

[31] Sapir, T., Shternhall, K., Meivar-Levy, I., Blumenfeld, I., Cohen, H., Skutelsky, E., Eventov-Friedman, S., Barshack, I., Goldberg, I., Pri-Chen, S., Ben-Dor, L., Polak-Charcon, S., Karasik, A., Shimon, I., Mor, E., & Ferber, S. (2005). Cell-replacement therapy for diabetes: generating functional insulin-producing tissue from adult hu‐

[32] Fodor, A., Harel, C., Fodor, L., Armoni, M., Salmon, P., Trono, D., & Karnielli, E. (2007). Adult rat liver cells transdifferentiated with lentiviral IPF1 vectors reverse

[33] Vollenweider, F., Irminger, J. C., Gross, D. J., Villa-Komaroff, L., & Halban, P. A. (1992). Processing of proinsulin by transfected hepatoma (FAO) cells. *J Biol Chem*,

diabetes in mice: an ex vivo gene therapy approach. *Diabetologia*, 50-121.

mary rat hepatocytes and human liver cell lines. *J Biol Chem*, 275-26050.

cholinergic control of pancreatic beta-cell function. *Endocr Rev*, 22-565.

*Physiol Rev*, 61-914.

724 Gene Therapy - Tools and Potential Applications

the mammalian CNS. *Neuron*, 7, 45-57.

lated insulin secretion. *FASEB J*, 17-1682.

man liver cells. *Proc Natl Acad Sci USA*, 102-7964.

22, 39-52.

*Islets*, 1, 60-70.

267-14629.

*docrinology*, 127-126.


[48] Gericke, M., Droogmans, G., & Nilius, B. (1993). Thapsigargin discharges intracellu‐ lar calcium stores and induces transmembrane currents in human endothelial cells. *Pflügers Arch*, 422-552.

**Chapter 30**

**Feasibility of Gene Therapy for Tooth**

Katsu Takahashi, Honoka Kiso, Kazuyuki Saito,

Additional information is available at the end of the chapter

Kazuhisa Bessho

**1. Introduction**

http://dx.doi.org/10.5772/52529

Yumiko Togo, Hiroko Tsukamoto, Boyen Huang and

**Regeneration by Stimulation of a Third Dentition**

The tooth is a complex biological organ that consists of multiple tissues, including enam‐ el, dentin, cementum, and pulp. Missing teeth is a common and frequently occurring problem in aging populations. To treat these defects, the current approach involves fixed or removable prostheses, autotransplantation, and dental implants. The exploration of new strategies for tooth replacement has become a hot topic. Using the foundations of ex‐ perimental embryology, developmental and molecular biology, and the principles of bio‐ mimetics, tooth regeneration is becoming a realistic possibility. Several different methods have been proposed to achieve biological tooth replacement[1-8]. These include scaffoldbased tooth regeneration, cell pellet engineering, chimeric tooth engineering, stimulation of the formation of a third dentition, and gene-manipulated tooth regeneration. The idea that a third dentition might be locally induced to replace missing teeth is an attractive concept[5,8,9]. This approach is generally presented in terms of adding molecules to in‐ duce *de novo* tooth initiation in the mouth. It might be combined with gene-manipulated tooth regeneration; that is, endogenous dental cells *in situ* can be activated or repressed by a gene-delivery technique to produce a tooth. Tooth development is the result of re‐ ciprocal and reiterative signaling between oral ectoderm-derived dental epithelium and cranial neural crest cell-derived dental mesenchyme under genetic control [10-12]. More than 200 genes are known to be expressed during tooth development (http://bite-it.helsin‐ ki.fi/). A number of mouse mutants are now starting to provide some insights into the mechanisms of supernumerary tooth formation. Multiple supernumerary teeth may have genetic components in their etiology and partially represent the third dentition in hu‐

> © 2013 Takahashi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


### **Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition**

Katsu Takahashi, Honoka Kiso, Kazuyuki Saito, Yumiko Togo, Hiroko Tsukamoto, Boyen Huang and Kazuhisa Bessho

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/52529

#### **1. Introduction**

[48] Gericke, M., Droogmans, G., & Nilius, B. (1993). Thapsigargin discharges intracellu‐ lar calcium stores and induces transmembrane currents in human endothelial cells.

[49] Parekh, A. B., Terlau, H., & Stühmer, W. (1993). Depletion of InsP3 stores activates a

[50] Randriamampita, C., & Tsien, R. Y. (1993). Emptying of intracellular Ca2+ stores re‐ leases a novel small messenger that stimulates Ca2+ influx. *Nature*, 364-809.

[51] Roe, M. W., Mertz, R. J., Lancaster, M. E., Worley, J. F. 3rd, & Dukes, I. D. (1994). Thapsigargin inhibits the glucose-induced decrease of intracellular Ca2+ in mouse is‐

[52] Miura, Y., Henquin, J. C., & Gilon, P. (1997). Emptying of intracellular Ca2+ stores stimulates Ca2+ entry in mouse pancreatic beta-cells by both direct and indirect mech‐

[53] Worley, J. F., Mc Intyre, M. S., Spencer, B., & Dukes, I. D. (1994a). Depletion of intra‐ cellular Ca2+ stores activates a maitotoxin-sensitive nonselective cationic current in

[54] Worley, J. F., Mc Intyre, M. S., Spencer, B., Mertz, R. J., Roe, M. W., & Dukes, I. D. (1994b). Endoplasmic reticulum calcium store regulates membrane potential in

[55] Meissner, G. (1986). Ryanodine activation of the Ca2+ release channel of sarcoplasmic

[56] Nevins, A. K., & Thurmond, D. C. (2003). Glucose regulates the cortical actin net‐ work through modulation of Cdc42 cycling to stimulate insulin secretion. *Am J Physi‐*

[57] Meda, P., Bosco, D., Chanson, M., Giordano, E., Vallar, L., Wollheim, C., & Orci, L. (1990). Rapid and reversible secretion changes during uncoupling of rat insulin-pro‐

lets of Langerhans. *Am J Physiol*, 266, E 852-862.

beta-cells. *J Biol Chem*, 269, 32055-32058.

reticulum. *J Biol Chem*, 261-6300.

*ol Cell Physiol*, 285, C698-710.

ducing cells. *J Clin Invest*, 86-759.

mouse islet beta-cells. *J Biol Chem*, 269, 14359-14362.

current by means of a phosphatase and a diffusible messenger. *Nature*,

*Pflügers Arch*, 422-552.

726 Gene Therapy - Tools and Potential Applications

anisms. *J Physiol*, 503-387.

Ca2+ and K+

364-814.

The tooth is a complex biological organ that consists of multiple tissues, including enam‐ el, dentin, cementum, and pulp. Missing teeth is a common and frequently occurring problem in aging populations. To treat these defects, the current approach involves fixed or removable prostheses, autotransplantation, and dental implants. The exploration of new strategies for tooth replacement has become a hot topic. Using the foundations of ex‐ perimental embryology, developmental and molecular biology, and the principles of bio‐ mimetics, tooth regeneration is becoming a realistic possibility. Several different methods have been proposed to achieve biological tooth replacement[1-8]. These include scaffoldbased tooth regeneration, cell pellet engineering, chimeric tooth engineering, stimulation of the formation of a third dentition, and gene-manipulated tooth regeneration. The idea that a third dentition might be locally induced to replace missing teeth is an attractive concept[5,8,9]. This approach is generally presented in terms of adding molecules to in‐ duce *de novo* tooth initiation in the mouth. It might be combined with gene-manipulated tooth regeneration; that is, endogenous dental cells *in situ* can be activated or repressed by a gene-delivery technique to produce a tooth. Tooth development is the result of re‐ ciprocal and reiterative signaling between oral ectoderm-derived dental epithelium and cranial neural crest cell-derived dental mesenchyme under genetic control [10-12]. More than 200 genes are known to be expressed during tooth development (http://bite-it.helsin‐ ki.fi/). A number of mouse mutants are now starting to provide some insights into the mechanisms of supernumerary tooth formation. Multiple supernumerary teeth may have genetic components in their etiology and partially represent the third dentition in hu‐

© 2013 Takahashi et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. mans. Such candidate molecules or genes might be those that are involved in embryonic tooth induction, in successional tooth formation, or in the control of the number of teeth. This means that it may be possible to induce *de novo* tooth formation by the in situ re‐ pression or activation of a single candidate gene. In this review, we present an overview of the collective knowledge of tooth regeneration, especially regarding the control of the number of teeth for gene therapy by the stimulation of a third dentition.

