**Electrically Mediated Gene Delivery : Basic and Translational Concepts**

J. Teissié

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54780

## **1. Introduction**

Naked plasmid (pDNA) electrotransfer is an approach for gene transfer that is very efficient for in vitro studies since its introduction in 1982. It was extended for a use in vivo in a per‐ spective for clinical gene therapy.

This chapter is a review on the state of the art. The molecular processes bringing the pDNA transfer and expression will be described. A critical view of the barriers preventing an effi‐ cient level of expression is given. This gives the tools to design the relevant protocols for the use on animals and the potential clinical trials.

Delivery of naked plasmids (pDNA) in tissue to obtain gene expression was facing the limit of a poor level of expression [1]. But the clinical advantage was that it was a safe approach for the patient. Improvement of the delivery and the resulting expression was known to be obtained at the cellular level by applying electric field pulses to the cellpDNA mixture [2]. It was shown that this boost in expression could be obtained on tis‐ sues [3]. During the last 15 years, many developments in this approach have been performed and Electrotransfection is now considered as a perspective for gene therapies [4-9]. A Phase I clinical trial using gene therapy by electrically mediated delivery has been performed (www.clinicaltrials.gov identifier NCT00323206)[10]. More recent data showed that the delivery and expression can be obtained on very sensitive organs [11-13]. Electropulsation mediated gene delivery appears now to be one of the effective contributors for the success of gene therapy

© 2013 Teissié; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

### **2. Basic processes**

#### **2.1. Electropermeabilization**

Biological membrane cohesion is known for almost 40 years to be affected by external electric field pulses [14]. A transient and reversible membrane permeabilization (Electropermeabili‐ zation) results from a controlled application of electric pulses to cells [15]. This can be induced not only in vitro but on living tissues as well. The key feature is that the structural transition is obtained only when the external field is larger than a critical value. A physical targeting of the effect is therefore present in tissues. This improves the safety of the approach as only a limited volume of the tissue is affected. This metastable membrane structural organization remains poorly understood. New properties are brought to the cell plasma membrane that, besides being permeabilized, becomes fusogenic and allows exogenous proteins to be inserted in it. Electropermeabilization is used to introduce a large variety of molecules into many different cells *in vitro* [16, 17]. The molecular transports, that result, are either due to an electrophoretic drift or/and to a concentration driven diffusion [18, 19]. The practical use for drug delivery remains rather empirical but results from more than 20 years of trials. Clinical applications of the method are now routinely used in many oncology centers (more than 100 in Europe only) as results from the EU funded Cliniporator and ESOPE programs. A local antitumoral drug delivery to patients (electrochemotherapy) results from the direct application of electric pulses to the patient [20-27]. In Europe, the treatment has been approved and patients can be reimbursed,

unclear [15]. The other main limit in gene electrotransfer is that the transport is not only across

Electrically Mediated Gene Delivery: Basic and Translational Concepts

http://dx.doi.org/10.5772/54780

35

The present review focuses on the processes supporting gene electrotransfer in vitro and their implications for the clinical applications. The events occurring before, during, after pulse application leading to gene electrotransfer will be described. Theoretical considerations about membrane structures involved in the plasmid uptake will be described in a (very) critical

Although the first pioneering report on gene electrotransfer in cells was published 30 years ago by E. Neumann, the molecular basis behind the process of gene electrotransfer

Most theories are based on the DNA sliding model [37]. Several steps are predicted: entry, electrophoretic translocation as long as the field was present, diffusion after the pulse delivery. An interesting simulation with predictive conclusions has just been published [38]. The DNA translocation was through a putative "electropore" assuming that the DNA was double stranded but linear (i.e open) meaning that the length was 2.4 μm. Indeed the sliding model needs the DNA to be linear to allow the "binding" of one end to the "electropore" entry. Experimental results just showed that the closed form was more effective for expression [39]. One speculative hypothesis in the simulation was that it always assumed that the binding step (entry) of the DNA to the "electropore" was present before the simulation started. De Gennes predicted that it was a very limiting step in the process (a crucial moment) when a chain end faces the electropore and enters it against the strong friction coefficient against the "pore" sides.

The major hypothesis in the sliding model is that "pores" must be present. Krassowska's model supports the simplest mechanism, in which plasmid enters the 5 nm thick membrane through stable electropores (up to 20 nm in diameter)[40, 41]. The electrically induced defects result from the field associated membrane potential changes. It predicts a post-pulse growth of "macropores" on the sub-second time scale fairly consistent with experimental evidence on pure lipid vesicles [42]. This model predicts "electropores" large enough to permit the plasmid uptake (under a linear form). These "electropores" are supposed to remain open for the entire duration of electrotransfer providing adequate time for the plasmid to enter the cell [43]. Indeed in Lin's simulation [38], they may remain present on a much longer duration than the

This model remains very attractive in spite of the existence of many experimental contradic‐ tions. Indeed, until now, no study made it possible to visualize these membrane pores. This

the plasma membrane but must target the nucleus volume.

manner as very few direct experimental data are available.

**3. Theories of DNA plasmid electroentry**

is still highly debated.

**3.1. The sliding model**

**3.2. The electropore**

A black box remains the "electropore".

pulse as a diffusion might follow a partial electrotransfer.

#### **2.2. Electrotransfection**

The most frequent application of electric field induced membrane permeabilization is the transfer and expression of gene into mammalian cells. Plasmid DNA (pDNA) can be transferred and expressed in mammalian cells when electropermeabilization is trig‐ gered [2]. This a complex process that involves not only the transport of pDNA into the cytoplasm, but also depends on subsequent cellular processes [27]. The transfer of naked DNA plasmid and the expression of the gene of interest are enhanced by elec‐ tropulsation into different tissues, including the skeletal muscle [28, 29], liver [3,30], skin [31, 32], lungs [33] and tumors [34, 35]. The transfection efficiency of this physical method *in vivo* must still be improved compared to the viral vectors. But it is ob‐ tained with naked pDNA avoiding the biological risks associated to the viral methods. Furthermore there is no theoretical restriction on the size of the pDNA to transfer. As a result, due to its easiness to perform, to the very fast expression after electric pulse delivery, reproducibility, limited costs (of the technology and logistics) and safety, gene electrotransfer is an attractive technology of gene therapy for clinical application. This is well illustrated by the increasing number of reviews covering the pre-clinical developments of the approach [4-9, 36].

As mentioned above, one of main limits of the widespread use of electropermeabilization is that very few is known on the biophysical mechanisms supporting the reorganization of the cell membrane (pore, electropore, defects?). The molecular target of the field effect remains unclear [15]. The other main limit in gene electrotransfer is that the transport is not only across the plasma membrane but must target the nucleus volume.

The present review focuses on the processes supporting gene electrotransfer in vitro and their implications for the clinical applications. The events occurring before, during, after pulse application leading to gene electrotransfer will be described. Theoretical considerations about membrane structures involved in the plasmid uptake will be described in a (very) critical manner as very few direct experimental data are available.

## **3. Theories of DNA plasmid electroentry**

Although the first pioneering report on gene electrotransfer in cells was published 30 years ago by E. Neumann, the molecular basis behind the process of gene electrotransfer is still highly debated.

#### **3.1. The sliding model**

**2. Basic processes**

34 Novel Gene Therapy Approaches

can be reimbursed,

**2.2. Electrotransfection**

developments of the approach [4-9, 36].

**2.1. Electropermeabilization**

Biological membrane cohesion is known for almost 40 years to be affected by external electric field pulses [14]. A transient and reversible membrane permeabilization (Electropermeabili‐ zation) results from a controlled application of electric pulses to cells [15]. This can be induced not only in vitro but on living tissues as well. The key feature is that the structural transition is obtained only when the external field is larger than a critical value. A physical targeting of the effect is therefore present in tissues. This improves the safety of the approach as only a limited volume of the tissue is affected. This metastable membrane structural organization remains poorly understood. New properties are brought to the cell plasma membrane that, besides being permeabilized, becomes fusogenic and allows exogenous proteins to be inserted in it. Electropermeabilization is used to introduce a large variety of molecules into many different cells *in vitro* [16, 17]. The molecular transports, that result, are either due to an electrophoretic drift or/and to a concentration driven diffusion [18, 19]. The practical use for drug delivery remains rather empirical but results from more than 20 years of trials. Clinical applications of the method are now routinely used in many oncology centers (more than 100 in Europe only) as results from the EU funded Cliniporator and ESOPE programs. A local antitumoral drug delivery to patients (electrochemotherapy) results from the direct application of electric pulses to the patient [20-27]. In Europe, the treatment has been approved and patients

The most frequent application of electric field induced membrane permeabilization is the transfer and expression of gene into mammalian cells. Plasmid DNA (pDNA) can be transferred and expressed in mammalian cells when electropermeabilization is trig‐ gered [2]. This a complex process that involves not only the transport of pDNA into the cytoplasm, but also depends on subsequent cellular processes [27]. The transfer of naked DNA plasmid and the expression of the gene of interest are enhanced by elec‐ tropulsation into different tissues, including the skeletal muscle [28, 29], liver [3,30], skin [31, 32], lungs [33] and tumors [34, 35]. The transfection efficiency of this physical method *in vivo* must still be improved compared to the viral vectors. But it is ob‐ tained with naked pDNA avoiding the biological risks associated to the viral methods. Furthermore there is no theoretical restriction on the size of the pDNA to transfer. As a result, due to its easiness to perform, to the very fast expression after electric pulse delivery, reproducibility, limited costs (of the technology and logistics) and safety, gene electrotransfer is an attractive technology of gene therapy for clinical application. This is well illustrated by the increasing number of reviews covering the pre-clinical

As mentioned above, one of main limits of the widespread use of electropermeabilization is that very few is known on the biophysical mechanisms supporting the reorganization of the cell membrane (pore, electropore, defects?). The molecular target of the field effect remains

Most theories are based on the DNA sliding model [37]. Several steps are predicted: entry, electrophoretic translocation as long as the field was present, diffusion after the pulse delivery. An interesting simulation with predictive conclusions has just been published [38]. The DNA translocation was through a putative "electropore" assuming that the DNA was double stranded but linear (i.e open) meaning that the length was 2.4 μm. Indeed the sliding model needs the DNA to be linear to allow the "binding" of one end to the "electropore" entry. Experimental results just showed that the closed form was more effective for expression [39]. One speculative hypothesis in the simulation was that it always assumed that the binding step (entry) of the DNA to the "electropore" was present before the simulation started. De Gennes predicted that it was a very limiting step in the process (a crucial moment) when a chain end faces the electropore and enters it against the strong friction coefficient against the "pore" sides. A black box remains the "electropore".

