**3.2 Liposomes and nanotechnology**

Liposomes are lipid particles that resemble the cell membrane. Liposome-based gene delivery was first reported by Felgner in 1987, and is still one of the major techniques for non viral gene delivery into cells (Niidome & Huang, 2002). Lipoplexes are formed by the interaction of anionic nucleic acids binding to the surface of cationic lipids, forming multilamellar lipid–nucleic acid complexes where the negatively charged nucleic acid remains trapped inside the lipid bilayer (Dass, 2004). Since its discovery, different lipid formulations have been tested and modified. For example, stealth liposomes sterically stabilized with methoxypoly(ethylene glycol)distearoylphosphatidylethanolamine conjugates (PEG-DSPE) have long circulation half-lives following intravenous injection (Moreira et al., 2001). In addition, linear polycations such as linear, branched and dendritic vectors based on poly(ethylenimine) (PEI), poly(L-lysine) (PLL) and a range of

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 155

et al (2008) for Mucopolysaccharidosis type I, although they used a plasmid bearing a fusion gene consisting of Transferrin (Tf) and α-L-iduronidase. The fusion product consisted of an enzymatically active protein that was transported into the CNS by TfR-mediated endocytosis. Short-term treatment resulted in a decrease in GAGs in the cerebellum of

**Stable Expression**

**Liposome Normal GM1**

**1 2 30 Time (days)**

Fig. 3. Schematic view of a Trojan horse liposome. A stealth PEGylated liposome is

complexed with monoclonal antibodies (MAb) that undergo receptor-mediated transcytosis

values of affected fibroblasts: 68 nmoles/h/mg prot. (Balestrin et al., 2008).

Fig. 2. Stable expression of β-Galactosidase in fibroblasts from G1 Gangliosidosis patients after *in vitro* liposome-based gene transfer. Mean values of liposome-treated cells: 300 nmoles/h/mg prot.; mean values of normal fibroblasts: 1,300 nmoles/h/mg prot.; mean

Mucopolysaccharidosis type I mice.

**Enzyme activity**

across the blood-brain barrier.

**(nmoles/h/mg prot.)**

poly(ethylene glycol) (PEG) were also developed (Hunter, 2006). Although linear PEI shows greater *in vivo* efficiency because of a dynamic structure change of the complex under high salt concentrations as found in blood (Niidome & Huang, 2002), it also possesses greater toxicity (Morille et al., 2008).

Cationic nanoemulsions have been more recently considered as potential systems for nucleic acid delivery. The interest in these systems is justified by the fact that they are biocompatible and able to form complexes with DNA protecting it from enzymatic degradation (Nam et al., 2009). Other practical advantages include ease of production and the potential for repeated administration (Al-Dorsari and Gao, 2009). We have investigated the influence of phospholipids on the properties of cationic nanoemulsions/pDNA complexes. Complexes containing the phospholipids DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DSPE (1,2- distearoyl-sn-glycero-3- phosphoethanolamine) were less toxic in comparison with the formulations obtained with lecithin, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In addition, higher amounts of reporter DNA were detected for the formulation obtained with the DSPC phospholipid (Fraga et al., in press).

These cationic macromolecules can readily condense DNA or RNA into stable nanostructures for use in gene delivery (Hunter, 2006) but also for other nanotechnology-based approaches useful for the treatment of LSDs (Muro, 2010). Nanomaterials, as a result of their small size and their large surface area offer great promise for neuro-therapeutics (Ragnail et al., 2011) and thus may be a valid option for a large number of LSDs that affect the CNS.

Liposome-mediated gene transfer for LSD has been performed *in vitro* using patient's fibroblasts as target cells. Estruch et al. (2001) delivered therapeutic genes by integrinmediated uptake into fibroblasts from patients with Fucosidosis and Fabry disease. The vectors consisted of a complex of lipofectin and a peptide containing an integrin-targeting domain and a poly-lysine domain to which plasmid DNA was bound. Transfected cells produced the corresponding enzyme at levels which were 10-40% of the total activity in cultures of normal fibroblasts. Although 95-98% of this activity was secreted, it did not appear to affect the viability of the cells. Our group used Lipofectamine to transduce fibroblasts from GM1 Gangliosidosis patients with the beta-galactosidase gene. Treated cells showed 33 to 100- fold increases in enzyme activity compared to untreated fibroblasts. However, after seven days enzyme activity was back to uncorrected values (Balestrin et al., 2008). When Geneticin was added to the medium (figure 2), stable expression at therapeutic levels was observed (mean 300 nmoles/h/mg prot) for 30 days, although at values lower than the normal range (mean 1,300 nmoles/h/mg prot).