#### **2. The third dentition**

It has been suggested that, in humans, a "third dentition" with one or more supernumerary teeth can occur in addition to the permanent dentition, and supernumerary teeth are some‐ times thought to represent a partial post-permanent dentition [13-15]. The basic dentition pattern observed in mammals is diphyodont, and consists of three incisors, one canine, four premolars, and three molars, while Human teeth are diphyodont excepting the permanent molars [16]. The deciduous teeth are, ontogenetically, the first generation of teeth. The per‐ manent teeth (except molar) belong to the second dentition. The term "third dentition" re‐ fers to the opinion that one more set of teeth can occur in addition to the permanent teeth (Figure 1). Human teeth are diphyodont excepting the permanent molars. The normal mouse dentition is monophyodont and composed of one incisor and three molars in each quadrant. The number of teeth is usually strictly determined. It was initially reported that there is an anlage of the third dentition in some mammals [17]. The presence of an epithelial anlage of the third dentition was also noticed in humans [18,19]. The teeth and anlagen that appear in third dentition in serial sections of infant jaws and some fetuses have been ana‐ lyzed. The epithelium which is considered as the anlagen of the third dentition develops lin‐ gual to all permanent tooth germs [15]. Furthermore, when it appears, the predecessor (permanent tooth germ) is in the bell-shaped stage [15]. The timing of appearance of the third dentition seems to be after birth (Table 1). This means that we have a chance to access the formation of the third dentition in the mouth.

**Figure 1.** Multiple impacted supernumerary teeth in a 13-year-old non-syndromic patient. The third dentition devel‐ ops lingual to the permanent tooth germ (D). All impacted supernumerary teeth in this patient are located to the lin‐ gual side of the permanent teeth (white arrow) (A-C). These multiple supernumerary teeth seem to be post-

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

729

Analysis of other model systems with continuous tooth replacement or secondary tooth for‐ mation, such as in the fish, snake, lizard, and ferret, is providing insights into the molecular and cellular mechanisms underlying successional tooth development, and will assist in studies on supernumerary tooth formation in humans. While some nonmammalian species have multi rowed dentition and replace their teeth regularly throughout life, mammalian vertebrates have one row of teeth and only renew their teeth once, or, in some rodents, show no replacement [20-23]. Detailed histological analysis of the tooth replacement in these mod‐ els indicates that the successional teeth are initiated from the dental lamina epithelium, which grows from the lingual side of the deciduous tooth enamel organ, and it later elon‐ gates and buds into the jaw mesenchyme, forming successional teeth. Jarvien et al. showed that, in the ferret, Sostdc1 (also known as USAG-1, ectodin, and Wise) is expressed in the elongating successional dental lamina at the interface between the lamina and deciduous tooth, as well as the buccal side of the dental lamina, suggesting that Sostdc1 plays a role in defining the identity of the dental lamina [20]. Handrigan et al. analyzed successional tooth formation in the snake and in lizard, and proposed that dental epithelium stem cells are re‐ sponsible for the formation of successional lamina, and Wnt signaling may regulate the stem cell fate in these cells [24]. Maintenance or reactivation of component dental lamina is thus

pivotal for the replacement tooth and supernumerary formation.

permanent dentition ("third dentition").


mans. Such candidate molecules or genes might be those that are involved in embryonic tooth induction, in successional tooth formation, or in the control of the number of teeth. This means that it may be possible to induce *de novo* tooth formation by the in situ re‐ pression or activation of a single candidate gene. In this review, we present an overview of the collective knowledge of tooth regeneration, especially regarding the control of the

It has been suggested that, in humans, a "third dentition" with one or more supernumerary teeth can occur in addition to the permanent dentition, and supernumerary teeth are some‐ times thought to represent a partial post-permanent dentition [13-15]. The basic dentition pattern observed in mammals is diphyodont, and consists of three incisors, one canine, four premolars, and three molars, while Human teeth are diphyodont excepting the permanent molars [16]. The deciduous teeth are, ontogenetically, the first generation of teeth. The per‐ manent teeth (except molar) belong to the second dentition. The term "third dentition" re‐ fers to the opinion that one more set of teeth can occur in addition to the permanent teeth (Figure 1). Human teeth are diphyodont excepting the permanent molars. The normal mouse dentition is monophyodont and composed of one incisor and three molars in each quadrant. The number of teeth is usually strictly determined. It was initially reported that there is an anlage of the third dentition in some mammals [17]. The presence of an epithelial anlage of the third dentition was also noticed in humans [18,19]. The teeth and anlagen that appear in third dentition in serial sections of infant jaws and some fetuses have been ana‐ lyzed. The epithelium which is considered as the anlagen of the third dentition develops lin‐ gual to all permanent tooth germs [15]. Furthermore, when it appears, the predecessor (permanent tooth germ) is in the bell-shaped stage [15]. The timing of appearance of the third dentition seems to be after birth (Table 1). This means that we have a chance to access

number of teeth for gene therapy by the stimulation of a third dentition.

**2. The third dentition**

728 Gene Therapy - Tools and Potential Applications

the formation of the third dentition in the mouth.

**Table 1.** Timing of appearance of the third dentition

**Figure 1.** Multiple impacted supernumerary teeth in a 13-year-old non-syndromic patient. The third dentition devel‐ ops lingual to the permanent tooth germ (D). All impacted supernumerary teeth in this patient are located to the lin‐ gual side of the permanent teeth (white arrow) (A-C). These multiple supernumerary teeth seem to be postpermanent dentition ("third dentition").

Analysis of other model systems with continuous tooth replacement or secondary tooth for‐ mation, such as in the fish, snake, lizard, and ferret, is providing insights into the molecular and cellular mechanisms underlying successional tooth development, and will assist in studies on supernumerary tooth formation in humans. While some nonmammalian species have multi rowed dentition and replace their teeth regularly throughout life, mammalian vertebrates have one row of teeth and only renew their teeth once, or, in some rodents, show no replacement [20-23]. Detailed histological analysis of the tooth replacement in these mod‐ els indicates that the successional teeth are initiated from the dental lamina epithelium, which grows from the lingual side of the deciduous tooth enamel organ, and it later elon‐ gates and buds into the jaw mesenchyme, forming successional teeth. Jarvien et al. showed that, in the ferret, Sostdc1 (also known as USAG-1, ectodin, and Wise) is expressed in the elongating successional dental lamina at the interface between the lamina and deciduous tooth, as well as the buccal side of the dental lamina, suggesting that Sostdc1 plays a role in defining the identity of the dental lamina [20]. Handrigan et al. analyzed successional tooth formation in the snake and in lizard, and proposed that dental epithelium stem cells are re‐ sponsible for the formation of successional lamina, and Wnt signaling may regulate the stem cell fate in these cells [24]. Maintenance or reactivation of component dental lamina is thus pivotal for the replacement tooth and supernumerary formation.

#### **3. Human syndromes associated with supernumerary teeth**

Supernumerary teeth can be associated with a syndrome (Table 2) or they can be found in non-syndromic patients [25-28]. Only 1% of non-syndromic cases have multiple supernum‐ erary teeth, which occur most frequently in the mandibular premolar area, followed by the molar and anterior regions, respectively [29-34]. There are special cases exhibiting perma‐ nent supernumerary teeth developing as supplementary teeth forming after the permanent teeth. These are thought to represent a third dentition, best known as manifestations of clei‐ docranial dysplasia (CCD).

ulates the proliferation of cells and may exert specific control on the dental lamina and forma‐ tion of successive dentitions. Runx2 heterozygous mutant mice mostly phenocopied the skeletal defects of CCD in humans, but with no supernumerary tooth formation [48] (Otto, 1997). Notably, in Runx2 homozygous and heterozygous mouse upper molars, a prominent epithelial bud regularly presents. This epithelial bud protrudes lingually with active Shh sig‐ naling, and it may represent the extension of the dental lamina for successional tooth formation in mice. Hence, although Runx2 is required for primary tooth development, it prevents the

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

731

Familial adenomatous polyposis (FAP), also named adenomatous polyposis of the colon (APC), is an autosomal dominant hereditary disorder characterized by the development of many precancerous colorectal adenomatous polyps, some of which will inevitably develop in‐ to cancer. In addition to colorectal neoplasm, individuals can develop variable extracolonic le‐ sions, including upper gastrointestinal polyposis, osteomas, congenital hypertrophy of the retinal pigment epithelium, soft tissue tumors, desmoid tumors, and dental anomalies [49-53]. Dental abnormalities include impacted teeth, congenital absence of one or more teeth, super‐ numerary teeth, dentigerous cysts associated with the crown of an unerupted tooth, and odontomas[50,52]. Gardner syndrome is a variant of FAP characterized by multiple adeno‐ mas of the colon and rectum typical of FAP together with osteomas and soft tissue tu‐ mors[49,51]. Supernumerary teeth and osteomas were originally described as a part of Gardner syndrome, but they can also occur in FAP patients with or without other extracolonic lesions [51,52]. FAP and Gardner syndrome are caused by a large number of germinal muta‐ tions in the *APC* gene [52,53]. *APC* is a tumor suppressor gene involved in the down-regula‐ tion of free intracellular ß-catenin, the major signal transducer of the canonical Wnt signaling pathway, as well as a central component of the E-cadherin adhesion complex [54,55]. In addi‐ tion, the *APC* protein may also play roles in chromosomal stability, the regulation of cell mi‐ gration up the colonic crypt and cell adhesion through association with GSK3ß, and other functions associated with microtubule bundles [55,56]. Inactivation of *APC* would lead to the stabilization and accumulation of the proto-oncogene ß-catenin, dysregulation of the cell cy‐ cle, and chromosomal instability [52]. Approximately 11-27% of patients have supernumerary teeth, but, so far, no specific codon mutation of the *APC* gene has been found to correlate with supernumerary teeth. Correlations seem to exist between dental abnormalities and the num‐ ber and type of osteomas, with the highest incidence of supernumerary teeth and odontomas being found in FAP patients with three or more osteomas[52]. Conditional knockout of the *Apc*-gene resulted in supernumerary teeth in mice [57-59]. Notably, adult oral tissues, espe‐ cially young adult tissues, are still responsive to the loss of *Apc*[60]. In old adult mice, super‐ numerary teeth can be induced on both labial and lingual sides of the incisors, which contain adult stem cells supporting the continuous growth of mouse incisors [60,61]. In young mice, supernumerary tooth germs were induced in multiple regions of the jaw in both incisor and molar regions. They can form directly from the oral epithelium, in the dental lamina connect‐ ing the developing molar or incisor tooth germ to the oral epithelium, in the crown region, as

growth of the dental lamina and successional tooth formation [47].