#### **3.2. The electropore**

The major hypothesis in the sliding model is that "pores" must be present. Krassowska's model supports the simplest mechanism, in which plasmid enters the 5 nm thick membrane through stable electropores (up to 20 nm in diameter)[40, 41]. The electrically induced defects result from the field associated membrane potential changes. It predicts a post-pulse growth of "macropores" on the sub-second time scale fairly consistent with experimental evidence on pure lipid vesicles [42]. This model predicts "electropores" large enough to permit the plasmid uptake (under a linear form). These "electropores" are supposed to remain open for the entire duration of electrotransfer providing adequate time for the plasmid to enter the cell [43]. Indeed in Lin's simulation [38], they may remain present on a much longer duration than the pulse as a diffusion might follow a partial electrotransfer.

This model remains very attractive in spite of the existence of many experimental contradic‐ tions. Indeed, until now, no study made it possible to visualize these membrane pores. This

validation appears impossible [27]. Moreover, the resealing time of pores appears to be shorter in this lipidic model than in experiments on cells (e.g. seconds rather than minutes) [44-47]. The conclusion is that pDNA transport across the membrane is always very fast. To date, theoretical models could predict stable pores of only a few nanometers in radius; larger pores are unstable while they are needed for the sliding pDNA transfer [48, 49]. These models are confirmed by some experiments, in which high-voltage pulses a few microseconds long are used that are supposed to have created a large number of very small pores (radii of about 1 nm, i.e. the size of a few phospholipids cluster) [50]. To reconcile these results with the experimental evidence of plasmid translocation after electropulsation, some researchers postulated that plasmid entry into cells relies on the plasmid/membrane interactions, which may be facilitated by a coalescence of many small, 1 nm defects [51-54, 43]. The slow transport of DNA across the electropermeabilized membrane reflects a highly interactive electrotransfer, where many small lipid defects coalesced into large DNA-lipids assemblies where the transmembrane transport occurs [55].

transfection, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (10 msec) in the cell membrane [62]. In addition, in the case of CHO cells, plasmid remains accessible to DNAase I in the minute, which follows the end of electropulsation. This shows that the plasmid

Electrically Mediated Gene Delivery: Basic and Translational Concepts

http://dx.doi.org/10.5772/54780

37

A molecular dynamic approach gives a mechanism, in which plasmids do not translocate across the membrane during the electropulsation [63]. The DNA/lipid system simulation was undertaken starting from a well-equilibrated 12bp-DNA duplex placed near a model POPC bilayer. The perturbation of the system under a 1.0 V.nm−1 transverse electric field (i.e. a transmembrane voltage of 5 V !) is followed during 2 ns. Under this high electric field, the DNA duplex diffuses towards the interior of the bilayer only after the creation of a pore beneath it, and within the same timescale, it remains at the interfacial region when no pore is present. Diffusion of the strand toward the interior of the membrane leads to a DNA/lipid complex in which the lipid head groups encapsulate the strand. The dipole carried by the zwitterionic phosphatidylcholine groups of the lipids is known to be efficient for neutralizing the charges carried by the DNA [64]. Such interactions between the plasmid and the lipids contribute to the effective screening of DNA charges and therefore to the stabilization of the complex. One should not forget that electropulsation-mediated gene delivery concerns much larger super‐

Most methods for chemically mediated gene transfer described the transport as an endocytotic complex formation between the DNA vesicles and the cell surface. Several studies investigated if this can occur in electrotransfection. Treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) yielded substantial and statistically significant reduc‐ tions in the electrotransfection mediated expression [62]. These findings suggest that electro‐ transfection depends on endocytosis of membrane-bound pDNA. [65]. Colocalization studies with endocytotic markers under a microscope showed that pDNA is internalized with concomitant clathrin- and caveolin/raft-mediated endocytosis [66]. But this cannot explain how the pDNA is released from the endocytic vesicles and why free PDNA was observed in the cytoplasm a few minutes after the pulse delivery [67]. A direct assay of the formation of endocytic vesicles brought the conclusion that endocytosis was not stimulated by applying electric pulses with intensities above the threshold value for gene electrotransfer. The conclu‐ sion was that electro-endocytosis is not a crucial mechanism for gene electrotransfer [68].

PDNA electrotransfer was observed at the single cell level by digitized high resolution fluorescence microscopy [67]. The introduction of DNA only occurs in the part of the mem‐ brane facing the cathode and requires a number of consecutive steps: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the mem‐

coiled plasmids than the 12 bp construct considered in the MD simulation.

transfer inside the cell occurs after the electropulsation [17].

**3.5. In silico electrotransfer**

**3.6. Endocytosis**

**3.7. The multistep model**

### **3.3. Electrophoresis across the micellar structures**

Other data report that gene electrotransfer through lipid bilayer could be mediated by transient complexes between plasmid and specific lipids in the edges of elongated, electropercolated hydrophilic membrane associated micellar structures [56]. The plasmid association with a lipid bilayer results in a facilitated transport of small ions. A locally conductive plasmid/lipid interaction zone is induced where parts of the plasmid may be inserted in the bilayer. Plasmid is transiently inserted in, and then electrophoretically pulled through the permeabilized zones onto the other membrane side [57]. With such a model, in the case of mammalian cells, the resting potential difference should be the driving force for plasmid translocation after the pulse induced insertion. The external field is only used to electrophoretically accumulate the pDNA on the cell surface. This has not been checked yet.

#### **3.4. An electrophoretic transfer**

Previous works suggested that electric pulses induce the membrane permeabilization, then plasmid molecules are concentrated near the membrane surface and pushed through by electrophoretic forces [58-60, 54]. The plasmid may interact with electropermeabilized membrane by three possible ways: (a) the plasmid coil is aligned in an electric field, and at the appropriate pulse polarity it moves toward the permeabilized membrane. Transfer is depend‐ ent on electrophoretic forces and is complete at the end of the pulse. Post pulse cell treatment should not affect the efficiency of transfer.

If the electrophoretic forces are the only driving forces of the plasmid transfer into the cell, similar transfection efficiencies should be obtained for equal E.T values (i.e. E, field strength and T, pulse duration). This is not supported by the experiments [61] When the E.N.T value is constant, transfection rate depends preferentially on T [52]. Therefore, the electrophoretic migration cannot be the only driving force of the plasmid transfer into the cells but clearly supports the formation of aggregates. Trypsin treatment of cells at 10 min post electrotrans‐ fection stripped off membrane-bound pDNA and resulted in a significant reduction in transfection, indicating that the time period for complete cellular uptake of pDNA (between 10 and 40 min) far exceeded the lifetime of electric field-induced transient pores (10 msec) in the cell membrane [62]. In addition, in the case of CHO cells, plasmid remains accessible to DNAase I in the minute, which follows the end of electropulsation. This shows that the plasmid transfer inside the cell occurs after the electropulsation [17].

#### **3.5. In silico electrotransfer**

validation appears impossible [27]. Moreover, the resealing time of pores appears to be shorter in this lipidic model than in experiments on cells (e.g. seconds rather than minutes) [44-47]. The conclusion is that pDNA transport across the membrane is always very fast. To date, theoretical models could predict stable pores of only a few nanometers in radius; larger pores are unstable while they are needed for the sliding pDNA transfer [48, 49]. These models are confirmed by some experiments, in which high-voltage pulses a few microseconds long are used that are supposed to have created a large number of very small pores (radii of about 1 nm, i.e. the size of a few phospholipids cluster) [50]. To reconcile these results with the experimental evidence of plasmid translocation after electropulsation, some researchers postulated that plasmid entry into cells relies on the plasmid/membrane interactions, which may be facilitated by a coalescence of many small, 1 nm defects [51-54, 43]. The slow transport of DNA across the electropermeabilized membrane reflects a highly interactive electrotransfer, where many small lipid defects coalesced into large DNA-lipids assemblies where the

Other data report that gene electrotransfer through lipid bilayer could be mediated by transient complexes between plasmid and specific lipids in the edges of elongated, electropercolated hydrophilic membrane associated micellar structures [56]. The plasmid association with a lipid bilayer results in a facilitated transport of small ions. A locally conductive plasmid/lipid interaction zone is induced where parts of the plasmid may be inserted in the bilayer. Plasmid is transiently inserted in, and then electrophoretically pulled through the permeabilized zones onto the other membrane side [57]. With such a model, in the case of mammalian cells, the resting potential difference should be the driving force for plasmid translocation after the pulse induced insertion. The external field is only used to electrophoretically accumulate the pDNA

Previous works suggested that electric pulses induce the membrane permeabilization, then plasmid molecules are concentrated near the membrane surface and pushed through by electrophoretic forces [58-60, 54]. The plasmid may interact with electropermeabilized membrane by three possible ways: (a) the plasmid coil is aligned in an electric field, and at the appropriate pulse polarity it moves toward the permeabilized membrane. Transfer is depend‐ ent on electrophoretic forces and is complete at the end of the pulse. Post pulse cell treatment

If the electrophoretic forces are the only driving forces of the plasmid transfer into the cell, similar transfection efficiencies should be obtained for equal E.T values (i.e. E, field strength and T, pulse duration). This is not supported by the experiments [61] When the E.N.T value is constant, transfection rate depends preferentially on T [52]. Therefore, the electrophoretic migration cannot be the only driving force of the plasmid transfer into the cells but clearly supports the formation of aggregates. Trypsin treatment of cells at 10 min post electrotrans‐ fection stripped off membrane-bound pDNA and resulted in a significant reduction in

transmembrane transport occurs [55].

36 Novel Gene Therapy Approaches

**3.3. Electrophoresis across the micellar structures**

on the cell surface. This has not been checked yet.

should not affect the efficiency of transfer.