*In vivo*, PEG-coated liposomes have been modified with monoclonal antibodies in order to reach the CNS. A liposome is coated with peptidomimetic monoclonal antibodies that undergo receptor-mediated transcytosis across the blood-brain barrier on the endogenous peptide receptor transporters (Pardridge, 2007). These Trojan horses (figure 3) may use the insulin or transferrin receptor, and since the MAb binding site is different from the binding site of the endogenous ligand, there is no interference of endogenous ligand transport (Skarlatos et al, 1995).

This approach has been used to deliver a non-viral plasmid DNA to the brain across the blood-brain-barrier after intravenous administration of liposomes coated with monoclonal antibody to the mouse transferrin receptor in a mouse model of Mucopolysaccharidosis type VII (Zhang et al, 2008). The enzyme activity was increased greater than ten-fold in brain, liver, spleen, lung, and kidney, but not in heart. A similar strategy has been used by Osborn

poly(ethylene glycol) (PEG) were also developed (Hunter, 2006). Although linear PEI shows greater *in vivo* efficiency because of a dynamic structure change of the complex under high salt concentrations as found in blood (Niidome & Huang, 2002), it also possesses greater

Cationic nanoemulsions have been more recently considered as potential systems for nucleic acid delivery. The interest in these systems is justified by the fact that they are biocompatible and able to form complexes with DNA protecting it from enzymatic degradation (Nam et al., 2009). Other practical advantages include ease of production and the potential for repeated administration (Al-Dorsari and Gao, 2009). We have investigated the influence of phospholipids on the properties of cationic nanoemulsions/pDNA complexes. Complexes containing the phospholipids DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) and DSPE (1,2- distearoyl-sn-glycero-3- phosphoethanolamine) were less toxic in comparison with the formulations obtained with lecithin, DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) and DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine). In addition, higher amounts of reporter DNA were detected for the formulation obtained with the DSPC phospholipid

These cationic macromolecules can readily condense DNA or RNA into stable nanostructures for use in gene delivery (Hunter, 2006) but also for other nanotechnology-based approaches useful for the treatment of LSDs (Muro, 2010). Nanomaterials, as a result of their small size and their large surface area offer great promise for neuro-therapeutics (Ragnail et al., 2011) and

Liposome-mediated gene transfer for LSD has been performed *in vitro* using patient's fibroblasts as target cells. Estruch et al. (2001) delivered therapeutic genes by integrinmediated uptake into fibroblasts from patients with Fucosidosis and Fabry disease. The vectors consisted of a complex of lipofectin and a peptide containing an integrin-targeting domain and a poly-lysine domain to which plasmid DNA was bound. Transfected cells produced the corresponding enzyme at levels which were 10-40% of the total activity in cultures of normal fibroblasts. Although 95-98% of this activity was secreted, it did not appear to affect the viability of the cells. Our group used Lipofectamine to transduce fibroblasts from GM1 Gangliosidosis patients with the beta-galactosidase gene. Treated cells showed 33 to 100- fold increases in enzyme activity compared to untreated fibroblasts. However, after seven days enzyme activity was back to uncorrected values (Balestrin et al., 2008). When Geneticin was added to the medium (figure 2), stable expression at therapeutic levels was observed (mean 300 nmoles/h/mg prot) for 30 days, although at values lower

*In vivo*, PEG-coated liposomes have been modified with monoclonal antibodies in order to reach the CNS. A liposome is coated with peptidomimetic monoclonal antibodies that undergo receptor-mediated transcytosis across the blood-brain barrier on the endogenous peptide receptor transporters (Pardridge, 2007). These Trojan horses (figure 3) may use the insulin or transferrin receptor, and since the MAb binding site is different from the binding site of the endogenous ligand, there is no interference of endogenous ligand transport

This approach has been used to deliver a non-viral plasmid DNA to the brain across the blood-brain-barrier after intravenous administration of liposomes coated with monoclonal antibody to the mouse transferrin receptor in a mouse model of Mucopolysaccharidosis type VII (Zhang et al, 2008). The enzyme activity was increased greater than ten-fold in brain, liver, spleen, lung, and kidney, but not in heart. A similar strategy has been used by Osborn

thus may be a valid option for a large number of LSDs that affect the CNS.

than the normal range (mean 1,300 nmoles/h/mg prot).

toxicity (Morille et al., 2008).

(Fraga et al., in press).

(Skarlatos et al, 1995).

et al (2008) for Mucopolysaccharidosis type I, although they used a plasmid bearing a fusion gene consisting of Transferrin (Tf) and α-L-iduronidase. The fusion product consisted of an enzymatically active protein that was transported into the CNS by TfR-mediated endocytosis. Short-term treatment resulted in a decrease in GAGs in the cerebellum of Mucopolysaccharidosis type I mice.