well as in the elongating and furcation area of the developing root [60].

tooth formation.

The identification of mutations in *RUNX2* causing an isolated dental phenotype in CCD and in *APC* causing FAP has attracted attention as a possible route towards inducing *de novo*


**Table 2.** Human syndromes associated with supernumerary teeth

Genetic mutations have been associated with the presence or absence of individual types of teeth. Supernumerary teeth are associated with more than 20 syndromes and developmental abnormalities like CCD, and Gardner syndrome [35]. The percentage occurrence in CCD is 22% in the maxillary incisor region and 5% in the molar region[36-38]. CCD is a dominantly inherit‐ ed skeletal dysplasia caused by mutations in *Runx2* [39-40]. It is characterized by persistently open sutures or the delayed closure of sutures, hypoplastic or aplastic clavicles, a short stature, delayed eruption of permanent dentition, supernumerary teeth, and other skeletal anomalies. There is a wide spectrum of phenotypic variability ranging from the full-blown phenotype to an isolated dental phenotype characterized by supernumerary tooth formation and/or the de‐ layed eruption of permanent teeth in CCD (Figure 1) [41-44]. A dose-related effect seems to be present, as the milder case of CCD, and those exhibiting primary dental anomalies, are related to mutations that reduce, but do not abolish, protein stability, DNA binding, and transactiva‐ tion [41,43-45]. Runx2-deficient mice were found to exhibit lingualbuds in front of the upper molars, and these were much more prominent than in wild-type mice[46,47].These buds pre‐ sumably represent the mouse secondary dentition, and it is likely that Runx2 acts to prevent the formation of these buds. Runx2 usually functions as a cell growth inhibitor[43]. Runx2 reg‐ ulates the proliferation of cells and may exert specific control on the dental lamina and forma‐ tion of successive dentitions. Runx2 heterozygous mutant mice mostly phenocopied the skeletal defects of CCD in humans, but with no supernumerary tooth formation [48] (Otto, 1997). Notably, in Runx2 homozygous and heterozygous mouse upper molars, a prominent epithelial bud regularly presents. This epithelial bud protrudes lingually with active Shh sig‐ naling, and it may represent the extension of the dental lamina for successional tooth formation in mice. Hence, although Runx2 is required for primary tooth development, it prevents the growth of the dental lamina and successional tooth formation [47].

**3. Human syndromes associated with supernumerary teeth**

docranial dysplasia (CCD).

730 Gene Therapy - Tools and Potential Applications

**Table 2.** Human syndromes associated with supernumerary teeth

Supernumerary teeth can be associated with a syndrome (Table 2) or they can be found in non-syndromic patients [25-28]. Only 1% of non-syndromic cases have multiple supernum‐ erary teeth, which occur most frequently in the mandibular premolar area, followed by the molar and anterior regions, respectively [29-34]. There are special cases exhibiting perma‐ nent supernumerary teeth developing as supplementary teeth forming after the permanent teeth. These are thought to represent a third dentition, best known as manifestations of clei‐

Genetic mutations have been associated with the presence or absence of individual types of teeth. Supernumerary teeth are associated with more than 20 syndromes and developmental abnormalities like CCD, and Gardner syndrome [35]. The percentage occurrence in CCD is 22% in the maxillary incisor region and 5% in the molar region[36-38]. CCD is a dominantly inherit‐ ed skeletal dysplasia caused by mutations in *Runx2* [39-40]. It is characterized by persistently open sutures or the delayed closure of sutures, hypoplastic or aplastic clavicles, a short stature, delayed eruption of permanent dentition, supernumerary teeth, and other skeletal anomalies. There is a wide spectrum of phenotypic variability ranging from the full-blown phenotype to an isolated dental phenotype characterized by supernumerary tooth formation and/or the de‐ layed eruption of permanent teeth in CCD (Figure 1) [41-44]. A dose-related effect seems to be present, as the milder case of CCD, and those exhibiting primary dental anomalies, are related to mutations that reduce, but do not abolish, protein stability, DNA binding, and transactiva‐ tion [41,43-45]. Runx2-deficient mice were found to exhibit lingualbuds in front of the upper molars, and these were much more prominent than in wild-type mice[46,47].These buds pre‐ sumably represent the mouse secondary dentition, and it is likely that Runx2 acts to prevent the formation of these buds. Runx2 usually functions as a cell growth inhibitor[43]. Runx2 reg‐ Familial adenomatous polyposis (FAP), also named adenomatous polyposis of the colon (APC), is an autosomal dominant hereditary disorder characterized by the development of many precancerous colorectal adenomatous polyps, some of which will inevitably develop in‐ to cancer. In addition to colorectal neoplasm, individuals can develop variable extracolonic le‐ sions, including upper gastrointestinal polyposis, osteomas, congenital hypertrophy of the retinal pigment epithelium, soft tissue tumors, desmoid tumors, and dental anomalies [49-53]. Dental abnormalities include impacted teeth, congenital absence of one or more teeth, super‐ numerary teeth, dentigerous cysts associated with the crown of an unerupted tooth, and odontomas[50,52]. Gardner syndrome is a variant of FAP characterized by multiple adeno‐ mas of the colon and rectum typical of FAP together with osteomas and soft tissue tu‐ mors[49,51]. Supernumerary teeth and osteomas were originally described as a part of Gardner syndrome, but they can also occur in FAP patients with or without other extracolonic lesions [51,52]. FAP and Gardner syndrome are caused by a large number of germinal muta‐ tions in the *APC* gene [52,53]. *APC* is a tumor suppressor gene involved in the down-regula‐ tion of free intracellular ß-catenin, the major signal transducer of the canonical Wnt signaling pathway, as well as a central component of the E-cadherin adhesion complex [54,55]. In addi‐ tion, the *APC* protein may also play roles in chromosomal stability, the regulation of cell mi‐ gration up the colonic crypt and cell adhesion through association with GSK3ß, and other functions associated with microtubule bundles [55,56]. Inactivation of *APC* would lead to the stabilization and accumulation of the proto-oncogene ß-catenin, dysregulation of the cell cy‐ cle, and chromosomal instability [52]. Approximately 11-27% of patients have supernumerary teeth, but, so far, no specific codon mutation of the *APC* gene has been found to correlate with supernumerary teeth. Correlations seem to exist between dental abnormalities and the num‐ ber and type of osteomas, with the highest incidence of supernumerary teeth and odontomas being found in FAP patients with three or more osteomas[52]. Conditional knockout of the *Apc*-gene resulted in supernumerary teeth in mice [57-59]. Notably, adult oral tissues, espe‐ cially young adult tissues, are still responsive to the loss of *Apc*[60]. In old adult mice, super‐ numerary teeth can be induced on both labial and lingual sides of the incisors, which contain adult stem cells supporting the continuous growth of mouse incisors [60,61]. In young mice, supernumerary tooth germs were induced in multiple regions of the jaw in both incisor and molar regions. They can form directly from the oral epithelium, in the dental lamina connect‐ ing the developing molar or incisor tooth germ to the oral epithelium, in the crown region, as well as in the elongating and furcation area of the developing root [60].

The identification of mutations in *RUNX2* causing an isolated dental phenotype in CCD and in *APC* causing FAP has attracted attention as a possible route towards inducing *de novo* tooth formation.