**3.4. An electrophoretic transfer**

A molecular dynamic approach gives a mechanism, in which plasmids do not translocate across the membrane during the electropulsation [63]. The DNA/lipid system simulation was undertaken starting from a well-equilibrated 12bp-DNA duplex placed near a model POPC bilayer. The perturbation of the system under a 1.0 V.nm−1 transverse electric field (i.e. a transmembrane voltage of 5 V !) is followed during 2 ns. Under this high electric field, the DNA duplex diffuses towards the interior of the bilayer only after the creation of a pore beneath it, and within the same timescale, it remains at the interfacial region when no pore is present. Diffusion of the strand toward the interior of the membrane leads to a DNA/lipid complex in which the lipid head groups encapsulate the strand. The dipole carried by the zwitterionic phosphatidylcholine groups of the lipids is known to be efficient for neutralizing the charges carried by the DNA [64]. Such interactions between the plasmid and the lipids contribute to the effective screening of DNA charges and therefore to the stabilization of the complex. One should not forget that electropulsation-mediated gene delivery concerns much larger super‐ coiled plasmids than the 12 bp construct considered in the MD simulation.

#### **3.6. Endocytosis**

Most methods for chemically mediated gene transfer described the transport as an endocytotic complex formation between the DNA vesicles and the cell surface. Several studies investigated if this can occur in electrotransfection. Treatment of cells with three endocytic inhibitors (chlorpromazine, genistein, dynasore) yielded substantial and statistically significant reduc‐ tions in the electrotransfection mediated expression [62]. These findings suggest that electro‐ transfection depends on endocytosis of membrane-bound pDNA. [65]. Colocalization studies with endocytotic markers under a microscope showed that pDNA is internalized with concomitant clathrin- and caveolin/raft-mediated endocytosis [66]. But this cannot explain how the pDNA is released from the endocytic vesicles and why free PDNA was observed in the cytoplasm a few minutes after the pulse delivery [67]. A direct assay of the formation of endocytic vesicles brought the conclusion that endocytosis was not stimulated by applying electric pulses with intensities above the threshold value for gene electrotransfer. The conclu‐ sion was that electro-endocytosis is not a crucial mechanism for gene electrotransfer [68].

#### **3.7. The multistep model**

PDNA electrotransfer was observed at the single cell level by digitized high resolution fluorescence microscopy [67]. The introduction of DNA only occurs in the part of the mem‐ brane facing the cathode and requires a number of consecutive steps: electrophoretic migration of DNA towards the cell, DNA insertion into the membrane, translocation across the mem‐ brane, migration of DNA towards the nucleus and, finally, transfer of DNA across the nuclear envelope. Only localized parts of the cell membrane brought to the permeabilized state are competent for transfer. The transport of plasmid follows an "anchoring step", connecting the plasmid to the permeabilized membrane, that takes place during the pulse. During the first pulsation, plasmids are electrophoretically drifted and interact with a limited number of sites on the membrane. These sites become highly conductive and attract the field lines giving an electrophoretic local accumulation [69]. PDNAs form a limited number of aggregates on the cell surface. Their sizes increase during the pulses or with successive pulses but not their number [70]. Transfer of membrane bound plasmids is certainly a complex process, that is not occurring during but after the pulse delivery. Two classes of DNA/membrane interactions result from the pulse: (i) a metastable DNA/membrane complex from which the DNA can leave and return to external medium and (ii) a stable DNA/membrane. Only DNA belonging to the second class may be effective for transmembrane transport and the resulting gene expression [71]. Nevertheless this model shows that the plasmid is stabilized in the millisecond following the pulse in the membrane core after electropulsation (in agreement with the overall experi‐ mentally observed process of DNA translocation).

**5. PDNA under electrotransfection conditions**

**5.1. Complexities in the determination of pDNA size evaluation**

The size of the plasmid can be a significant modulator of the efficiency of transfer. The sliding model assumed that the pDNA was linear. This is not relevant of the experiments where a closed form was used in almost all reported cases. The gyration volume appears more appropriated. It is known that this diameter is highly sensitive to the compaction factors that are present. A tightly packed form is found in viral capsids. During the last 20 years, biotech‐ nologists have been playing with chemical additives to obtain more compact forms. Indeed adding NaCl and/or MgCl2 is affecting the diameters. In [82] the authors wrote: "conforma‐ tional and thermodynamic properties of supercoiled DNA depend strongly on ionic condi‐ tions. The effective double-helix diameter increases from 3 to 15 nm as the salt concentration is reduced from 1.00 to 0.01 M.". It was later observed with pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris, that the superhelix diameter from the simulated conformations decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration[83]. This value did not significantly change to lower values at higher Na+ concentration. And in [84] upon addition of 0.122 M NaCl, the radius of gyration (RG) decreased substantially, which indicates that p30 delta adopts a more compact structure. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased. Using Polymethacrylate monoliths,[85], size evaluations are described on a model plasmid, consisting of 4.9 kbp, Under physiological conditions, a 45 nm radius was evaluated. But the pore size distributions in these samples (see below) are broad: as such, the changes in the median pore diameter measured by mercury intrusion porosimetry reflect general shifts in the position of the pore distribution envelope, rather than the position of a well-defined, sharp peak [86](in http://www.liv.ac.uk/~aicooper/AKH\_monolith.pdf).In [87] it was found for covalently closed supercoiled ColE1-plasmid DNA in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, a gyration radius about 100 nm but with EDTA meaning with no divalent ions but in 0.2 M NaCl. Finally, in [88] for pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms, RG = 90 +/- 3 nm,, and RG = 82 +/- 2.5 nm were obtained.

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39

As a conclusion, a large distribution of gyration radii is described in the literature but, they are all larger that the compact form reported by Krassowska and that she used to predict the need of "electropores" of about 10 nm for DNA translocation during the pulse. Even larger sizes are requested with linearized form, that are known to be effective for expression after their electrotransfer [39]. For a closed form (highly effective for expression after the electro‐ transfer), in a condensed form under physiological conditions (Na> 20 mM, MgCl2 about 1 mM), the diameter of the structures supporting a free transmembrane transfer needs to be at

As NaCl concentration decreases, the superhelix becomes less regular and more compact. In the presence of just 10 mM MgCl2, supercoiled DNA adopts essentially the same set of

Simulations give shapes of supercoiled closed form of PDNA (7 kb) [89]

conformations as in moderate to high concentrations of NaCl.

least 100 nm.

## **4. The cytoplasmic transfer**

Transfer from the membrane to the nucleus is mediated by the cell (cytoskeleton with molec‐ ular motors?). The final step should take place through the nuclear pore complex [72-74]. No direct biophysical method to alter the nuclear envelope or pore has been reported (yet). But imaging methods [75, 76] support the occurrence of a direct effect of the field on organelles [77].

For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. 2 steps are therefore present involving 2 classes of barriers. The cytoskeletal meshwork prevents pDNA (larger than 1 kb) movement in the cytoplasm. Actin patches colocalizing with the DNA at the plasma membrane were observed several minutes after pulse delivery with characteristics similar to those of the DNA aggre‐ gates, that are formed during the early stages of electrotransfection [78]. The microtubule network is required for directed plasmid trafficking to the nucleus [79]. Microtubule–DNA interactions can be enhanced due to sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), such as the cytomegalovirus immediate early promoter (CMViep). Insertion of cytoplasmic adapter proteins transcription factors (TFs) binding sites within plasmids permits cytoplasmic trafficking of plasmids and an effective expression.

NLS sequences can help for the transfer inside the nucleus. In non-dividing cells, the nuclear envelope is an especially problematic hurdle to gene transfer. A successful approach is in modifying plasmid (pDNA) vectors to enhance nuclear import through the Nuclear Pore Complex [80]. Proteomics tools have been used to study DNA nuclear entry telling that Transcription factor-binding sites promote DNA nuclear translocation and Cell-specific transcription factors drive cell-specific DNA nuclear entry [81]. NLS peptides or nuclear proteins complexed with plasmids may enhance DNA nuclear translocation.

## **5. PDNA under electrotransfection conditions**

brane, migration of DNA towards the nucleus and, finally, transfer of DNA across the nuclear envelope. Only localized parts of the cell membrane brought to the permeabilized state are competent for transfer. The transport of plasmid follows an "anchoring step", connecting the plasmid to the permeabilized membrane, that takes place during the pulse. During the first pulsation, plasmids are electrophoretically drifted and interact with a limited number of sites on the membrane. These sites become highly conductive and attract the field lines giving an electrophoretic local accumulation [69]. PDNAs form a limited number of aggregates on the cell surface. Their sizes increase during the pulses or with successive pulses but not their number [70]. Transfer of membrane bound plasmids is certainly a complex process, that is not occurring during but after the pulse delivery. Two classes of DNA/membrane interactions result from the pulse: (i) a metastable DNA/membrane complex from which the DNA can leave and return to external medium and (ii) a stable DNA/membrane. Only DNA belonging to the second class may be effective for transmembrane transport and the resulting gene expression [71]. Nevertheless this model shows that the plasmid is stabilized in the millisecond following the pulse in the membrane core after electropulsation (in agreement with the overall experi‐

Transfer from the membrane to the nucleus is mediated by the cell (cytoskeleton with molec‐ ular motors?). The final step should take place through the nuclear pore complex [72-74]. No direct biophysical method to alter the nuclear envelope or pore has been reported (yet). But imaging methods [75, 76] support the occurrence of a direct effect of the field on organelles [77]. For non-viral gene delivery to be successful, plasmids must move through the cytoplasm to the nucleus in order to be transcribed. 2 steps are therefore present involving 2 classes of barriers. The cytoskeletal meshwork prevents pDNA (larger than 1 kb) movement in the cytoplasm. Actin patches colocalizing with the DNA at the plasma membrane were observed several minutes after pulse delivery with characteristics similar to those of the DNA aggre‐ gates, that are formed during the early stages of electrotransfection [78]. The microtubule network is required for directed plasmid trafficking to the nucleus [79]. Microtubule–DNA interactions can be enhanced due to sequence specificity with promoters containing binding sites for cyclic AMP response-element binding protein (CREB), such as the cytomegalovirus immediate early promoter (CMViep). Insertion of cytoplasmic adapter proteins transcription factors (TFs) binding sites within plasmids permits cytoplasmic trafficking of plasmids and an

NLS sequences can help for the transfer inside the nucleus. In non-dividing cells, the nuclear envelope is an especially problematic hurdle to gene transfer. A successful approach is in modifying plasmid (pDNA) vectors to enhance nuclear import through the Nuclear Pore Complex [80]. Proteomics tools have been used to study DNA nuclear entry telling that Transcription factor-binding sites promote DNA nuclear translocation and Cell-specific transcription factors drive cell-specific DNA nuclear entry [81]. NLS peptides or nuclear

proteins complexed with plasmids may enhance DNA nuclear translocation.

mentally observed process of DNA translocation).