Fig. 2. Stable expression of β-Galactosidase in fibroblasts from G1 Gangliosidosis patients after *in vitro* liposome-based gene transfer. Mean values of liposome-treated cells: 300 nmoles/h/mg prot.; mean values of normal fibroblasts: 1,300 nmoles/h/mg prot.; mean values of affected fibroblasts: 68 nmoles/h/mg prot. (Balestrin et al., 2008).

Fig. 3. Schematic view of a Trojan horse liposome. A stealth PEGylated liposome is complexed with monoclonal antibodies (MAb) that undergo receptor-mediated transcytosis across the blood-brain barrier.

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 157

implanted in the peritoneum of the MPS II mouse model. An increase in IDS activity in plasma was observed, along with a reduction on urinary GAG between the fourth and the sixth week of treatment. After 8 weeks, a reduction of 30% in the amount of GAG

accumulated in the liver and 38% in the kidney were shown (Friso et al 2005).

Fig. 4. Traffic of lysosomal enzymes throughout the encapsulated cells. The nascent lysosomal enzymes are glycosylated in the endoplasmic reticulum (ER) of the genetically modified cells. (A) The enzymes are phosphorylated at the residue of mannose-6 in the Golgi apparatus. (B) Most enzymes are transported to mature lysosomes. (C) Some, however, are secreted to the extracellular environment and (D) to outside of the microcapsules. (E) Phosphorylated enzymes bind to mannose-6-phosphate receptors (M6PR) of the enzyme-deficient cells (F) where they are endocyted and subsequently

To evaluate the usefulness of this technique to treat MPS VII, Ross et al (2000a) injected APA encapsulated non-autologous cells overexpressing Gusb in the peritoneum of MPS VII mice. The results showed the presence of Gusb in the plasma 24 hours after implantation, reaching 66% of physiological levels by 2 weeks post implantation. Activity of Gusb was also detected in liver and spleen for the duration of the 8-week experiment. Accumulation of GAG was significantly reduced in liver and spleen sections and urinary GAG content reached normal levels. In another study, enzyme released by APA encapsulated 2A50 fibroblasts implanted directly into the lateral ventricles of the brain of MPS VII mice was delivered throughout most of the CNS, reversing the histological pathology (Ross et al., 2000b). *In vitro* studies were performed by Nakama et al (2006) who encapsulated immortalized recombinant human amniotic epithelial cells with MPS VII human and mouse fibroblasts and high GUSB

targeted to the lysosomes (Matte et al., 2011).

#### **3.3 Cell microencapsulation**

Cell microencapsulation is an approach in which cells are trapped in a semipermeable membrane, allowing the exchange of metabolites and nutrients between them and the external environment. The membrane prevents the access of the immune system to the cells, without the need for continued immunosuppression of the host (Uludag et al., 2000). Furthermore, this technique allows the localized and controlled release, and long term duration of therapeutic products derived from the microencapsulated cells (Orive et al., 2002). Microencapsulation has become an important system for cellular preservation (Mayer et al., 2010) and a potential strategy for the controlled delivery of therapeutic products (Orive et al., 2003). Alginate has been the most important encapsulation polymer due to its abundance, easy gelling properties and biocompatibility. Agarose, chitosan, and hyaluronic acid are other polymers used for microencapsulation (Orive et al., 2003).

Cell microencapsulation presents the potential to deliver the therapeutic product of interest directly to the Central Nervous System (CNS). This has been achieved by different groups for brain tumors (Kuijlen et al 2007) and neurodegenerative diseases (Spuch et al 2010). A phase I clinical trial was conducted in Huntington's patients without signs of toxicity (Bloch et al 2004). This approach delivers the gene of interest in the spinal fluid, similar to the intrathecal enzyme replacement therapy. Thus, cell encapsulation can be suitable for the treatment of LSD, once the deficient enzyme could be released for long term directly in the CNS (Matte et al., 2011).

In order to obtain larger amounts of secreted enzyme, the encapsulated cells should be genetically modified to over-express the enzyme of interest. This enzyme would then be released to the extracapsular space (Bressel et al 2008) and uptaken by adjacent deficient cells (figure 4). This strategy has been used experimentally for different LSD, especially the Mucopolysaccharidosis (MPS).

Three *in vitro* studies were performed in LSDs other than the MPS, one in Fabry disease and the other two in Metachromatic Leukodystrophy (MLD). Naganawa et al (2002) co-cultured fibroblasts from patients with Fabry disease with microencapsulated recombinant Chinese Hamster Ovary cells (CHO) over-expressing alpha-galactosidase. The deficient cells were able to uptake the enzyme decreasing their levels of globotriaosylceramide storage. A similar approach was used by our group to test the ability of Baby Hamster Kidney (BHK) cells over-expressing ARSA to correct the deficiency of this enzyme in human skin fibroblasts from MLD patients. Fibroblasts co-cultured with the encapsulated cells for four weeks showed levels of enzyme activity higher than normal. Transmission electron microscopy showed evidence of normalization of the lysosomal ultra structure, suggesting that the secreted enzyme was able to degrade the substrate (Lagranha et al., 2008). Consiglio et al (2007) collected the conditioned media of C2C12 cells over-expressing ARSA encapsulated in polyether-sulfone polymer and used it to treat oligodendrocytes from MLD mice. The deficient cells internalized the enzyme and it was normally sorted to the lysosomal compartment, reaching 80% of physiological levels and restoring sulfatide metabolism.