#### **4. Supernumerary tooth formation in a mouse model**

The number of teeth is usually strictly determined. Whereas evidence supporting a genetic etiology for tooth agenesis is well established, the etiology of supernumerary tooth forma‐ tion is only partially understood in the mouse model (Table 3). Unlike humans, mice have only molars and incisors separated by a toothless region called the diastema. In addition, mice only have a single primary dentition and their teeth are not replaced. Therefore, mice may not be an optimal model for studying tooth replacement and supernumerary tooth for‐ mation [62]. Most of the reported mouse supernumerary teeth are located in the diastema region. This is not a *de novo* tooth formation but the rescue of vestigial tooth rudiments. Dur‐ ing the early stages of tooth development, many transient vestigial dental buds develop in the diastema area. Some of them can develop into the bud stage, but later regress and disap‐ pear by apoptosis, or merge with the mesial crown of the first molar tooth [63-68]. Major sig‐ naling pathways regulating tooth development are also expressed in these vestigial dental buds. Modulation of these signals can rescue these vestigial tooth rudiments to develop into supernumerary diastema teeth [23]. A number of mutant mouse strains have been reported exhibiting supernumerary diastema teeth. Although the rudimentary tooth buds form in the embryonic diastema, they regress apoptically [69]. Transgenic mice in which the keratin 14 promoter directs Ectodysplasin (Eda), a member of the tumor necrosis factor (TNF) family of signaling molecules, or Eda receptor expression to the epithelium had supernumerary teeth mesial to the first molar as a result of diastema tooth development [70-72]. It has also been reported that Sprouty2 (Spry2) or Spry4 (which encode negative feedback regulators of fibroblast growth factor (FGF)) deficient mice showed supernumerary tooth formation as a result of diastema tooth development[73]. Hypomorphic Polaris mice and *Wnt-Cre* (Polaris conditional mutant mice with affected Shh signaling) [73-74], Pax6 mutant mice [75] and Gas1 null mutants [73] were also included. Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We reported that USAG-1-deficient mice have supernumerary teeth (Figure 2).

**Table 3.** Mutant mouse associated with supernumerary teeth

down-stream molecule Lef-1 are essential for tooth development [77].

The supernumerary maxillary incisor appears to form as a result of the successive develop‐ ment of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells [14]. BMP signaling in the rudimentary maxillary incisor, as‐ sessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was signifi‐ cantly enhanced. Wnt signaling, as demonstrated by the nuclear localization of β-catenin, was also up-regulated. The inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signal‐ ing results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1-deficient mouse model (Figure 3) [76]. Canonical Wnt/β-catenin signaling and its

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

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733

**Figure 2.** Supernumerary teeth formation in Sostdc 1 (USAG-1) (A-C) and CEBPB (D-H) adult mutant mice. A: Oblique view of the maxillary incisors. B: Occlusal view of the mandibular incisors. C: Occlusal view of the mandibular molars. Micro-CT images (D-F) and HE-staining (G,H) of the murine head. A frontal view (D), a sagittal view (E) and a horizontal view (F) showed supernumerary tooth (red arrow). Two supernumerary teeth and an odontoma were seen in a low (G) and a high (H) magnification.


**Table 3.** Mutant mouse associated with supernumerary teeth

**4. Supernumerary tooth formation in a mouse model**

732 Gene Therapy - Tools and Potential Applications

mice have supernumerary teeth (Figure 2).

(G) and a high (H) magnification.

The number of teeth is usually strictly determined. Whereas evidence supporting a genetic etiology for tooth agenesis is well established, the etiology of supernumerary tooth forma‐ tion is only partially understood in the mouse model (Table 3). Unlike humans, mice have only molars and incisors separated by a toothless region called the diastema. In addition, mice only have a single primary dentition and their teeth are not replaced. Therefore, mice may not be an optimal model for studying tooth replacement and supernumerary tooth for‐ mation [62]. Most of the reported mouse supernumerary teeth are located in the diastema region. This is not a *de novo* tooth formation but the rescue of vestigial tooth rudiments. Dur‐ ing the early stages of tooth development, many transient vestigial dental buds develop in the diastema area. Some of them can develop into the bud stage, but later regress and disap‐ pear by apoptosis, or merge with the mesial crown of the first molar tooth [63-68]. Major sig‐ naling pathways regulating tooth development are also expressed in these vestigial dental buds. Modulation of these signals can rescue these vestigial tooth rudiments to develop into supernumerary diastema teeth [23]. A number of mutant mouse strains have been reported exhibiting supernumerary diastema teeth. Although the rudimentary tooth buds form in the embryonic diastema, they regress apoptically [69]. Transgenic mice in which the keratin 14 promoter directs Ectodysplasin (Eda), a member of the tumor necrosis factor (TNF) family of signaling molecules, or Eda receptor expression to the epithelium had supernumerary teeth mesial to the first molar as a result of diastema tooth development [70-72]. It has also been reported that Sprouty2 (Spry2) or Spry4 (which encode negative feedback regulators of fibroblast growth factor (FGF)) deficient mice showed supernumerary tooth formation as a result of diastema tooth development[73]. Hypomorphic Polaris mice and *Wnt-Cre* (Polaris conditional mutant mice with affected Shh signaling) [73-74], Pax6 mutant mice [75] and Gas1 null mutants [73] were also included. Uterine sensitization associated gene-1 (USAG-1) is a BMP antagonist, and also modulates Wnt signaling. We reported that USAG-1-deficient

**Figure 2.** Supernumerary teeth formation in Sostdc 1 (USAG-1) (A-C) and CEBPB (D-H) adult mutant mice. A: Oblique view of the maxillary incisors. B: Occlusal view of the mandibular incisors. C: Occlusal view of the mandibular molars. Micro-CT images (D-F) and HE-staining (G,H) of the murine head. A frontal view (D), a sagittal view (E) and a horizontal view (F) showed supernumerary tooth (red arrow). Two supernumerary teeth and an odontoma were seen in a low The supernumerary maxillary incisor appears to form as a result of the successive develop‐ ment of the rudimentary upper incisor. USAG-1 abrogation rescued apoptotic elimination of odontogenic mesenchymal cells [14]. BMP signaling in the rudimentary maxillary incisor, as‐ sessed by expressions of Msx1 and Dlx2 and the phosphorylation of Smad protein, was signifi‐ cantly enhanced. Wnt signaling, as demonstrated by the nuclear localization of β-catenin, was also up-regulated. The inhibition of BMP signaling rescues supernumerary tooth formation in E15 incisor explant culture. Based upon these results, we conclude that enhanced BMP signal‐ ing results in supernumerary teeth and BMP signaling was modulated by Wnt signaling in the USAG-1-deficient mouse model (Figure 3) [76]. Canonical Wnt/β-catenin signaling and its down-stream molecule Lef-1 are essential for tooth development [77].

showed a supernumerary tooth in the upper left quadrant. Another CEBPB-/- adult mouse did not display any supernumerary teeth in either jaw, but an odontoma in the lower-right quadrant. All of the CEBPB-/- adults appeared with a normal number of erupted incisors and molars. Nevertheless, 20%of the CEBPB+/- 12-month-olds hada missing lower third molar. Dental anomalies such as supernumerary teeth, odontomas, or hypodontia were not found

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

735

These mouse models clearly demonstrated that it was possible to induce *de novo* tooth for‐ mation by the in situ repression or activation of single candidate gene such as USAG-1.

Gene therapy provides a unique tool for the delivery of previously identified signaling molecules in both time and space that may significantly augment our progress toward clin‐ ical tooth regeneration. Stimulation of the formation of a third dentition and gene-manipu‐ lated tooth regeneration comprise an attractive concept (Figure 4). This approach is generally presented in terms of adding molecules to induce *de novo* tooth initiation in the mouth. It might be combined with gene-manipulated tooth regeneration; that is, endoge‐ nous dental cells *in situ* can be activated or repressed by a gene-delivery technique to make a tooth. We have a chance to access the formation of the third dentition in the mouth, be‐ cause the time of appearance of the third dentition seems to be after birth. As the half-life of targeted proteins in vivo is transient, tooth regeneration is not a common outcome fol‐ lowing conventional therapy. Typically, high concentrations are required to promote regen‐ eration [82]). Therefore, supplemental local production via gene transfer could be superior

**Figure 4.** *In vivo* gene delivery approach for the tooth regeneration by stimulation of a third dentition.

in mice of any other genotypes and/or age[81].

**5. Gene therapy approaches**

to bolus delivery methods.