**4. The cytoplasmic transfer**

38 Novel Gene Therapy Approaches

effective expression.

#### **5.1. Complexities in the determination of pDNA size evaluation**

The size of the plasmid can be a significant modulator of the efficiency of transfer. The sliding model assumed that the pDNA was linear. This is not relevant of the experiments where a closed form was used in almost all reported cases. The gyration volume appears more appropriated. It is known that this diameter is highly sensitive to the compaction factors that are present. A tightly packed form is found in viral capsids. During the last 20 years, biotech‐ nologists have been playing with chemical additives to obtain more compact forms. Indeed adding NaCl and/or MgCl2 is affecting the diameters. In [82] the authors wrote: "conforma‐ tional and thermodynamic properties of supercoiled DNA depend strongly on ionic condi‐ tions. The effective double-helix diameter increases from 3 to 15 nm as the salt concentration is reduced from 1.00 to 0.01 M.". It was later observed with pUC18 (2686 bp), in dilute aqueous solution at salt concentrations between 0 and 1.5 M Na+ in 10 mM Tris, that the superhelix diameter from the simulated conformations decreased from 18.0 +/- 1.5 nm at 10 mM to 9.4 +/- 1.5 nm at 100 mM salt concentration[83]. This value did not significantly change to lower values at higher Na+ concentration. And in [84] upon addition of 0.122 M NaCl, the radius of gyration (RG) decreased substantially, which indicates that p30 delta adopts a more compact structure. When 4 mM Mg2+ was added to native supercoiled p30 delta in 0.1 M NaCl, Rg decreased.

Using Polymethacrylate monoliths,[85], size evaluations are described on a model plasmid, consisting of 4.9 kbp, Under physiological conditions, a 45 nm radius was evaluated. But the pore size distributions in these samples (see below) are broad: as such, the changes in the median pore diameter measured by mercury intrusion porosimetry reflect general shifts in the position of the pore distribution envelope, rather than the position of a well-defined, sharp peak [86](in http://www.liv.ac.uk/~aicooper/AKH\_monolith.pdf).In [87] it was found for covalently closed supercoiled ColE1-plasmid DNA in 0.2 M NaCl, 0.002 M NaPO4 pH 7.0,0.002 M EDTA, a gyration radius about 100 nm but with EDTA meaning with no divalent ions but in 0.2 M NaCl. Finally, in [88] for pGem1a plasmids (3730 base pairs) in the relaxed circular (nicked) and supercoiled forms, RG = 90 +/- 3 nm,, and RG = 82 +/- 2.5 nm were obtained.

As a conclusion, a large distribution of gyration radii is described in the literature but, they are all larger that the compact form reported by Krassowska and that she used to predict the need of "electropores" of about 10 nm for DNA translocation during the pulse. Even larger sizes are requested with linearized form, that are known to be effective for expression after their electrotransfer [39]. For a closed form (highly effective for expression after the electro‐ transfer), in a condensed form under physiological conditions (Na> 20 mM, MgCl2 about 1 mM), the diameter of the structures supporting a free transmembrane transfer needs to be at least 100 nm.

Simulations give shapes of supercoiled closed form of PDNA (7 kb) [89]

As NaCl concentration decreases, the superhelix becomes less regular and more compact. In the presence of just 10 mM MgCl2, supercoiled DNA adopts essentially the same set of conformations as in moderate to high concentrations of NaCl.

The size of a supercoiled plasmid is difficult to access. As shown in [89], the bulk size is large. This explains why many reports are giving data around 100 nm. But in fact, it is not a sphere (a coil) but a rather elongated thread-like shape that is more relevant. The DNA topology is described quantitatively by the twist of double helix and by the num‐ ber of times the helix crosses over on itself (plectoneme). Plectonemic structures are typi‐ cally formed by bacterial plasmids. Then in one direction we got a cross section close to the 20 nm used by Krassowska. A larger value is indeed observed under the low salt (Mg free) solution [90, 91].

**6. Physical controls in optimizing the protocols**

by the cumulated pulse duration is present[100, 101].

inter-pulse cooling of the pulsed sample.

penetration remains rather limited

**7.1. Reactive Oxygen Species (ROS)**

**7. Cellular responses and controls**

**6.2. Electrodes**

electrical parameters must be chosen to preserve the cell viability.

tissue is controlled by the geometry of the electrodes [106].

from the permeabilizing pulse. This protocol is better to preserve the viability.

Cell electropermeabilization must occur and an efficient electrophoretic accumulation of pDNA must be applied. This means that the field strength must be larger than a critical value (permeabilizing threshold at the level of the target in the tissue). Again the advantage of a targeted effect in tissues is present. But a modulation of transfer in this well defined volume

Electrically Mediated Gene Delivery: Basic and Translational Concepts

http://dx.doi.org/10.5772/54780

41

The pulse duration can be short (0.1 ms) but longer pulses are more efficient as they are associated to a longer and therefore more efficient electrophoresis of the pDNA [102, 104]. The

A double pulse method (a short high voltage pulse followed after a short delay by a long low voltage one)(HV LV) was therefore described [105]. The electrophoretic drift can be delayed

A destructive Joule effect can be present bringing limits in the parameters of the protocol. Intra pulse delay choice in a train of pulses can help to reduce this damaging effect by allowing a

Getting an optimized field distribution of the field intensities at the level of the target in the

Most trials are performed by using needle electrodes that are penetrating inside the tar‐ get tissues. Many designs have been reported (number of needles, diameters, distances, number, depth of penetration (see [107]). One major concern with these systems (that al‐ low a deep penetration of the field) is the damaging effects of the electrodes (not only due to the perforation of the tissue but linked to the local effects at the electrode surface (local heating [108], electrochemical reactions [109]). Contact electrodes appear less de‐ structive as the skin to electrodes contact is due to a conductive gel [110] but the field

Cellular responses are present under electrotransfection. The field induced membrane reorganization is a stress for the molecular assembly. A defense mechanism is present as shown by the generation of reactive oxygen species at the surface of the permeabilized cell [111-113]. ROS are highly destructive for DNA and reduce the number of copies that remains intact and therefore effective for expression. Protective effects are brought by the addition of anti-

**6.1. Electrical pulse parameters**

The final conclusion is that the general conformation of pDNA used for electrotransfec‐ tion is "complex" and do not support the model used for the sliding model. The lack of knowledge on the theoretical processes supporting the transmembrane transport brings the need of a rather empirical approach in the optimization of the technology for gene therapy.[92].

#### **5.2. Smaller plasmids are more effective**

The basic protocols are using Naked plasmid DNA under a Double strand closed form. No advantage is brought by preparing the linear form by restriction enzymes digestion.

As size controls the efficiency of transfer, minicircle forms (MC) are more efficient [93]. Minicircle DNA lacks the bacterial backbone sequence consisting of an antibiotic resist‐ ance gene, an origin of replication, and inflammatory sequences intrinsic to bacterial DNA that represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. Expression following electrotransfer is improved with MC constructs over full-length plasmid (same promoter, same coding cassette) with different reporter genes. This great efficiency of MC was correlated to more efficient vec‐ tor uptake by cells. Nevertheless, one should keep in mind that huge pDNA have been transferred and that decreasing the size of the plasmid is just bringing improvement but is not needed [94].

#### **5.3. Field effect on pDNA conformation**

Under electrotransfection protocols, besides the ionic content of the buffer that can be easily adjusted under in vitro protocols (but remains poorly controlled for gene therapy) [95], a critical parameter is present. An electric field is present that may affect the confor‐ mation of the pDNA.

Concerning DNA in electric fields, conflicting observations and predictions are present in the literature. Low DC field do not greatly perturb the conformation of large DNA. In [96], it is reported that larger fields give rise to chain orientation and stretching. This is in agreement with a simulation [97]. In fact, at high concentrations, strong intermolecular aggregation was observed even under 100V/cm [98, 99]. This can be considered as an ex‐ planation for the formation of the stable spots that we observed as an early stage in the multistep process.

## **6. Physical controls in optimizing the protocols**

#### **6.1. Electrical pulse parameters**

The size of a supercoiled plasmid is difficult to access. As shown in [89], the bulk size is large. This explains why many reports are giving data around 100 nm. But in fact, it is not a sphere (a coil) but a rather elongated thread-like shape that is more relevant. The DNA topology is described quantitatively by the twist of double helix and by the num‐ ber of times the helix crosses over on itself (plectoneme). Plectonemic structures are typi‐ cally formed by bacterial plasmids. Then in one direction we got a cross section close to the 20 nm used by Krassowska. A larger value is indeed observed under the low salt

The final conclusion is that the general conformation of pDNA used for electrotransfec‐ tion is "complex" and do not support the model used for the sliding model. The lack of knowledge on the theoretical processes supporting the transmembrane transport brings the need of a rather empirical approach in the optimization of the technology for gene

The basic protocols are using Naked plasmid DNA under a Double strand closed form. No

As size controls the efficiency of transfer, minicircle forms (MC) are more efficient [93]. Minicircle DNA lacks the bacterial backbone sequence consisting of an antibiotic resist‐ ance gene, an origin of replication, and inflammatory sequences intrinsic to bacterial DNA that represent a potential risk for safe clinical application and reduce gene transfer rates as well as transgene expression. Expression following electrotransfer is improved with MC constructs over full-length plasmid (same promoter, same coding cassette) with different reporter genes. This great efficiency of MC was correlated to more efficient vec‐ tor uptake by cells. Nevertheless, one should keep in mind that huge pDNA have been transferred and that decreasing the size of the plasmid is just bringing improvement but

Under electrotransfection protocols, besides the ionic content of the buffer that can be easily adjusted under in vitro protocols (but remains poorly controlled for gene therapy) [95], a critical parameter is present. An electric field is present that may affect the confor‐

Concerning DNA in electric fields, conflicting observations and predictions are present in the literature. Low DC field do not greatly perturb the conformation of large DNA. In [96], it is reported that larger fields give rise to chain orientation and stretching. This is in agreement with a simulation [97]. In fact, at high concentrations, strong intermolecular aggregation was observed even under 100V/cm [98, 99]. This can be considered as an ex‐ planation for the formation of the stable spots that we observed as an early stage in the

advantage is brought by preparing the linear form by restriction enzymes digestion.