Both *in vitro* and *in vivo* studies have been performed in the MPS types I, II and VII. For MPS II two studies were performed. The first was a proof of principle in which Hunter primary fibroblasts were co-cultured with alginate microcapsules containing C2C12 cell clones overexpressing IDS. After 5 days of co-culture this strategy was able to increase IDS activity inside the deficient fibroblasts to levels similar to normal (Tomanin et al 2002). The second study was a pre-clinical experiment in which APA (alginate-poli-L-lysine-alginate) microcapsules containing 1.5 x 106 allogeneic C2C12 myoblasts over-expressing IDS were

Cell microencapsulation is an approach in which cells are trapped in a semipermeable membrane, allowing the exchange of metabolites and nutrients between them and the external environment. The membrane prevents the access of the immune system to the cells, without the need for continued immunosuppression of the host (Uludag et al., 2000). Furthermore, this technique allows the localized and controlled release, and long term duration of therapeutic products derived from the microencapsulated cells (Orive et al., 2002). Microencapsulation has become an important system for cellular preservation (Mayer et al., 2010) and a potential strategy for the controlled delivery of therapeutic products (Orive et al., 2003). Alginate has been the most important encapsulation polymer due to its abundance, easy gelling properties and biocompatibility. Agarose, chitosan, and hyaluronic

Cell microencapsulation presents the potential to deliver the therapeutic product of interest directly to the Central Nervous System (CNS). This has been achieved by different groups for brain tumors (Kuijlen et al 2007) and neurodegenerative diseases (Spuch et al 2010). A phase I clinical trial was conducted in Huntington's patients without signs of toxicity (Bloch et al 2004). This approach delivers the gene of interest in the spinal fluid, similar to the intrathecal enzyme replacement therapy. Thus, cell encapsulation can be suitable for the treatment of LSD, once the deficient enzyme could be released for long term directly in the

In order to obtain larger amounts of secreted enzyme, the encapsulated cells should be genetically modified to over-express the enzyme of interest. This enzyme would then be released to the extracapsular space (Bressel et al 2008) and uptaken by adjacent deficient cells (figure 4). This strategy has been used experimentally for different LSD, especially the

Three *in vitro* studies were performed in LSDs other than the MPS, one in Fabry disease and the other two in Metachromatic Leukodystrophy (MLD). Naganawa et al (2002) co-cultured fibroblasts from patients with Fabry disease with microencapsulated recombinant Chinese Hamster Ovary cells (CHO) over-expressing alpha-galactosidase. The deficient cells were able to uptake the enzyme decreasing their levels of globotriaosylceramide storage. A similar approach was used by our group to test the ability of Baby Hamster Kidney (BHK) cells over-expressing ARSA to correct the deficiency of this enzyme in human skin fibroblasts from MLD patients. Fibroblasts co-cultured with the encapsulated cells for four weeks showed levels of enzyme activity higher than normal. Transmission electron microscopy showed evidence of normalization of the lysosomal ultra structure, suggesting that the secreted enzyme was able to degrade the substrate (Lagranha et al., 2008). Consiglio et al (2007) collected the conditioned media of C2C12 cells over-expressing ARSA encapsulated in polyether-sulfone polymer and used it to treat oligodendrocytes from MLD mice. The deficient cells internalized the enzyme and it was normally sorted to the lysosomal

compartment, reaching 80% of physiological levels and restoring sulfatide metabolism.

Both *in vitro* and *in vivo* studies have been performed in the MPS types I, II and VII. For MPS II two studies were performed. The first was a proof of principle in which Hunter primary fibroblasts were co-cultured with alginate microcapsules containing C2C12 cell clones overexpressing IDS. After 5 days of co-culture this strategy was able to increase IDS activity inside the deficient fibroblasts to levels similar to normal (Tomanin et al 2002). The second study was a pre-clinical experiment in which APA (alginate-poli-L-lysine-alginate) microcapsules containing 1.5 x 106 allogeneic C2C12 myoblasts over-expressing IDS were

acid are other polymers used for microencapsulation (Orive et al., 2003).

**3.3 Cell microencapsulation** 

CNS (Matte et al., 2011).

Mucopolysaccharidosis (MPS).

implanted in the peritoneum of the MPS II mouse model. An increase in IDS activity in plasma was observed, along with a reduction on urinary GAG between the fourth and the sixth week of treatment. After 8 weeks, a reduction of 30% in the amount of GAG accumulated in the liver and 38% in the kidney were shown (Friso et al 2005).