**Figure 3.** Diagrammatic representation of the Sostdc (USAG-1) pathway during development

Overexpression of Lef-1 under the control of the K14 promoter in transgenic mice leads to the development abnormal invaginations of the dental epithelium in the mesenchyme and formation of a tooth-like structure [78]. *De novo* supernumerary teeth arising directly from the primary tooth germ or dental lamina have been reported in *Apc* loss-of-function (as dis‐ cussed in the previous section) or β-catenin gain-of-function mice, and *Sp6* (*Epiprofin*)-defi‐ cient mice. It was demonstrated that mouse tooth buds expressing stabilized β-catenin give rise to extra teeth[58] (Jarvinen et al., 2006). More recently, Epiprofin (Epfn) (a zinc finger transcription factor belonging to the Sp transcription factor superfamily)-deficient mice de‐ veloped an excess number of teeth[79]. Mammals only have one row of teeth in each jaw. Interestingly, in the *Osr2* null mutant mouse embryo, supernumerary tooth germs were found developing directly from the oral epithelium lingual to their molar tooth germs [80]. More recently, we also demonstrated that CEBPB deficiency was related to the formation of supernumerary teeth[81]. A total of 66.7% of CEBPB-/- 12-month-olds sustained supernumer‐ ary teeth and/or odontomas in the diastema between the incisor and the first molar. Two su‐ pernumerary teeth accompanied with a complex odontoma near the root of the upper right incisor were identified in a CEBPB-/- adult (Figure 2), whilst two other CEBPB-/- mice simply showed a supernumerary tooth in the upper left quadrant. Another CEBPB-/- adult mouse did not display any supernumerary teeth in either jaw, but an odontoma in the lower-right quadrant. All of the CEBPB-/- adults appeared with a normal number of erupted incisors and molars. Nevertheless, 20%of the CEBPB+/- 12-month-olds hada missing lower third molar. Dental anomalies such as supernumerary teeth, odontomas, or hypodontia were not found in mice of any other genotypes and/or age[81].

These mouse models clearly demonstrated that it was possible to induce *de novo* tooth for‐ mation by the in situ repression or activation of single candidate gene such as USAG-1.

#### **5. Gene therapy approaches**

**Figure 3.** Diagrammatic representation of the Sostdc (USAG-1) pathway during development

734 Gene Therapy - Tools and Potential Applications

Overexpression of Lef-1 under the control of the K14 promoter in transgenic mice leads to the development abnormal invaginations of the dental epithelium in the mesenchyme and formation of a tooth-like structure [78]. *De novo* supernumerary teeth arising directly from the primary tooth germ or dental lamina have been reported in *Apc* loss-of-function (as dis‐ cussed in the previous section) or β-catenin gain-of-function mice, and *Sp6* (*Epiprofin*)-defi‐ cient mice. It was demonstrated that mouse tooth buds expressing stabilized β-catenin give rise to extra teeth[58] (Jarvinen et al., 2006). More recently, Epiprofin (Epfn) (a zinc finger transcription factor belonging to the Sp transcription factor superfamily)-deficient mice de‐ veloped an excess number of teeth[79]. Mammals only have one row of teeth in each jaw. Interestingly, in the *Osr2* null mutant mouse embryo, supernumerary tooth germs were found developing directly from the oral epithelium lingual to their molar tooth germs [80]. More recently, we also demonstrated that CEBPB deficiency was related to the formation of supernumerary teeth[81]. A total of 66.7% of CEBPB-/- 12-month-olds sustained supernumer‐ ary teeth and/or odontomas in the diastema between the incisor and the first molar. Two su‐ pernumerary teeth accompanied with a complex odontoma near the root of the upper right incisor were identified in a CEBPB-/- adult (Figure 2), whilst two other CEBPB-/- mice simply

Gene therapy provides a unique tool for the delivery of previously identified signaling molecules in both time and space that may significantly augment our progress toward clin‐ ical tooth regeneration. Stimulation of the formation of a third dentition and gene-manipu‐ lated tooth regeneration comprise an attractive concept (Figure 4). This approach is generally presented in terms of adding molecules to induce *de novo* tooth initiation in the mouth. It might be combined with gene-manipulated tooth regeneration; that is, endoge‐ nous dental cells *in situ* can be activated or repressed by a gene-delivery technique to make a tooth. We have a chance to access the formation of the third dentition in the mouth, be‐ cause the time of appearance of the third dentition seems to be after birth. As the half-life of targeted proteins in vivo is transient, tooth regeneration is not a common outcome fol‐ lowing conventional therapy. Typically, high concentrations are required to promote regen‐ eration [82]). Therefore, supplemental local production via gene transfer could be superior to bolus delivery methods.

**Figure 4.** *In vivo* gene delivery approach for the tooth regeneration by stimulation of a third dentition.

Simply stated, gene therapy consists of the insertion of genes into an individual's cells di‐ rectly or indirectly with a matrix to promote a specific biological effect. Gene therapy can be used to induce a more favorable host response. Targeting cells for gene therapy re‐ quires the use of vectors or direct delivery methods to transfect them. To overcome the short half-lives of peptides in vivo, gene therapy that uses a vector that encodes the candi‐ date genes is utilized to stimulate the formation of the third dentition. The two main strat‐ egies of gene vector delivery have been applied. Gene vectors can be introduced directly to the target site (*in vivo* gene delivery) [83] or selected cells can be harvested, expanded, ge‐ netically transduced, and then reimplanted (*ex vivo* gene delivery). *In vivo* gene transfer in‐ volves the insertion of the gene of interest directly into the body, anticipating the genetic modification of the target cells. *Ex vivo* gene transfer includes the incorporation of genetic material into cells exposed from a tissue biopsy with subsequent reimplantation into the recipient. So far, *in vivo* gene delivery has been a suitable gene therapy approach in tooth regeneration by stimulation of the third dentition, but *ex vivo* gene delivery is not realistic because of the poor availability of ideal cells.

**6. Conclusion**

**Acknowledgement**

**Author details**

Katsu Takahashi1

Boyen Huang2

**References**

Aid for JSPS Fellows:02109741.

versity, Sakyo-ku, Kyoto, Japan

versity, Cairns, Australia

1599-1610.

, Honoka Kiso1

and Kazuhisa Bessho1

murine teeth. J Dent Res 2004;83(7):518-522.

We have a chance to access the formation of the third dentition in the mouth, because the timing of the appearance of the third dentition seems to be after birth. The identification of mutations in *RUNX2* causing an isolated dental phenotype in CCD and supernumerary tooth formation in the mouse model clearly demonstrated that it was possible to induce *de novo* tooth formation by the in situ repression or activation of a single candidate gene. These results support the idea that the *de novo* repression or activation of candidate genes such as RUNX2 or USAG-1 might be used to stimulate the third dentition in order to induce new tooth formation in the mouse (Figure 4). *In vivo* gene delivery seems to be a suitable gene

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

This work was supported by Grant-in-Aid for Scientific Research(C):22592213 and Grant-in-

1 Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto Uni‐

2 Department of Paediatric Dentistry, School of Medicine and Dentistry, James Cook Uni‐

[1] Ohazama A, Modino SA, Miletich I, Sharpe PT. Stem-cell-based tissue engineering of

[2] Duailibi MT, Duailibi SE, Young CS, Bartlett JD, Vacanti JP, Yelick PC. Bioengineered

[3] Young CS, Abukawa H, Asrican R, Ravens M, Troulis MJ, Kaban LB, Vacanti JP, Ye‐ lick PC. Tissue-engineered hybrid tooth and bone. Tissue Eng 2005;11(9-10):

teeth from cultured rat tooth bud cells. J Dent Res 2004;83(7):523-528.

, Yumiko Togo1

, Hiroko Tsukamoto1

http://dx.doi.org/10.5772/52529

737

,

, Kazuyuki Saito1

therapy approach in tooth regeneration by stimulation of the third dentition.

Gene transfer is accomplished through the use of viral and nonviral vectors. The three main classes of virus used for gene therapy are the retrovirus, adenovirus, and adenoas‐ sociated viruses. Retroviruses are ideal for long-term gene therapy since, once intro‐ duced, their DNA integrates and becomes part of the genome of the host cells. Indeed, the current human genome contains up to 5 to 8% of endogenous retroviral sequences that have been acquired over the course of evolution [84]. Adenoviruses are more com‐ monly suited for short-term gene delivery and are highly targeted for tissue engineering strategies that desire protein production over the course of several weeks. Efficient ade‐ novirus-directed gene delivery to odontogenic mesenchymal cells derived from cranial neural crest cells was reported [85,86]. In addition, because the adenovirus is well-known as the "virus of the common cold," infection is generally nontoxic and self-limiting. However, determination of the genotoxicity for each specific application is necessary to keep the safety profile within acceptable parameters. Adenoassociated viruses have be‐ come the focus of much research in recent years because of their complete inability to replicate without a helper virus, potential for tissue-specific targeting, and gene expres‐ sion in the order of months to years. The ability to specifically target one tissue type without adverse effects on neighboring tissues is highly desired in fields such as tooth regeneration. On the other hand, nonviral methods are safe and do not require immuno‐ suppression for successful gene delivery, but suffer from lower transfection efficiencies. DNA injection followed by application of electric fields (electroporation) has been more effective for introducing DNA than the use of simple DNA injection [87]. However, this method involves the concern that the electric pulse causes tissue damage. Recently, we reported that gene transfer using an ultra-fine needle [88], in addition to microbubbles enhanced transcutaneous sonoporation [87].

*In vivo* gene delivery seems to be a suitable gene therapy approach in tooth regeneration by stimulation of the third dentition.