(Mg free) solution [90, 91].

40 Novel Gene Therapy Approaches

**5.2. Smaller plasmids are more effective**

**5.3. Field effect on pDNA conformation**

therapy.[92].

is not needed [94].

mation of the pDNA.

multistep process.

Cell electropermeabilization must occur and an efficient electrophoretic accumulation of pDNA must be applied. This means that the field strength must be larger than a critical value (permeabilizing threshold at the level of the target in the tissue). Again the advantage of a targeted effect in tissues is present. But a modulation of transfer in this well defined volume by the cumulated pulse duration is present[100, 101].

The pulse duration can be short (0.1 ms) but longer pulses are more efficient as they are associated to a longer and therefore more efficient electrophoresis of the pDNA [102, 104]. The electrical parameters must be chosen to preserve the cell viability.

A double pulse method (a short high voltage pulse followed after a short delay by a long low voltage one)(HV LV) was therefore described [105]. The electrophoretic drift can be delayed from the permeabilizing pulse. This protocol is better to preserve the viability.

A destructive Joule effect can be present bringing limits in the parameters of the protocol. Intra pulse delay choice in a train of pulses can help to reduce this damaging effect by allowing a inter-pulse cooling of the pulsed sample.

#### **6.2. Electrodes**

Getting an optimized field distribution of the field intensities at the level of the target in the tissue is controlled by the geometry of the electrodes [106].

Most trials are performed by using needle electrodes that are penetrating inside the tar‐ get tissues. Many designs have been reported (number of needles, diameters, distances, number, depth of penetration (see [107]). One major concern with these systems (that al‐ low a deep penetration of the field) is the damaging effects of the electrodes (not only due to the perforation of the tissue but linked to the local effects at the electrode surface (local heating [108], electrochemical reactions [109]). Contact electrodes appear less de‐ structive as the skin to electrodes contact is due to a conductive gel [110] but the field penetration remains rather limited

## **7. Cellular responses and controls**

#### **7.1. Reactive Oxygen Species (ROS)**

Cellular responses are present under electrotransfection. The field induced membrane reorganization is a stress for the molecular assembly. A defense mechanism is present as shown by the generation of reactive oxygen species at the surface of the permeabilized cell [111-113]. ROS are highly destructive for DNA and reduce the number of copies that remains intact and therefore effective for expression. Protective effects are brought by the addition of antioxydants as long as they are not interfering with the transport.[114, 115]. There is a need to find biocompatible additive to reduce the ROS generation. Co-block polymers appear as a promising pathway.It was shown that postshock poloxamer administration reduced tissue inflammation and damage in comparison with dextran-treated or control tissues [116].

dynamic and takes place during the pulse delivery. This remains under technical investiga‐ tions by using simulations in electrical engineering [123, 124]. This time dependence of the electrical properties of the pulse tissue is an important parameter for the proper choice of the

Electrically Mediated Gene Delivery: Basic and Translational Concepts

http://dx.doi.org/10.5772/54780

43

But clearly the biotechnological contributions cannot be neglected. Optimization in the plasmid constructs is strongly needed. The use of minicircles is promising to get a better transfer. But a key problem remains the design of the promoter that is shown to be an active

The work was supported by grants from the seventh framework programme OncomiR (# 201102) and from the region Midi-Pyrénées (#11052700). The author wants to thank Dr JM

1 CNRS;IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France

[1] Wolff, J. A, Malone, R. W, Williams, P, Chong, W, Acsadi, G, & Jani, A. Felgner PL Direct gene transfer into mouse muscle in vivo. Science. (1990). 247(4949 Pt 1), 1465-8.

[2] Neumann, E, Shaefer-ridder, M, & Wang, Y. Hofschneider PH Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J (1982). , 1, 841-845. [3] Heller, R, Jaroszeski, M, Atkin, A, Moradpour, D, Gilbert, R, Wands, J, & Nicolau, C. In vivo gene electroinjection and expression in rat liver.FEBS Lett. (1996). , 389(3), 225-8.

[4] Satkauskas, S, Ruzgys, P, & Venslauskas, M. S. Towards the mechanisms for efficient gene transfer into cells and tissues by means of cell electroporation. Expert Opin Biol

[5] Gothelf, A, & Gehl, J. Gene electrotransfer to skin; review of existing literature and

clinical perspectives.Curr Gene Ther. (2010). , 10(4), 287-99.

sequence of electrical pulses that must be delivered [125].

partner in the cytoplasmic traffic to the nuclear volume.

Address all correspondence to: justin.teissie@ipbs.fr

2 Université de Toulouse, Toulouse, France

Ther. (2012). , 12(3), 275-86.

**Acknowledgements**

**Author details**

J. Teissié1,2\*

**References**

Escoffre for many discussions.

### **7.2. Extracellular matrix (ECM)**

Uniform DNA distribution in tumors is a prerequisite step for an homogeneous transfection efficiency in solid tumors. This is of course valid for other target tissues (skin, muscles). The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. The characteristic electromobility behavior, under most electrotransfection pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, but a decisive role is played by the pore size of the fibrous matrix (the extracellular matrix in tumors) through which the DNA migrated [117, 118]. The intratumoral field, which determines the efficiency of electric field-mediated gene delivery, can differ significantly from the applied field at the surface of the tumor.[119]. This local field is under the control of the geometry of the electrodes as described above. The field strengths in tumor tissues were significantly lower (down to 50%) than the applied field due to the multicellular organization. But when the external field was uniform (plate parallel electrodes) the electric fields in the center region of tumors were macroscopically uniform on ex Vivo slices.

Indeed tumor histological properties strongly affected transfection efficiency. Soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected than rigid tumors with high proteoglycan and collagen content, small spindle-shaped cells and high cell density [120].

Electrotransfection in tissue can be improved by modulation of the extracellular matrix, using collagenase and/or hyaluronidase in tumors [121] as well as in muscles, a major target organ for DNA vaccination, a great topic for gene therapy [122].

## **8. Conclusion**

Even if our knowledge on the molecular mechanisms governing the transfer of pDNA due to the delivery of pulsed electric field remains limited, it gives recommandations for an optimal choice of the protocols.

Pulse generators should provide the largest flexibility in the choice of the electrical parameters (voltage, duration, delay, number, current intensity, sharpness of the pulse onset) and offer an internal monitoring of the delivered pulse. Very few products on the market meet these specifications.

The local field on the tissue target is a complex function of the choice of the electrodes and on the electrical changes of the tissue due to the electrical treatment. This last parameter is highly dynamic and takes place during the pulse delivery. This remains under technical investiga‐ tions by using simulations in electrical engineering [123, 124]. This time dependence of the electrical properties of the pulse tissue is an important parameter for the proper choice of the sequence of electrical pulses that must be delivered [125].

But clearly the biotechnological contributions cannot be neglected. Optimization in the plasmid constructs is strongly needed. The use of minicircles is promising to get a better transfer. But a key problem remains the design of the promoter that is shown to be an active partner in the cytoplasmic traffic to the nuclear volume.

## **Acknowledgements**

The work was supported by grants from the seventh framework programme OncomiR (# 201102) and from the region Midi-Pyrénées (#11052700). The author wants to thank Dr JM Escoffre for many discussions.

## **Author details**

J. Teissié1,2\*

oxydants as long as they are not interfering with the transport.[114, 115]. There is a need to find biocompatible additive to reduce the ROS generation. Co-block polymers appear as a promising pathway.It was shown that postshock poloxamer administration reduced tissue inflammation and damage in comparison with dextran-treated or control tissues [116].

Uniform DNA distribution in tumors is a prerequisite step for an homogeneous transfection efficiency in solid tumors. This is of course valid for other target tissues (skin, muscles). The interstitial space is a rate limiting physiological barrier to non-viral gene delivery. External pulsed electric fields have been proposed to increase DNA transport in the interstitium, thereby improving non-viral gene delivery. The characteristic electromobility behavior, under most electrotransfection pulsing conditions, consisted of three distinct phases: stretching, reptation, and relaxation. Electromobility depended strongly on the field magnitude, pulse duration, but a decisive role is played by the pore size of the fibrous matrix (the extracellular matrix in tumors) through which the DNA migrated [117, 118]. The intratumoral field, which determines the efficiency of electric field-mediated gene delivery, can differ significantly from the applied field at the surface of the tumor.[119]. This local field is under the control of the geometry of the electrodes as described above. The field strengths in tumor tissues were significantly lower (down to 50%) than the applied field due to the multicellular organization. But when the external field was uniform (plate parallel electrodes) the electric fields in the

Indeed tumor histological properties strongly affected transfection efficiency. Soft tumors with larger spherical cells, low proteoglycan and collagen content, and low cell density are more effectively transfected than rigid tumors with high proteoglycan and collagen content, small

Electrotransfection in tissue can be improved by modulation of the extracellular matrix, using collagenase and/or hyaluronidase in tumors [121] as well as in muscles, a major target organ

Even if our knowledge on the molecular mechanisms governing the transfer of pDNA due to the delivery of pulsed electric field remains limited, it gives recommandations for an optimal

Pulse generators should provide the largest flexibility in the choice of the electrical parameters (voltage, duration, delay, number, current intensity, sharpness of the pulse onset) and offer an internal monitoring of the delivered pulse. Very few products on the market meet these

The local field on the tissue target is a complex function of the choice of the electrodes and on the electrical changes of the tissue due to the electrical treatment. This last parameter is highly

center region of tumors were macroscopically uniform on ex Vivo slices.

spindle-shaped cells and high cell density [120].

**8. Conclusion**

specifications.

choice of the protocols.

for DNA vaccination, a great topic for gene therapy [122].