Fig. 4. Traffic of lysosomal enzymes throughout the encapsulated cells. The nascent lysosomal enzymes are glycosylated in the endoplasmic reticulum (ER) of the genetically modified cells. (A) The enzymes are phosphorylated at the residue of mannose-6 in the Golgi apparatus. (B) Most enzymes are transported to mature lysosomes. (C) Some, however, are secreted to the extracellular environment and (D) to outside of the microcapsules. (E) Phosphorylated enzymes bind to mannose-6-phosphate receptors (M6PR) of the enzyme-deficient cells (F) where they are endocyted and subsequently targeted to the lysosomes (Matte et al., 2011).

To evaluate the usefulness of this technique to treat MPS VII, Ross et al (2000a) injected APA encapsulated non-autologous cells overexpressing Gusb in the peritoneum of MPS VII mice. The results showed the presence of Gusb in the plasma 24 hours after implantation, reaching 66% of physiological levels by 2 weeks post implantation. Activity of Gusb was also detected in liver and spleen for the duration of the 8-week experiment. Accumulation of GAG was significantly reduced in liver and spleen sections and urinary GAG content reached normal levels. In another study, enzyme released by APA encapsulated 2A50 fibroblasts implanted directly into the lateral ventricles of the brain of MPS VII mice was delivered throughout most of the CNS, reversing the histological pathology (Ross et al., 2000b). *In vitro* studies were performed by Nakama et al (2006) who encapsulated immortalized recombinant human amniotic epithelial cells with MPS VII human and mouse fibroblasts and high GUSB

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 159

Fig. 6. Microcapsules used for the treatment of MPS I mice. Left upper panel:

attached to the intestine.

**3.4 Transposon-based systems** 

photomicrography of alginate beads containing recombinant BHK that overexpress IDUA (small round dots). Right upper panel: Macroscopic aspect of the microcapsules (black dots) in a 96-well plate. Capsules were ink stained to help visualization. Lower panel: Aspect of the capsules in the peritoneum ten days after implantation. Note that some capsules are

For human gene therapy we can enumerate some important aspects of transposon systems (special emphasis in this chapter will be given to the *sleeping beauty* transposon) that make them appealing as a vector: (1) the integrated gene has stable expression, providing longlasting expression of a therapeutic gene, which, as already mentioned, is essential for

activity was detected in the medium. Addition of mannose-6-phosphate led to decreased enzyme activity, suggesting that enzyme uptake was mediated by mannose-6-phosphate receptor.

Our group has shown that the correction of MPS I fibroblasts by recombinant encapsulated BHK cells is also mediated by mannose-6-phosphate receptor. The effect of the ratio fibroblasts:encapsulated cells was also analysed (figure 5). The amount of enzyme uptaken by the fibroblasts is essentially the same under the different ratios (5:1; 1:1; 1:5) although the enzyme activity in the medium increases, as more enzyme is released.

Fig. 5. IDUA enzyme activity in MPS I fibroblasts co-cultured with recombinant BHK cells overexpressing IDUA at different ratios.

Our results also showed an increase in IDUA activity in MPS I fibroblasts after 15, 30 and 45 days of co-culture with the capsules. Cytological analysis showed a marked reduction in GAG storage within MPS I cells (Baldo et al., 2011). Ongoing experiments are under way in the MPS I mouse model. The capsules were implanted in the peritoneum (figure 6) and animals were sacrificed at 4 months later. Histological analysis showed a reduction on GAG storage although plasma and tissue enzyme activity levels were not increased.

These results are quite different from those of Barsoum et al (2003) who implanted genetically modified Madin-Darby canine kidney cells (MDCK) over-expressing canine Idua in the brain parenchyma of one MPS I dog. Enzyme in plasma and cerebrospinal fluid was low but detectable for 21 days. Immunohistochemistry with anti-IDUA antibody showed the presence of the enzyme in various brain regions, however an extensive inflammatory reaction was noted, both at the sites of implantation and in the immediate vicinity. This may be the reason why histological correction of lysosomal inclusions has not been observed.

activity was detected in the medium. Addition of mannose-6-phosphate led to decreased enzyme activity, suggesting that enzyme uptake was mediated by mannose-6-phosphate

Our group has shown that the correction of MPS I fibroblasts by recombinant encapsulated BHK cells is also mediated by mannose-6-phosphate receptor. The effect of the ratio fibroblasts:encapsulated cells was also analysed (figure 5). The amount of enzyme uptaken by the fibroblasts is essentially the same under the different ratios (5:1; 1:1; 1:5) although the

Activity inside fibroblasts

Activity in the extracellular media

Fig. 5. IDUA enzyme activity in MPS I fibroblasts co-cultured with recombinant BHK cells