#### **6. Conclusion**

Simply stated, gene therapy consists of the insertion of genes into an individual's cells di‐ rectly or indirectly with a matrix to promote a specific biological effect. Gene therapy can be used to induce a more favorable host response. Targeting cells for gene therapy re‐ quires the use of vectors or direct delivery methods to transfect them. To overcome the short half-lives of peptides in vivo, gene therapy that uses a vector that encodes the candi‐ date genes is utilized to stimulate the formation of the third dentition. The two main strat‐ egies of gene vector delivery have been applied. Gene vectors can be introduced directly to the target site (*in vivo* gene delivery) [83] or selected cells can be harvested, expanded, ge‐ netically transduced, and then reimplanted (*ex vivo* gene delivery). *In vivo* gene transfer in‐ volves the insertion of the gene of interest directly into the body, anticipating the genetic modification of the target cells. *Ex vivo* gene transfer includes the incorporation of genetic material into cells exposed from a tissue biopsy with subsequent reimplantation into the recipient. So far, *in vivo* gene delivery has been a suitable gene therapy approach in tooth regeneration by stimulation of the third dentition, but *ex vivo* gene delivery is not realistic

Gene transfer is accomplished through the use of viral and nonviral vectors. The three main classes of virus used for gene therapy are the retrovirus, adenovirus, and adenoas‐ sociated viruses. Retroviruses are ideal for long-term gene therapy since, once intro‐ duced, their DNA integrates and becomes part of the genome of the host cells. Indeed, the current human genome contains up to 5 to 8% of endogenous retroviral sequences that have been acquired over the course of evolution [84]. Adenoviruses are more com‐ monly suited for short-term gene delivery and are highly targeted for tissue engineering strategies that desire protein production over the course of several weeks. Efficient ade‐ novirus-directed gene delivery to odontogenic mesenchymal cells derived from cranial neural crest cells was reported [85,86]. In addition, because the adenovirus is well-known as the "virus of the common cold," infection is generally nontoxic and self-limiting. However, determination of the genotoxicity for each specific application is necessary to keep the safety profile within acceptable parameters. Adenoassociated viruses have be‐ come the focus of much research in recent years because of their complete inability to replicate without a helper virus, potential for tissue-specific targeting, and gene expres‐ sion in the order of months to years. The ability to specifically target one tissue type without adverse effects on neighboring tissues is highly desired in fields such as tooth regeneration. On the other hand, nonviral methods are safe and do not require immuno‐ suppression for successful gene delivery, but suffer from lower transfection efficiencies. DNA injection followed by application of electric fields (electroporation) has been more effective for introducing DNA than the use of simple DNA injection [87]. However, this method involves the concern that the electric pulse causes tissue damage. Recently, we reported that gene transfer using an ultra-fine needle [88], in addition to microbubbles

*In vivo* gene delivery seems to be a suitable gene therapy approach in tooth regeneration by

because of the poor availability of ideal cells.

736 Gene Therapy - Tools and Potential Applications

enhanced transcutaneous sonoporation [87].

stimulation of the third dentition.

We have a chance to access the formation of the third dentition in the mouth, because the timing of the appearance of the third dentition seems to be after birth. The identification of mutations in *RUNX2* causing an isolated dental phenotype in CCD and supernumerary tooth formation in the mouse model clearly demonstrated that it was possible to induce *de novo* tooth formation by the in situ repression or activation of a single candidate gene. These results support the idea that the *de novo* repression or activation of candidate genes such as RUNX2 or USAG-1 might be used to stimulate the third dentition in order to induce new tooth formation in the mouse (Figure 4). *In vivo* gene delivery seems to be a suitable gene therapy approach in tooth regeneration by stimulation of the third dentition.

#### **Acknowledgement**

This work was supported by Grant-in-Aid for Scientific Research(C):22592213 and Grant-in-Aid for JSPS Fellows:02109741.

#### **Author details**

Katsu Takahashi1 , Honoka Kiso1 , Kazuyuki Saito1 , Yumiko Togo1 , Hiroko Tsukamoto1 , Boyen Huang2 and Kazuhisa Bessho1

1 Department of Oral and Maxillofacial Surgery, Graduate School of Medicine, Kyoto Uni‐ versity, Sakyo-ku, Kyoto, Japan

2 Department of Paediatric Dentistry, School of Medicine and Dentistry, James Cook Uni‐ versity, Cairns, Australia

#### **References**


[4] Edwards PC, Mason JM. Gene-enhanced tissue engineering for dental hard tissue re‐ generation: (1) overview and practical considerations. Head Face Med2006;2:12.

[21] Koussoulakou DS, Margaritis LH, Koussoulakos SL. A curriculum vitae of teeth: evo‐

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

739

[23] Tummers M, Thesleff I. The importance of signal pathway modulation in all aspects

[24] Handrigan GR, Leung KJ, Richman JM. Identification of putative dental epithelial stem cells in a lizard with life-long tooth replacement. Development2010;137(21):

[25] Garvey MT, Barry HJ, Blake M. Supernumerary teeth--an overview of classification,

[26] Liu DG, Zhang WL, Zhang ZY, Wu YT, Ma XC. Three-dimensional evaluations of su‐ pernumerary teeth using cone-beam computed tomography for 487 cases. Oral Surg

[27] Díaz A, Orozco J, Fonseca M. Multiple hyperodontia: report of a case with 17 super‐ numerary teeth with non syndromic association. Med Oral Patol Oral Cir Bu‐

[28] Ferrés-Padró E, Prats-Armengol J, Ferrés-Amat E. A descriptive study of 113 uner‐ upted supernumerary teeth in 79 pediatric patients in Barcelona. Med Oral Patol Or‐

[29] Yusof WZ. Non-syndrome multiple supernumerary teeth: literature review. J Can

[30] Batra P, Duggal R, Parkash H. Non-syndromic multiple supernumerary teeth trans‐ mitted as an autosomal dominant trait. J Oral Pathol Med2005;34(10):621-625.

[31] Orhan AI, Ozer L, Orhan K. Familial occurrence of nonsyndromal multiple super‐

[32] Hyun HK, Lee SJ, Ahn BD, Lee ZH, Heo MS, Seo BM, Kim JW. Nonsyndromic multi‐ ple mandibular supernumerary premolars. J Oral MaxillofacSurg2008;66(7):

[33] Yagüe-García J, Berini-Aytés L, Gay-Escoda C. Multiple supernumerary teeth not as‐ sociated with complex syndromes: a retrospective study.Med Oral Patol Oral Cir Bu‐

[34] Inchingolo F, Tatullo M, Abenavoli FM, Marrelli M, Inchingolo AD, Gentile M, In‐ chingolo AM, Dipalma G. Non-syndromic multiple supernumerary teeth in a family

[35] Zhu JF, Marcushamer M, King DL, Henry RJ. Supernumerary and congenitally ab‐

unit with a normal karyotype: case report.Int J Med Sci 2010;7(6):378-384.

sent teeth: a literature review. J ClinPediatr Dent1996;20(2):87-95.

numerary teeth. A rare condition. Angle Orthod 2006;76(5):891-897.

[22] Mikkola ML. Controlling the number of tooth rows. Sci Signal2009;2(85):pe53.

of tooth development. J ExpZool B MolDevEvol2009;312B(4):309-319

diagnosis and management. J Can Dent Assoc1999;65(11):612-616

Oral Med Oral Pathol Oral RadiolEndod2007;103(3):403-11.

lution, generation, regeneration.Int J BiolSci2009;5(3):226-243.

3545-3549.

cal2009;14(5):E229-31.

1366-1369.

cal 2009;14(7):E331-336

al Cir Bucal2009;14(3):E146-52

Dent Assoc1990;56(2):147-149.


[4] Edwards PC, Mason JM. Gene-enhanced tissue engineering for dental hard tissue re‐ generation: (1) overview and practical considerations. Head Face Med2006;2:12.

[6] Nakao K, Morita R, Saji Y, Ishida K, Tomita Y, Ogawa M, Saitoh M, Tomooka Y, Tsuji T. The development of a bioengineered organ germ method. Nat Methods.2007;4(3):

[7] Ferreira CF, Magini RS, Sharpe PT. Biological tooth replacement and repair. J Oral

[8] Yu J, Shi J, Jin Y. Current Approaches and Challenges in Making a Bio-Tooth. Tissue

[9] Takahashi, K., Sakata, T., Murashima-Suginami, A., Tsukamoto, H., Kiso, H. and Bes‐ sho, K. Tooth regeneration: Potential for stimulation of the formation of a third denti‐

[10] Thesleff I, Sharpe P. Signalling networks regulating dental development. MechDev

[11] Chai Y, Slavkin HC. Prospects for tooth regeneration in the 21st century: a perspec‐

[12] ThesleffI. The genetic basis of tooth development and dental defects. Am J Med Gen‐

[13] Jensen BL, Kreiborg S. Development of the dentition in cleidocranial dysplasia. J Oral

[14] Murashima-Suginami A, Takahashi K, Kawabata T, Sakata T, Tsukamoto H, Sugai M, Yanagita M, Shimizu A, Sakurai T, Slavkin HC, Bessho K. Rudiment incisors sur‐ vive and erupt as supernumerary teeth as a result of USAG-1 abrogation. Biochem.