**7.2. Extracellular matrix (ECM)**

42 Novel Gene Therapy Approaches

Address all correspondence to: justin.teissie@ipbs.fr

1 CNRS;IPBS (Institut de Pharmacologie et de Biologie Structurale), Toulouse, France

2 Université de Toulouse, Toulouse, France

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**Chapter 3**

**Solid Lipid Nanoparticles:**

Tranum Kaur and Roderick Slavcev

http://dx.doi.org/10.5772/54781

**1. Introduction**

Additional information is available at the end of the chapter

**Tuneable Anti-Cancer Gene/Drug Delivery Systems**

With the advent of multifunctional nano delivery systems, simultaneous imaging and therapy aspires to detect and treat tumors at a very early stage with promising out‐ comes. In this context, numerous anti-cancer drug/gene delivery systems have been ex‐ plored with the primary aim to increase the treatment efficacy without compromising safety. Secondary goals include enhancing bioavailability, specific targeting, apart from the enhanced stability of the formulation [1]. The multifaceted applications of nanoparti‐ cles are the direct result of their ability to deliver high pay loads of drugs or biomarkers to the desired sites within the body. Design and development of tumor specific nanopar‐ ticles could significantly amplify the delivering capacity to a specific target of interest, without affecting healthy cells [2]. Technological advances in nanomaterials and nano‐ technology have paved the way for several carriers such as liposomes [3], dendrimers [4], and micelles [5], solid lipid nanoparticles (SLN) [6] and recently nanostructured lipid carriers [1, 7]. Polymeric micelles, or nanosized (~10–100 nm) supramolecular constructs composed of amphiphilic block-copolymers, are emerging as powerful drug delivery ve‐ hicles for hydrophobic drugs. Liposomes are currently the most popular nanosized drug delivery systems, with one or several lipid bilayers enclosing an aqueous core. Liposomeencapsulated formulations of doxorubicin earlier approved for the treatment of Kaposi's sarcoma, are now used against breast cancer and refractory ovarian cancer. Breast cancer in particular has been the focus of many studies involving liposome-based chemothera‐ peutics, in part due to the clinical success of various drugs such as Doxil, which is a lip‐ osomal formulation currently used to treat recurrent breast cancer [7]. The anthracycline doxorubicin is the active cytotoxic agent and is contained within the internal aqueous core of the liposome. The encapsulation of doxorubicin within liposomes significantly re‐

> © 2013 Kaur and Slavcev; 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 reproduction in any medium, provided the original work is properly cited.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,


## **Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems**

Tranum Kaur and Roderick Slavcev

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54781

### **1. Introduction**

[116] Lee, R. C, River, L. P, Pan, F. S, & Ji, L. Wollmann RL Surfactant-induced sealing of electropermeabilized skeletal muscle membranes in vivo. Proc Natl Acad Sci U S A.

[117] Henshaw, J. W, Zaharoff, D. A, Mossop, B. J, & Yuan, F. A single molecule detection method for understanding mechanisms of electric field-mediated interstitial transport

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[119] Mossop, B. J, Barr, R. C, Henshaw, J. W, & Zaharoff, D. A. YuanF Electric Fields in Tumors Exposed to External Voltage Sources: Implication for Electric Field-Mediated Drug and Gene Delivery Annals of Biomedical Engineering, (2006). C 2006):1564-1572

[120] Mesojednik, S, Pavlin, D, Sersa, G, Coer, A, Kranjc, S, Grosel, A, & Tevz, G. Cemazar M The effect of the histological properties of tumors on transfection efficiency of electrically assisted gene delivery to solid tumors in mice. Gene Ther. (2007). , 14(17),

[121] Cemazar, M, Golzio, M, Sersa, G, Escoffre, J. M, Coer, A, Vidic, S, & Teissie, J. Hyalur‐ onidase and collagenase increase the transfection efficiency of gene electrotransfer in

[122] Mcmahon, J. M, Signori, E, Wells, K. E, Fazio, V. M, & Wells, D. J. Optimisation of electrotransfer of plasmid into skeletal muscle by pretreatment with hyaluronidase- increased expression with reduced muscle damage. Gene Ther. (2001). , 8(16), 1264-70.

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electrotransfer into muscle tissue. Biomed Eng Online. (2010)., 9():66.

various murine tumors. Hum Gene Ther. (2012). , 23(1), 128-37.

electroporation. Phys Med Biol. (2012). , 57(17), 5425-40.

of genes Bioelectrochemistry. (2006). , 69(2), 248-53.

(1992). , 89(10), 4524-8.

52 Novel Gene Therapy Approaches

1261-9.

(2007). , 70(2), 501-7.

With the advent of multifunctional nano delivery systems, simultaneous imaging and therapy aspires to detect and treat tumors at a very early stage with promising out‐ comes. In this context, numerous anti-cancer drug/gene delivery systems have been ex‐ plored with the primary aim to increase the treatment efficacy without compromising safety. Secondary goals include enhancing bioavailability, specific targeting, apart from the enhanced stability of the formulation [1]. The multifaceted applications of nanoparti‐ cles are the direct result of their ability to deliver high pay loads of drugs or biomarkers to the desired sites within the body. Design and development of tumor specific nanopar‐ ticles could significantly amplify the delivering capacity to a specific target of interest, without affecting healthy cells [2]. Technological advances in nanomaterials and nano‐ technology have paved the way for several carriers such as liposomes [3], dendrimers [4], and micelles [5], solid lipid nanoparticles (SLN) [6] and recently nanostructured lipid carriers [1, 7]. Polymeric micelles, or nanosized (~10–100 nm) supramolecular constructs composed of amphiphilic block-copolymers, are emerging as powerful drug delivery ve‐ hicles for hydrophobic drugs. Liposomes are currently the most popular nanosized drug delivery systems, with one or several lipid bilayers enclosing an aqueous core. Liposomeencapsulated formulations of doxorubicin earlier approved for the treatment of Kaposi's sarcoma, are now used against breast cancer and refractory ovarian cancer. Breast cancer in particular has been the focus of many studies involving liposome-based chemothera‐ peutics, in part due to the clinical success of various drugs such as Doxil, which is a lip‐ osomal formulation currently used to treat recurrent breast cancer [7]. The anthracycline doxorubicin is the active cytotoxic agent and is contained within the internal aqueous core of the liposome. The encapsulation of doxorubicin within liposomes significantly re‐

© 2013 Kaur and Slavcev; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

duces the cardiotoxicity that commonly results from the use of unencapsulated anthracy‐ clines by decreasing the amount of the drug being delivered to the heart [7]. As such, patients can receive much higher doses of the chemotherapeutic in the liposomal formu‐ lation compared to unencapsulated, thereby allowing tumor tissue to potentially be ex‐ posed to a lethal dose of the drug while minimizing deleterious side effects. This inherent advantage associated with the use of liposomes as drug delivery vehicles also serves to minimize the many other toxic side effects associated with doxorubicin includ‐ ing gastrointestinal toxicity and complications arising from myelosuppression.

**2. Solid lipid nanoparticles**

**Figure 1.** Schematics of Functionalized Solid Lipid Nanoparticles

Solid lipid nanoparticles [17] or lipospheres are rapidly emerging as new class of safer and efficient gene/drug delivery vectors. SLNs are sub-micron colloidal carriers, ranging from 50 nm to 1 μm, that are composed of physiological lipid dispersed in water or in aqueous surfactant solution (Figure 1). SLNs function as an alternative drug carrier system to other novel delivery approaches such as emulsions, liposomes, and polymeric nanoparticles. SLNs offer several advantages conferred by their colloidal dimensions including: i) feasibility of incorporation of lipophilic and hydrophilic drugs; ii) improved physical stability; iii) control‐ led release; iv) improved biocompatibility; v) potential for site specific drug delivery; vi) improved drug stability; vii) better formulation stability; viii) the ability to freeze dry and reconstitute; ix) high drug payload; x) controllable particle size; xi) the avoidance of carrier toxicity; xii) low production cost; and xiii) easy scale-up and manufacturing [18]. In addition, significant toxicity and acidity associated with a number of biodegradable polymeric materials are not observed with SLNs. And, in contrast to emulsions and liposomes, the particle matrix of SLNs is composed of solid lipids. SLNs can be prepared using wide variety of lipids including lipid acids, mono- (glycerol monostearate), di- (glycerol bahenate) or triglycerides (tristearin), glyceride mixtures or waxes (e.g. cetyl palmitate) and stabilized by the biocom‐ patible surfactants(s) of choice (non-ionic or ionic). Lipids most commonly used are triglycer‐ ide esters of hydrogenated fatty acids, including hydrogenated cottonseed oil (Lubritab™ or Sterotex™), hydrogenated palm oil (Dynasan™ P60 or Softisan™ 154), hydrogenated castor oil (Cutina™ HR), and hydrogenated soybean oil (Sterotex™ HM, or Lipo™) as typical examples [19]. Various emulsifiers and their combination (Pluronic F 68, F 127) have also been added to stabilize the lipid dispersion by more efficiently preventing particle agglomeration.

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55

Surface Functionalization

Surfactant Layer

Drug

The disavantages associated with SLNs relate mostly to their preparation, which generally involves high pressure and rapid temperature changes that can lead to high pressure-induced

Solid Core

Each delivery system however, has its advantages and limitations. Advantages afforded for drug delivery include the presence of an inner core for lipophilic drug entrapment, as well as a hydrophilic outer shell that prevents particle aggregation and opsonisation [8]. This complexation prevents uptake by the reticuloendothelial system (RES), thereby improving circulation times which, combined with nanoscale sizing, confers preferential accumulation in tumor tissue. In general, nanovectors can be targeted to tumors by pas‐ sive and active targeting approaches, where a passive strategy takes advantage of a nan‐ vector's small size permitting it to penetrate and accumulate in the tumor. Most solid tumors are sustained by extensive angiogenesis leading to hypervascular tissue with an incomplete vascular architecture. They also have an impaired lymphatic drainage and an increased production of permeability factors resulting in the accumulation and inefficient clearance of nanoparticles leading to the enhanced permeability and retention effect [9]. The hyperpermeable nature of tumor vasculature is characterized by a pore cut off size ranging between 380 and 780 nm allowing particles less than 780 nm to extravasate into the tumor interstitium [10]. In addition, active targeting to various tissues may be ach‐ ieved *via* utilization of ligands on the surface of nanoparticles, reducing the side effects to the normal tissue by limiting drug/gene distribution to the target organ [11]. An excel‐ lent example is Abraxane, an albumin bound nanoparticle formulation of Paclitaxel (PTX), approved by FDA in January 2005 for the treatment of metastatic breast cancer. Abraxane has been shown to outperform standard PTX in the treatment of breast cancer [12]. Utility of this drug was initially limited due to its poor aqueous solubility [13], re‐ quiring use of an excipient, Cremophor, which is satisfied by novel engineered nanovec‐ tors. A recent Gynecologic Oncology Group Phase II evaluation of albumin-bound paclitaxel nanoparticles to treat recurrent or persistent platinum-resistant ovarian, fallopi‐ an tube, or primary peritoneal cancer, concluded that these nanoparticles are as effective and tolerable in their cohort of refractory ovarian cancer patients previously treated with paclitaxel [14]. Nanoparticles fabricated with albumin [15], poly(lactic-co-glycolic acid) [16] and poly lactic acid have also been loaded with PTX and used to passively target tumors. Albumin has been shown to be nontoxic, non-immunogenic, biocompatible and biodegradable making it an ideal candidate to fabricate nanoparticles for drug delivery. Site-specific drug delivery allows for the clinical translation of chemotherapeutic agents with safer targeted cell killing, that are otherwise abandoned due to insolubility, toxicity and safety concerns. Moreover, these new delivery devices can preferentially confine treatments to tumors within the nodal space while sparing healthy tissues.