Ratio MPS I fibroblasts/ Encapsulated cells Ratio 5 to 1 ratio 1 to 1 Ratio 1 to 5

Our results also showed an increase in IDUA activity in MPS I fibroblasts after 15, 30 and 45 days of co-culture with the capsules. Cytological analysis showed a marked reduction in GAG storage within MPS I cells (Baldo et al., 2011). Ongoing experiments are under way in the MPS I mouse model. The capsules were implanted in the peritoneum (figure 6) and animals were sacrificed at 4 months later. Histological analysis showed a reduction on GAG

These results are quite different from those of Barsoum et al (2003) who implanted genetically modified Madin-Darby canine kidney cells (MDCK) over-expressing canine Idua in the brain parenchyma of one MPS I dog. Enzyme in plasma and cerebrospinal fluid was low but detectable for 21 days. Immunohistochemistry with anti-IDUA antibody showed the presence of the enzyme in various brain regions, however an extensive inflammatory reaction was noted, both at the sites of implantation and in the immediate vicinity. This may be the reason why histological correction of lysosomal inclusions has

storage although plasma and tissue enzyme activity levels were not increased.

overexpressing IDUA at different ratios.

not been observed.

enzyme activity in the medium increases, as more enzyme is released.

receptor.

IDUA activity (nmol/h/ mg protein)

0

30

60

90

120

Fig. 6. Microcapsules used for the treatment of MPS I mice. Left upper panel: photomicrography of alginate beads containing recombinant BHK that overexpress IDUA (small round dots). Right upper panel: Macroscopic aspect of the microcapsules (black dots) in a 96-well plate. Capsules were ink stained to help visualization. Lower panel: Aspect of the capsules in the peritoneum ten days after implantation. Note that some capsules are attached to the intestine.

### **3.4 Transposon-based systems**

For human gene therapy we can enumerate some important aspects of transposon systems (special emphasis in this chapter will be given to the *sleeping beauty* transposon) that make them appealing as a vector: (1) the integrated gene has stable expression, providing longlasting expression of a therapeutic gene, which, as already mentioned, is essential for

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 161

Fig. 7. SB transposon system. This simplified version of the SB transposon system shows the cut-and-paste system used by the SB transposase to insert the DNA into the host genome. The gene of interest is flanked by two inverted repeats regions (IRs, arrows) which will be recognized by the transposase (usually given in *trans* in a second plasmid) and allow the transgene to be inserted into the host genome. This way only the transgene will have prolonged expression, as transposase gene expression is transient. The SB transposon is a non-viral method of gene delivery that allows integration of the transgene in the host cell.

Sustained *in vivo* transgene expression from plasmids can be difficult to achieve due to gene silencing. The mechanism by which this process occurs was postulated to be due to the deposition of repressive heterochromatin on the noncoding bacterial backbone sequences required for plasmid bacterial preparation and propagation (Chen et al, 2008; Riu et al, 2005).

**3.5 Minicircle gene therapy** 

lysosomal storage diseases; (2) the transposase directs the integration of single copies of a DNA sequence into chromatin and (3) the system is binary (the transposon is not autonomous or able to transpose on its own) (Hacket et al, 2005).

The most studied transposon system, which has been used in pre-clinical studies for treatment of lysosomal disorders, is the *Sleeping beauty (SB)* system. Sleeping beauty transposon is a type of mobile element that belongs to the *Tc1/Mariner* class and that is able to transpose via movement of a DNA element in a simple cut-and-paste manner. For that, a precise piece of DNA is excised from one DNA molecule and moved to another site in the same or in a different DNA molecule (Plasterk, 1993). This reaction is catalyzed by the protein transposase, which can be supplied *in trans* by another plasmid for gene therapy purposes.

The SB transposon system consists of two components: (i) a transposon, made up of the gene of interest flanked by inverted repeats (IRs), and (ii) a source of transposase (figure 7). For Sleeping Beauty-mediated transposition, the transposase can recognize the ends of the IRs, excises the gene of interest from the delivered plasmid DNA, and then inserts it into another DNA site. Based on studies in about 2,000 integration events in either mouse or human genomes, transposons seem to integrate into random sites, including exons, introns and intergenic sequences (Carlson et al, 2003; Horie et al, 2003; Hacket et al, 2005). This is a potential problem, since it may lead to an event of insertional mutagenesis. A complete list of insertion positions of the SB tranposon can be found at the Mouse Transposon Insertion database at http://mouse.ccgb.umn.edu/transposon/ (Roberg-Perez et al, 2003).

The use of SB transposons as gene therapy approach in LSD is still recent. The first published work was conducted by Aronovich et al (2007) who studied the effects of intravenous hydrodynamic injection of the SB transposon into mice with Mucopolysaccharidosis types I or VII. Without immunomodulation, initial enzyme activities in plasma reached levels higher than 100-fold that of wild-type (WT). However, both GUSB (MPS VII) and IDUA (MPS I) levels fell to background within 4 weeks postinjection.