[15] Ooë T. Epithelial anlagen of human third dentition and their migrations in the man‐

[17] Leche W. Studienuber die Entwicklung des Zahnsstemsbei den Saugetieren. Morph

[18] Rose C. Uberesteeinervorzeitigenpralaktealen und einerviertenZahnreihebeim Men‐

[20] Järvinen E, Tummers M, Thesleff I. The role of the dental lamina in mammalian tooth

[19] Ahrens H. Entwicklung der menschlichenZahne. AnatHefte 1913;48:169-267

replacement. J ExpZool B MolDevEvol2009 ;312B(4):281-291.

tion by one gene. Current Topics in Genetics 2008;3: 77-82.

[5] Sartaj R, Sharpe P. Biological tooth replacement. J Anat2006;209(4):503-509.

227-230.

738 Gene Therapy - Tools and Potential Applications

Rehabil2007;34(12):933-939.

1997;67(2):111-123.

et A2006;140(23):2530-2535.

Pathol Med 1990;19(2):89-93.

Jb 1893;19: 502-574.

Eng Part B Rev2008;14(3):307-319.

tive. Microsc Res Tech2003;60(5):469-479.

Biophys. Res. Commun 2007;359(3):549-555.

dible and maxilla. OkajimasFolAnatJap 1969; 46(5):243-251.

[16] Hillson S. Teeth. Cambridge:Cambridge University Press; 1986.

schen. Oester-ungarViertjschrZhik 1895;11:45-50.


[36] Ida, M., Nakamura, T., and Utsunomiya, J. Osteomatous changes and tooth abnor‐ malities found in the jaw of patients with adenomatosis coli. Oral Surg. Oral Med. Oral Pathol 1981;52(1), 2-11.

[48] Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

741

[49] Gardner EJ, Richards RC. Multiple cutaneous and subcutaneous lesions occurring si‐ multaneously with hereditary polyposis and osteomatosis. Am J Hum Gen‐

[50] Chimenos-Küstner E, Pascual M, Blanco I, Finestres F. Hereditary familial polyposis and Gardner's syndrome: contribution of the odonto-stomatology examination in its diagnosis and a case description. Med Oral Patol Oral Cir Bucal2005;10(5):402-409. [51] Ramaglia L, Morgese F, Filippella M, Colao A. Oral and maxillofacial manifestations of Gardner's syndrome associated with growth hormone deficiency: case report and literature review. Oral Surg Oral Med Oral Pathol Oral RadiolEn‐

[52] Wijn MA, Keller JJ, Giardiello FM, Brand HS. Oral and maxillofacial manifestations

[53] Okamoto M, Sato C, Kohno Y, Mori T, Iwama T, Tonomura A, Miki Y, Utsunomiya J, Nakamura Y, White R, et al. Molecular nature of chromosome 5q loss in colorectal tumors and desmoids from patients with familial adenomatous polyposis. Hum Gen‐

[54] Groden J, Thliveris A, Samowitz W, Carlson M, Gelbert L, Albertsen H, Joslyn G, Ste‐ vens J, Spirio L, Robertson M, et al. Identification and characterization of the familial

[55] Heinen CD. Genotype to phenotype: analyzing the effects of inherited mutations in

[56] Phelps RA, Broadbent TJ, Stafforini DM, Jones DA. New perspectives on APC control of cell fate and proliferation in colorectal cancer. Cell Cycle2009;8(16):2549-2556 [57] Kuraguchi M, Wang XP, Bronson RT, Rothenberg R, Ohene-Baah NY, Lund JJ, Ku‐ cherlapati M, Maas RL, Kucherlapati R. Adenomatous polyposis coli (APC) is re‐ quired for normal development of skin and thymus. PLoS Genet2006;2(9):e146. [58] Järvinen E, Salazar-Ciudad I, Birchmeier W, Taketo MM, Jernvall J, Thesleff I. Con‐ tinuous tooth generation in mouse is induced by activated epithelial Wnt/beta-cate‐

[59] Liu F, Chu EY, Watt B, Zhang Y, Gallant NM, Andl T, Yang SH, Lu MM, Piccolo S, Schmidt-Ullrich R, Taketo MM, Morrisey EE, Atit R, Dlugosz AA, Millar SE. Wnt/ beta-catenin signaling directs multiple stages of tooth morphogenesis. Dev‐

[60] Wang XP, O'Connell DJ, Lund JJ, Saadi I, Kuraguchi M, Turbe-Doan A, Cavallesco R, Kim H, Park PJ, Harada H, Kucherlapati R, Maas RL. Apc inhibition of Wnt signaling

of familial adenomatous polyposis. Oral Dis2007;13(4):360-365.

adenomatous polyposis coli gene. Cell 1991;66(3):589-600.

colorectal cancer families. Mutat Res2010;693(1-2):32-45.

nin signaling. ProcNatlAcadSci U S A 2006;103(49):18627-18632.

bone development. Cell1997;89(5):765-771.

et1953;5(2):139-147.

dod2007;103(6):e30-34.

et1990;85(6):595-599.

Biol2008;313(1):210-224.


[48] Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell1997;89(5):765-771.

[36] Ida, M., Nakamura, T., and Utsunomiya, J. Osteomatous changes and tooth abnor‐ malities found in the jaw of patients with adenomatosis coli. Oral Surg. Oral Med.

[37] Shafer, W. G., Hine, M. K., and Levi, B. M.Textbook of oral pathology 4th Ed. Phila‐

[38] Jensen, B. L., and Kreiborg, S. Craniofacial growth in cleidocranial dysplasia--a roent‐

[39] Lee B, Thirunavukkarasu K, Zhou L, Pastore L, Baldini A, Hecht J, Geoffroy V, Ducy P, Karsenty G. Misense mutations abolishing DNA binding of the osteoblast-specific transcription factor OSF2/CBFA1 in cleidocranial dysplasia, Nature Genetics 1997 ;

[40] Mundlos S, Otto F, Mundlos C, Mulliken JB, Aylsworth AS, Albright S, Lindhout D, Cole WG, Henn W, Knoll JH, Owen MJ, Mertelsmann R, Zabel BU, Olsen BR. Muta‐ tions involving the transcription factor CBFA1 cause cleidocranial dysplasia. Cell

[41] Zhou G, Chen Y, Zhou L, Thirunavukkarasu K, Hecht J, Chitayat D, Gelb BD, Pirinen S, Berry SA, Greenberg CR, Karsenty G, Lee B. CBFA1 mutation analysis and func‐ tional correlation with phenotypic variability in cleidocranial dysplasia, Hum Mol

[42] Quack I, Vonderstrass B, Stock M, Aylsworth AS, Becker A, Brueton L, Lee PJ, Ma‐ jewski F, Mulliken JB, Suri M, Zenker M, Mundlos S, Otto F. Mutation analysis of core biding factor A1 in patients with cleidocranial dysplasia. Am J hum Genet 1999;

[43] Yoshida T, Kanegane H, Osato M, Yanagida M, Miyawaki T, Ito Y, Shigesada K. Functional analysis of RUNX2 mutations in Japanese patients with cleidocranial dys‐ plasia demonstrates novel genotype-phenotype correlations. Am J Hum Gen‐

[44] Baumert U, Golan I, Redlich M, Aknin JJ, Muessig D. Cleidocranial dysplasia: molec‐ ular genetic analysis and phenotypic-based description of a Middle European patient

[45] Yoshida T, Kanegane H, Osato M, Yanagida M, Miyawaki T, Ito Y, Shigesada K. Functional analysis of RUNX2 mutations in cleidocranial dysplasia: novel insights in‐

[46] [46] Aberg T, Cavender A, Gaikwad JS, Bronckers AL, Wang X, Waltimo-Sirén J, The‐ sleff I, D'Souza RN. Phenotypic changes in dentition of Runx2 homozygote-null mu‐

[47] Wang XP, Aberg T, James MJ, Levanon D, Groner Y, Thesleff I. Runx2 (Cbfa1) inhib‐ its Shh signaling in the lower but not upper molars of mouse embryos and prevents

the budding of putative successional teeth. J Dent Res2005;84(2):138-143.

to genotype-phenotype correlations. Blood Cells Mol Dis 2003;30(2):184-193.

gencephalometric study. J Craniofac Genet DeveBiol 1995;15(1): 35-43.

Oral Pathol 1981;52(1), 2-11.

740 Gene Therapy - Tools and Potential Applications

delphia:WB Saunders Co;1983.

16(3): 307-310.

1997; 89(5): 773-779.

65(5): 1268-1278.

et2002;71(4):724-738.

group. Am J Med Genet A2005;139A(2):78-85.

tant mice. J HistochemCytochem2004;52(1):131-139.

Genet 1999; 8(12): 2311-2316.


regulates supernumerary tooth formation during embryogenesis and throughout adulthood. Development2009;136(11):1939-1949.