## **2. Solid lipid nanoparticles**

duces the cardiotoxicity that commonly results from the use of unencapsulated anthracy‐ clines by decreasing the amount of the drug being delivered to the heart [7]. As such, patients can receive much higher doses of the chemotherapeutic in the liposomal formu‐ lation compared to unencapsulated, thereby allowing tumor tissue to potentially be ex‐ posed to a lethal dose of the drug while minimizing deleterious side effects. This inherent advantage associated with the use of liposomes as drug delivery vehicles also serves to minimize the many other toxic side effects associated with doxorubicin includ‐

Each delivery system however, has its advantages and limitations. Advantages afforded for drug delivery include the presence of an inner core for lipophilic drug entrapment, as well as a hydrophilic outer shell that prevents particle aggregation and opsonisation [8]. This complexation prevents uptake by the reticuloendothelial system (RES), thereby improving circulation times which, combined with nanoscale sizing, confers preferential accumulation in tumor tissue. In general, nanovectors can be targeted to tumors by pas‐ sive and active targeting approaches, where a passive strategy takes advantage of a nan‐ vector's small size permitting it to penetrate and accumulate in the tumor. Most solid tumors are sustained by extensive angiogenesis leading to hypervascular tissue with an incomplete vascular architecture. They also have an impaired lymphatic drainage and an increased production of permeability factors resulting in the accumulation and inefficient clearance of nanoparticles leading to the enhanced permeability and retention effect [9]. The hyperpermeable nature of tumor vasculature is characterized by a pore cut off size ranging between 380 and 780 nm allowing particles less than 780 nm to extravasate into the tumor interstitium [10]. In addition, active targeting to various tissues may be ach‐ ieved *via* utilization of ligands on the surface of nanoparticles, reducing the side effects to the normal tissue by limiting drug/gene distribution to the target organ [11]. An excel‐ lent example is Abraxane, an albumin bound nanoparticle formulation of Paclitaxel (PTX), approved by FDA in January 2005 for the treatment of metastatic breast cancer. Abraxane has been shown to outperform standard PTX in the treatment of breast cancer [12]. Utility of this drug was initially limited due to its poor aqueous solubility [13], re‐ quiring use of an excipient, Cremophor, which is satisfied by novel engineered nanovec‐ tors. A recent Gynecologic Oncology Group Phase II evaluation of albumin-bound paclitaxel nanoparticles to treat recurrent or persistent platinum-resistant ovarian, fallopi‐ an tube, or primary peritoneal cancer, concluded that these nanoparticles are as effective and tolerable in their cohort of refractory ovarian cancer patients previously treated with paclitaxel [14]. Nanoparticles fabricated with albumin [15], poly(lactic-co-glycolic acid) [16] and poly lactic acid have also been loaded with PTX and used to passively target tumors. Albumin has been shown to be nontoxic, non-immunogenic, biocompatible and biodegradable making it an ideal candidate to fabricate nanoparticles for drug delivery. Site-specific drug delivery allows for the clinical translation of chemotherapeutic agents with safer targeted cell killing, that are otherwise abandoned due to insolubility, toxicity and safety concerns. Moreover, these new delivery devices can preferentially confine

ing gastrointestinal toxicity and complications arising from myelosuppression.

54 Novel Gene Therapy Approaches

treatments to tumors within the nodal space while sparing healthy tissues.

Solid lipid nanoparticles [17] or lipospheres are rapidly emerging as new class of safer and efficient gene/drug delivery vectors. SLNs are sub-micron colloidal carriers, ranging from 50 nm to 1 μm, that are composed of physiological lipid dispersed in water or in aqueous surfactant solution (Figure 1). SLNs function as an alternative drug carrier system to other novel delivery approaches such as emulsions, liposomes, and polymeric nanoparticles. SLNs offer several advantages conferred by their colloidal dimensions including: i) feasibility of incorporation of lipophilic and hydrophilic drugs; ii) improved physical stability; iii) control‐ led release; iv) improved biocompatibility; v) potential for site specific drug delivery; vi) improved drug stability; vii) better formulation stability; viii) the ability to freeze dry and reconstitute; ix) high drug payload; x) controllable particle size; xi) the avoidance of carrier toxicity; xii) low production cost; and xiii) easy scale-up and manufacturing [18]. In addition, significant toxicity and acidity associated with a number of biodegradable polymeric materials are not observed with SLNs. And, in contrast to emulsions and liposomes, the particle matrix of SLNs is composed of solid lipids. SLNs can be prepared using wide variety of lipids including lipid acids, mono- (glycerol monostearate), di- (glycerol bahenate) or triglycerides (tristearin), glyceride mixtures or waxes (e.g. cetyl palmitate) and stabilized by the biocom‐ patible surfactants(s) of choice (non-ionic or ionic). Lipids most commonly used are triglycer‐ ide esters of hydrogenated fatty acids, including hydrogenated cottonseed oil (Lubritab™ or Sterotex™), hydrogenated palm oil (Dynasan™ P60 or Softisan™ 154), hydrogenated castor oil (Cutina™ HR), and hydrogenated soybean oil (Sterotex™ HM, or Lipo™) as typical examples [19]. Various emulsifiers and their combination (Pluronic F 68, F 127) have also been added to stabilize the lipid dispersion by more efficiently preventing particle agglomeration.

**Figure 1.** Schematics of Functionalized Solid Lipid Nanoparticles

The disavantages associated with SLNs relate mostly to their preparation, which generally involves high pressure and rapid temperature changes that can lead to high pressure-induced drug degradation, lipid crystallization, gelation phenomena and the co-existence of several colloidal species [20]. The drug loading capacity of a conventional SLN is limited by the solubility of drug in the lipid melt, the structure of the lipid matrix and the polymeric state of the lipid matrix. If the lipid matrix consists of highly similar molecules (i.e. tristearin or tripalmitin), a perfect crystal with few imperfections is formed. Since incorporated drugs are located between fatty acid chains, between the lipid layers and also in crystal imperfections, a highly ordered crystal lattice cannot accommodate large amounts of drug. This may also lead to the fast release of a large dose of drug initially, generally known as "burst effect", followed by slow and incomplete release of drug. Since high lipid crystallinity is the major cause of burst release of drug from SLNs, this undesirable phenomenon may be minimized by choosing lipids that do not form good crystals, including mono- or di-glycerides, or triglycerides with chains of different lengths. For this reason, in formulation design use of more complex lipids is recommended for higher drug loading. Nanostructured lipid carriers or NLCs were designed to overcome these disadvantages with the main goal to increase drug loading and prevent drug expulsion [21]. For NLCs, the highest drug load could be achieved by mixing solid lipids with small amounts of liquid lipids (oils). These types of NLCs are called multiple types NLC, and are analogous to w/o/w emulsions since it is an oil-in-solid lipid-in-water dispersion.

micrometers. HPH of the pre-emulsion is carried out at temperatures above the melting point of the lipid. Usually, lower particle sizes are obtained at higher processing temperatures

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Hot homogenisation is the most frequently applied technique in which even temperature sensitive compounds can be processed because of the short exposure time to the elevated temperatures [25]. However, high temperatures increase the degradation rate of the drug and the carrier. Increasing the homogenization pressure or the number of cycles often results in an increase of the particle size due to high kinetic energy of the particles. The cold homogenisation technique is therefore recommended for extremely temperature sensitive compounds and hydrophilic compounds, which might partition from the liquid lipid phase to the water phase

During cold homogenization, the drug containing lipid melt is cooled and, after solidifica‐ tion, the lipidic mass is ground to yield lipid microparticles [26]. The lipid microparticles are dispersed in cold surfactant solution by stirring, yielding a macro-suspension. This suspen‐ sion is then passed through a high-pressure homogeniser at or below room temperature, where the microparticles are broken down to solid lipid nanoparticles. However, compared to hot homogenization, larger particle sizes and a broader size distribution are typical of

SLNs are also developed by high speed stirring or sonication [27]. The ultrasonic dispersion may offer an appropriate alternative for laboratory scale productions due to its rapid nature and the relatively low cost of required apparatus. So far, its suitability has only been evaluated for SLN [17, 28]. The primary disadvantage of this method is the broader particle size distribution that is yielded, ranging into the micrometer range. Potential metal contamination due to ultra‐ sonication is another issue presented by this method. To generate more stable formulations, high speed stirring and ultrasonication may be used in combination at high temperature.

In this method, the lipidic material, such as glyceride is dissolved in an organic solvent (e.g. chloroform, cyclohexane) and the solution is emulsified in an aqueous phase [29]. After evaporation of the solvent the lipid precipitates to form nanoparticles with a mean diameter of around 30 nm using cholesterol acetate as a model drug and lecithin/sodium glycocholate blend as an emulsifier [30]. The solution is emulsified in an aqueous phase by high pressure homogenization and the organic solvent is removed from the emulsion by evaporation un‐

This platform technology, with several variations for powder and nanoparticle preparation, is a relatively new technique for SLN production and offers the advantage of solvent-less

because of lowered viscosity of the lipid phase [24].

during the hot homogenisation.

*3.1.2. Cold homogenization*

cold homogenized samples.

*3.1.3. Ultrasonication or high speed homogenization*

**3.2. Solvent emulsification/evaporation**

der reduced pressure (40–60 mbar).