A second group of animals was performed with immunomodulation only in MPS I mice. Plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of the mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, which can be seen as a potential side-effect.

A posterior study by the same group (Aronovich et al, 2009) was performed in another MPS I strain, which is immunodeficient (NOD/SCID mice). Using the same approach from the previous experiment, they were able to show a persistent elevation (100-fold normal) in the plasma IDUA levels for 18 weeks. Also, IDUA activity was present in all organs analyzed, including the brain. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I mice, including bone abnormalities, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium. In 2008 the first human clinical trial using the SB transposon was approved in the USA for treatment of cancer (Williams, 2008), however although promising no clinical trials have been conducted in LSDs so far.

lysosomal storage diseases; (2) the transposase directs the integration of single copies of a DNA sequence into chromatin and (3) the system is binary (the transposon is not

The most studied transposon system, which has been used in pre-clinical studies for treatment of lysosomal disorders, is the *Sleeping beauty (SB)* system. Sleeping beauty transposon is a type of mobile element that belongs to the *Tc1/Mariner* class and that is able to transpose via movement of a DNA element in a simple cut-and-paste manner. For that, a precise piece of DNA is excised from one DNA molecule and moved to another site in the same or in a different DNA molecule (Plasterk, 1993). This reaction is catalyzed by the protein transposase, which can be supplied *in trans* by another plasmid for gene therapy

The SB transposon system consists of two components: (i) a transposon, made up of the gene of interest flanked by inverted repeats (IRs), and (ii) a source of transposase (figure 7). For Sleeping Beauty-mediated transposition, the transposase can recognize the ends of the IRs, excises the gene of interest from the delivered plasmid DNA, and then inserts it into another DNA site. Based on studies in about 2,000 integration events in either mouse or human genomes, transposons seem to integrate into random sites, including exons, introns and intergenic sequences (Carlson et al, 2003; Horie et al, 2003; Hacket et al, 2005). This is a potential problem, since it may lead to an event of insertional mutagenesis. A complete list of insertion positions of the SB tranposon can be found at the Mouse Transposon Insertion database at http://mouse.ccgb.umn.edu/transposon/ (Roberg-

The use of SB transposons as gene therapy approach in LSD is still recent. The first published work was conducted by Aronovich et al (2007) who studied the effects of intravenous hydrodynamic injection of the SB transposon into mice with Mucopolysaccharidosis types I or VII. Without immunomodulation, initial enzyme activities in plasma reached levels higher than 100-fold that of wild-type (WT). However, both GUSB (MPS VII) and IDUA (MPS I) levels fell to background within 4 weeks post-

A second group of animals was performed with immunomodulation only in MPS I mice. Plasma IDUA persisted for over 3 months at up to 100-fold WT activity in one-third of the mice, which was sufficient to reverse lysosomal pathology in the liver and, partially, in distant organs. Histological and immunohistochemical examination of liver sections in IDUA transposon-treated WT mice revealed inflammation 10 days post-injection consisting predominantly of mononuclear cells, which can be seen as a potential

A posterior study by the same group (Aronovich et al, 2009) was performed in another MPS I strain, which is immunodeficient (NOD/SCID mice). Using the same approach from the previous experiment, they were able to show a persistent elevation (100-fold normal) in the plasma IDUA levels for 18 weeks. Also, IDUA activity was present in all organs analyzed, including the brain. The SB transposon system proved efficacious in correcting several clinical manifestations of MPS I mice, including bone abnormalities, hepatomegaly, and accumulation of foamy macrophages in bone marrow and synovium. In 2008 the first human clinical trial using the SB transposon was approved in the USA for treatment of cancer (Williams, 2008), however although promising no clinical trials have been conducted

autonomous or able to transpose on its own) (Hacket et al, 2005).

purposes.

Perez et al, 2003).

injection.

side-effect.

in LSDs so far.

Fig. 7. SB transposon system. This simplified version of the SB transposon system shows the cut-and-paste system used by the SB transposase to insert the DNA into the host genome. The gene of interest is flanked by two inverted repeats regions (IRs, arrows) which will be recognized by the transposase (usually given in *trans* in a second plasmid) and allow the transgene to be inserted into the host genome. This way only the transgene will have prolonged expression, as transposase gene expression is transient. The SB transposon is a non-viral method of gene delivery that allows integration of the transgene in the host cell.

#### **3.5 Minicircle gene therapy**

Sustained *in vivo* transgene expression from plasmids can be difficult to achieve due to gene silencing. The mechanism by which this process occurs was postulated to be due to the deposition of repressive heterochromatin on the noncoding bacterial backbone sequences required for plasmid bacterial preparation and propagation (Chen et al, 2008; Riu et al, 2005).