[73] OD. Klein, G. Minowada, R. Peterkova, A. Kangas, BD. Yu, H. Lesot , M. Peterka, J. Jernvall, GR. Martin, Sprouty genes control diastema tooth development via bidirev‐ tional antagonism of epithelial-messenchymal FGF signaling,Dev.Cell 2006;11(2):

Feasibility of Gene Therapy for Tooth Regeneration by Stimulation of a Third Dentition

http://dx.doi.org/10.5772/52529

743

[74] Ohazama A, Haycraft CJ, Seppala M, Blackburn J, Ghafoor S, Cobourne M, Martinelli DC, Fan CM, Peterkova R, Lesot H, Yoder BK, Sharpe PT. Primary cilia regulate Shh activity in the control of molar tooth number. Development2009;136(6):897-903. [75] Zhang Q, Murcia NS, Chittenden LR, Richards WG, Michaud EJ, Woychik RP, Yoder BK. Loss of the Tg737 protein results in skeletal patterning defects.DevDyn2003 ;

[76] Kaufman MH, Chang HH, Shaw JP. Craniofacial abnormalities in homozygous Small

[77] Murashima-Suginami A, Takahashi K, Sakata T, Tsukamoto H, Sugai M, Yanagita M, Shimizu A, Sakurai T, Slavkin HC, Bessho K. Enhanced BMP signaling results in su‐ pernumerary tooth formation in USAG-1 deficient mouse. BiochemBiophys Res

[78] Kratochwil K, Dull M, Farinas I, Galceran J, Grosschedl R. Lef1 expression is activat‐ ed by BMP-4 and regulates inductive tissue interactions in tooth and hair develop‐

[79] Zhou P, Byrne C, Jacobs J, Fuchs E. Lymphoid enhancer factor 1 directs hair follicle

[80] Nakamura T, de Vega S, Fukumoto S, Jimenez L, Unda F, Yamada Y. Transcription factor epiprofin is essential for tooth morphogenesis by regulating epithelial cell fate

[81] Zhang Z, Lan Y, Chai Y, Jiang R. Antagonistic actions of Msx1 and Osr2 pattern

[82] Huang B, Takahashi K, Sakata-Goto T, Kiso H, Togo Y, Saito K, Tsukamoto H, Sugai M, Akira A, Shimizu A, Bessho K. Phenotypes of CEBPB Deficiency: Supernumerary

[83] Fang J, Zhu YY, Smiley E, Bonadio J, Rouleau JP, Goldstein SA, McCauley LK, Da‐ vidson BL, Roessler BJ. Stimulation of new bone formation by direct transfer of os‐

[84] Jin Q, Anusaksathien O, Webb SA, Printz MA, Giannobile WV. Engineering of toothsupporting structures by delivery of PDGF gene therapy vectors. MolTher2004 ;9(4):

[85] Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, Funke R, Gage D, Harris K, Heaford A, et al. Initial se‐

quencing and analysis of the human genome. Nature2001;409(6822):860-921.

mammalian teeth into a single row. Science2009;323(5918):1232-1234.

teogenic plasmid genes. ProcNatlAcadSci USA 1996;93(12):5753-5758.

Teeth and Elongated Coronoid Process. Oral Dis 2012; in press,

patterning and epithelial cell fate. Genes Dev1995;9(6):700-713.

and tooth number. J Biolchem2008;283(8):4825-4833.

eye (Sey/Sey) embryos and newborn mice. JAnat1995;186 ( Pt 3):607-617.

181-190.

227(1):78-90.

519-526.

Commun2008;369(4):1012-1016.

ment. Genes Dev1996;10(11):1382-1394.


[73] OD. Klein, G. Minowada, R. Peterkova, A. Kangas, BD. Yu, H. Lesot , M. Peterka, J. Jernvall, GR. Martin, Sprouty genes control diastema tooth development via bidirev‐ tional antagonism of epithelial-messenchymal FGF signaling,Dev.Cell 2006;11(2): 181-190.

regulates supernumerary tooth formation during embryogenesis and throughout

[61] Liu F, Dangaria S, Andl T, Zhang Y, Wright AC, Damek-Poprawa M, Piccolo S, Nagy A, Taketo MM, Diekwisch TG, Akintoye SO, Millar SE beta-Catenin initiates tooth

[62] Huysseune A, Thesleff I. Continuous tooth replacement: the possible involvement of

[63] Peterková R, Peterka M, Viriot L, Lesot H. Development of the vestigial tooth pri‐ mordia as part of mouse odontogenesis. Connect Tissue Res2002;43(2-3):120-128.

[64] Peterková R, Lesot H, Viriot L, Peterka M. The supernumerary cheek tooth in tabby/EDA mice-a reminiscence of the premolar in mouse ancestors. Arch Oral Biol

[65] Peterkova R, Churava S, Lesot H, Rothova M, Prochazka J, Peterka M, Klein OD. Re‐ vitalization of a diastemal tooth primordium in Spry2 null mice results from in‐ creased proliferation and decreased apoptosis. J ExpZool B MolDevEvol2009;312B(4):

[66] Prochazka J, Pantalacci S, Churava S, Rothova M, Lambert A, Lesot H, Klein O, Pe‐ terka M, Laudet V, Peterkova R. Patterning by heritage in mouse molar row develop‐

[67] Viriot L, Peterková R, Peterka M, Lesot H. Evolutionary implications of the occur‐ rence of two vestigial tooth germs during early odontogenesis in the mouse lower

[68] Witter K, Lesot H, Peterka M, Vonesch JL, Mísek I, Peterková R Origin and develop‐ mental fate of vestigial tooth primordia in the upper diastema of the field vole (Mi‐

[69] Keranen SV, Kettunen P, Aberg T,Thesleff T,Jernvall T. Gene expression patterns as‐ sociated with suppression of odontogenesis in mouse and vole diastema region, Dev.

[70] Mustonen T, Pispa J, Mikkola ML, Pummila M, Kangas AT, Pakkasjärvi L, Jaatinen R, Thesleff I. Stimulation of ectodermal organ development by Ectodysplasin-A1.

[71] Pispa J, Mustonen T, Mikkola ML, Kangas AT, Koppinen P, Lukinmaa PL, Jernvall J, Thesleff I. Tooth patterning and enamel formation can be manipulated by misexpres‐

[72] Tucker AS, Headon DJ, Courtney JM, Overbeek P, Sharpe PT. The activation level of the TNF family receptor, Edar, determines cusp number and tooth number during

neogenesis in adult rodent incisors. J Dent Res2010;89(9):909-914.

adulthood. Development2009;136(11):1939-1949.

epithelial stem cells. Bioessays2004;26(6):665-671.

ment. ProcNatlAcadSci U S A2010;107(35):15497-15502

crotusagrestis, Rodentia). Arch Oral Biol2005;50(4):401-409.

sion of TNF receptor Edar. DevDyn2004;231(2):432-440.

tooth development. DevBiol2004;268(1):185-194.

jaw. Connect Tissue Res 2002;43(2-3):129-133.

Genes Evol 1999;209(8): 495-506.

DevBiol2003;259(1):123-136.

2005;50(2):219-225.

742 Gene Therapy - Tools and Potential Applications

292-308.


[86] Takahashi, K., Nuckolls, G.H., Tanaka, O., Semba, I., Takahashi, I., Dashner,R., Shum, L. Slavkin, H.C. Adenovirus mediated ectopic expression of Msx2 in evennumbered rhombomeres cause apoptotic elimination of cranial neural crest cells en

[87] Takahashi K, Nuckolls GH, Takahashi I, Nonaka K, Nagata M, Ikura T, Slavkin HC and Shum L. Msx2 is a repressor of chondrogenic differentiation in migratory crainal

[88] Aihara H, Miyazaki J. Gene transfer into muscle by electroporation in vivo. Nat Bio‐

[89] Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. Osteoinduction by microbubbleenhanced transcutaneous sonoporation of human bone morphogenetic protein-2. J

[90] Osawa K, Okubo Y, Nakao K, Koyama N, Bessho K. Feasibility of BMP-2 gene thera‐ py using an ultra-fine needle. In: You Y. (ed.) Targets in Gene Therapy. Rijeka:In‐

ovo. Development 1998;125(9), 1627-1635.

technol 1998;16(9):867-870.

744 Gene Therapy - Tools and Potential Applications

Gene Med2009;11(7):633-641.

Tech; 2011.p159-166

neural crest cells. DevDyn 2001, 222(2), 252-262.

### *Edited by Francisco Martin Molina*

The Gene Therapy field is living exciting times after more than 20 years of poor results. Scientist and clinicians working in the gene therapy field have encountered many problems in the past that are now starting to be solved. The development of safer and more efficient gene transfer vectors and the advances on the cell therapy field have open new opportunities to tackle different diseases. The aim of this book is to bring together information about the different gene therapy tools, the clinical successes of gene therapy and the future applications.

Photo by Natali\_Mis / iStock

Gene Therapy - Tools and Potential Applications

Gene Therapy

Tools and Potential Applications

*Edited by Francisco Martin Molina*