Non Viral Gene Transfer Approaches for Lysosomal Storage Disorders 163

The limitations of non-viral gene transfer, i.e., transient expression of the transgene and low transfection efficiency, are being slowly overcome in the last decade using improved vector design and techniques, such as nanotechnology, transposons, and minicircle approaches (to name a few) as demonstrated throughout this chapter. Novel mechanisms to help the DNA to escape endosomal degradation and pass through the nuclear envelope are also under development but were not in the scope of this chapter. Nevertheless, these improvements help non-viral gene therapy to move towards clinical trials in LSDs, which are expected to happen in the years to come. Non-viral vectors are safer than viral particles, which make them an appealing alternative for treatment of lysosomal storage disorders and even other monogenic diseases. Yet, there is a long way to clinical application but the road is paved and

The authors are thankful to Conselho Nacional de Desenvolvimento Cientifico- CNPq and

We also would like to thank our colleagues Fabiana Quoos Mayer, Carlos Oscar Kieling, Valeska Lizzi Lagranha and Raquel Balestrin for contributing with data to this paper.

Al-Dosari, M.S., Gao X. (2009). Nonviral gene delivery: principle, limitations, and recent

Anson, D.S., Bielicki, J., Hopwood, J.J. (1992). Correction of mucopolysaccharidosis type I

Aronovich, E.L., Bell, J.B., Belur, L.R., Gunther, R., Koniar, B., Erickson, D.C., Schachern, P.A.,

Aronovich EL, Bell JB, Khan SA, Belur LR, Gunther R, Koniar B, Schachern PA, Parker JB,

Baiotto, C., Sperb, F., Matte ,U., Silva, C.D., Sano, R., Coelho, J.C., Giugliani, R. (2011) Population analysis of the GLB1 gene in south Brazil. *Gen Mol Biol*, Vol 34, No 1, pp 45-48. Baldo, G., Quoos Mayer, F., Burin, M., Carrillo-Farga, J., Matte, U., Giugliani, R. (2011)

Balestrin, R.C., Baldo, G., Vieira, M.B., Sano, R., Coelho, J.C., Giugliani, R., Matte, U. (2008)

Barsoum, S.C., Milgram, W., Mackay, W., Coblentz, C., Delaney, K.H., Kwiecien, J.M., Kruth,

the use of microencapsulation. *J Lab Clin Med,* Vol 142, No 6, pp 399-413.

fibroblasts by retroviral-mediated transfer of the human alpha-L-iduronidase gene.

Matise, I., McIvor, R.S., Whitley, C.B., Hackett, P.B. (2007) Prolonged expression of a lysosomal enzyme in mouse liver after Sleeping Beauty transposon-mediated gene delivery: implications for non-viral gene therapy of mucopolysaccharidoses. *J Gene* 

Carlson CS, Whitley CB, McIvor RS, Gupta P, Hackett PB. (2009) Systemic correction of storage disease in MPS I NOD/SCID mice using the sleeping beauty transposon

Recombinant Encapsulated Cells Overexpressing Alpha-L-Iduronidase Correct Enzyme Deficiency in Human Mucopolysaccharidosis Type I Cells. *Cells Tissues* 

Transient high-level expression of beta-galactosidase after transfection of fibroblasts from GM1 gangliosidosis patients with plasmid DNA. *Braz J Med Biol Res,* Vol 41, No

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the scientific community advances steadily.

**5. Acknowledgements** 

**6. References** 

Based on those findings, a new technology has emerged, known as the minicircle (MC) gene therapy. This system uses a phiC31 integrase recombination event to remove the bacterial backbone elements of the plasmid resulting in a DNA circle (the MC), encoding the mammalian expression cassette of choice and a small attR footprint (Chen et al, 2003). This has proven to be resistant to gene silencing *in vivo*, is maintained as an extrachromosomal episome, and therefore represents an interesting platform for gene replacement strategies for lysosomal storage disorders (figure 8).

This technology was recently used for treatment of MPS I mice in a proof-of-concept study (Osborn et al, 2011). In this study, the researchers performed a hydrodynamic injection of a minicircle plasmid containing the IDUA gene combined with immunomodulation, achieving stable expression of the transgene, increased IDUA tissue levels and reduction in GAG storage. As a recent technology, this is the only study performed on LSDs so far, but results are encouraging and should be tested on other diseases soon.

Fig. 8. Production of a minicircle plasmid. This simplified version of the process shows the parental plasmid containing both the gene of interest and the bacterial components, including origin of replication (ORI), genes that confer resistance to antibiotic (Ab resist) and sites that allow attachment of the integrase (att B and P). After activation of the integrase, a cis-recombination event occurs, separating the gene of interest and its regulatory elements from the bacterial backbone, which then is degraded.