**3.3. Supercritical fluid**

## **3. SLN preparation methods**

There are two main established SLN synthesis techniques, namely, the high-pressure homog‐ enisation technique described by Müller and Lucks [21], and the microemulsion-based technique described by Gasco [22, 23]. SLNs are prepared from lipid, emulsifier and water/ solvent using different methods, discussed below.

#### **3.1. High Pressure Homogenization (HPH)**

High Pressure Homogenization (HPH) is a very reliable technique in the production of SLNs. High pressure homogenizers are employed to push a liquid with high pressure (100–2000 bar) and the fluid accelerates on a very short distance to very high velocity (>1000 Km/h).. Very high shear stress and cavitation forces disrupt the particles down to the submicron range. Generally 5-10% lipid content is used but up to 40% lipid content has also been investigated. Typical SLNs production conditions are 500 bar and two or three homogenisation cycles. Two general approaches of HPH are hot and cold homogenization, both working on the same concept of mixing the drug in bulk of lipid melt.

#### *3.1.1. Hot homogenization*

Hot homogenization is carried out at temperatures above the melting point of the lipid and can therefore be regarded as the homogenization of an emulsion (Figure 2). A pre-emulsion of the drug loaded lipid melt and the aqueous emulsifier phase (same temperature) is obtained by high-shear mixing device. The quality of the pre-emulsion affects the quality of the final product to a great extent and it is desirable to obtain droplets in the size range of a few micrometers. HPH of the pre-emulsion is carried out at temperatures above the melting point of the lipid. Usually, lower particle sizes are obtained at higher processing temperatures because of lowered viscosity of the lipid phase [24].

Hot homogenisation is the most frequently applied technique in which even temperature sensitive compounds can be processed because of the short exposure time to the elevated temperatures [25]. However, high temperatures increase the degradation rate of the drug and the carrier. Increasing the homogenization pressure or the number of cycles often results in an increase of the particle size due to high kinetic energy of the particles. The cold homogenisation technique is therefore recommended for extremely temperature sensitive compounds and hydrophilic compounds, which might partition from the liquid lipid phase to the water phase during the hot homogenisation.

#### *3.1.2. Cold homogenization*

drug degradation, lipid crystallization, gelation phenomena and the co-existence of several colloidal species [20]. The drug loading capacity of a conventional SLN is limited by the solubility of drug in the lipid melt, the structure of the lipid matrix and the polymeric state of the lipid matrix. If the lipid matrix consists of highly similar molecules (i.e. tristearin or tripalmitin), a perfect crystal with few imperfections is formed. Since incorporated drugs are located between fatty acid chains, between the lipid layers and also in crystal imperfections, a highly ordered crystal lattice cannot accommodate large amounts of drug. This may also lead to the fast release of a large dose of drug initially, generally known as "burst effect", followed by slow and incomplete release of drug. Since high lipid crystallinity is the major cause of burst release of drug from SLNs, this undesirable phenomenon may be minimized by choosing lipids that do not form good crystals, including mono- or di-glycerides, or triglycerides with chains of different lengths. For this reason, in formulation design use of more complex lipids is recommended for higher drug loading. Nanostructured lipid carriers or NLCs were designed to overcome these disadvantages with the main goal to increase drug loading and prevent drug expulsion [21]. For NLCs, the highest drug load could be achieved by mixing solid lipids with small amounts of liquid lipids (oils). These types of NLCs are called multiple types NLC, and are analogous to w/o/w emulsions since it is an oil-in-solid lipid-in-water dispersion.

There are two main established SLN synthesis techniques, namely, the high-pressure homog‐ enisation technique described by Müller and Lucks [21], and the microemulsion-based technique described by Gasco [22, 23]. SLNs are prepared from lipid, emulsifier and water/

High Pressure Homogenization (HPH) is a very reliable technique in the production of SLNs. High pressure homogenizers are employed to push a liquid with high pressure (100–2000 bar) and the fluid accelerates on a very short distance to very high velocity (>1000 Km/h).. Very high shear stress and cavitation forces disrupt the particles down to the submicron range. Generally 5-10% lipid content is used but up to 40% lipid content has also been investigated. Typical SLNs production conditions are 500 bar and two or three homogenisation cycles. Two general approaches of HPH are hot and cold homogenization, both working on the same

Hot homogenization is carried out at temperatures above the melting point of the lipid and can therefore be regarded as the homogenization of an emulsion (Figure 2). A pre-emulsion of the drug loaded lipid melt and the aqueous emulsifier phase (same temperature) is obtained by high-shear mixing device. The quality of the pre-emulsion affects the quality of the final product to a great extent and it is desirable to obtain droplets in the size range of a few

**3. SLN preparation methods**

56 Novel Gene Therapy Approaches

solvent using different methods, discussed below.

concept of mixing the drug in bulk of lipid melt.

*3.1.1. Hot homogenization*

**3.1. High Pressure Homogenization (HPH)**

During cold homogenization, the drug containing lipid melt is cooled and, after solidifica‐ tion, the lipidic mass is ground to yield lipid microparticles [26]. The lipid microparticles are dispersed in cold surfactant solution by stirring, yielding a macro-suspension. This suspen‐ sion is then passed through a high-pressure homogeniser at or below room temperature, where the microparticles are broken down to solid lipid nanoparticles. However, compared to hot homogenization, larger particle sizes and a broader size distribution are typical of cold homogenized samples.

### *3.1.3. Ultrasonication or high speed homogenization*

SLNs are also developed by high speed stirring or sonication [27]. The ultrasonic dispersion may offer an appropriate alternative for laboratory scale productions due to its rapid nature and the relatively low cost of required apparatus. So far, its suitability has only been evaluated for SLN [17, 28]. The primary disadvantage of this method is the broader particle size distribution that is yielded, ranging into the micrometer range. Potential metal contamination due to ultra‐ sonication is another issue presented by this method. To generate more stable formulations, high speed stirring and ultrasonication may be used in combination at high temperature.

#### **3.2. Solvent emulsification/evaporation**

In this method, the lipidic material, such as glyceride is dissolved in an organic solvent (e.g. chloroform, cyclohexane) and the solution is emulsified in an aqueous phase [29]. After evaporation of the solvent the lipid precipitates to form nanoparticles with a mean diameter of around 30 nm using cholesterol acetate as a model drug and lecithin/sodium glycocholate blend as an emulsifier [30]. The solution is emulsified in an aqueous phase by high pressure homogenization and the organic solvent is removed from the emulsion by evaporation un‐ der reduced pressure (40–60 mbar).

### **3.3. Supercritical fluid**

This platform technology, with several variations for powder and nanoparticle preparation, is a relatively new technique for SLN production and offers the advantage of solvent-less processing [31]. SLNs can be prepared by the rapid expansion of supercritical carbon dioxide solutions (RESS) method, where carbon dioxide (99.99%) is a good choice as solvent.

**4.1. Spray drying method**

mixtures (10/90 v/v).

**4.2. Double emulsion method**

es is however, clathrin-dependent [38].

Spray drying is an alternative procedure to lyophilization in the transformation of an aqueous SLN dispersion into a solid drug product. This method results in particle aggregation due to high temperature, shear forces and partial melting of the particle. The use of lipid with melting point >70°C for spray drying is recommended [33]. Best results are obtained with an SLN concentration of 1% in a solution of trehalose in water or 20% trehalose in ethanol-water

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For the preparation of hydrophilic loaded SLN, double emulsion method, a novel approach based on solvent emulsification-evaporation can be employed. Here, the drug is encapsulated with a stabilizer to prevent drug partitioning to external water phase during solvent evapo‐

Research on cellular uptake mechanisms has repeatedly demonstrated that endocytosis is the preferred route of internalization of non-viral gene vectors via a number of distinct endocytic processes. The most studied mechanisms include macropinocytosis, circular dorsal ruffles, clathrin-mediated endocytosis and several clathrin-independent endocytic pathways [35]. Endocytic uptake mechanisms are highly dependent on cell type and on the nature of gene vectors [36]. Clathrin-mediated processes are limited to particles un‐ der 200 nm in size, whereas caveolae-dependent uptake prevails for particles between 200 and 500 nm [37]. The prevalent pathway for the cell internalization of PEI polyplex‐

Apart from overcoming cellular barriers of uptake, an **anticancer** drug must be specifi‐ cally targeted to the tumor in order to maximize its therapeutic effect, and therefore biodistribution studies are critical to assess the safety of a nanomedicine. However, since most groups work on healthy instead of tumor-bearing animals, it is difficult to confirm whether SLNs can lead to increased tumor drug concentrations by way of en‐ hanced permeability and retention [9]. Recently, Zhang *et al.* (2010) evaluated antitu‐ mor efficacy of docetaxel-loaded **solid lipid nanoparticles** (DSN) in a murine ovarian **cancer** model [39]. In this study, SLN biodistribution from RES more toward the circu‐ lation system was observed. Moreover, SLNs in comparison to the free drug demon‐ strated more potent *in vivo* anti-ovarian **cancer** activity with improved pharmacokinetics. In contrast, paclitaxel loaded in pegylated **solid lipid** nanoparticles were mainly taken up by the RES after intravenous administration in rats, showing 8 fold and 3-fold higher levels in liver and spleen, respectively, 8 h after administration compared to paclitaxel in Taxol® [40]. Moreover, paclitaxel levels in kidney, heart and lung were indistinguishable between the two formulations. The difference in biodistri‐

ration in the external water phase of w/o/w double emulsion [34]

**5. SLNs cellular uptake, pharmacokinetics and bio-distribution**

## **4. Microemulsion method**

This method is based on the dilution of microemulsions that are two-phase systems composed of an inner and outer phase (e.g. o/w microemulsions) [32]. They are made by stirring an optical‐ ly transparent mixture at 65-70°C, which typically composed of a low melting fatty acid (e.g. stearic acid), an emulsifier (e.g. polysorbate 20), co-emulsifiers (e.g. butanol) and water. The hot microemulsion is dispersed in cold water (2-3°C) with stirring. SLN dispersion can be used as granulation fluid for transferring into solid product (tablets, pellets) by granulation process, but in case of low particle content, excess water must first be removed. High-temperature gradients facilitate rapid lipid crystallization and prevent aggregation. Due to the dilution step, achieva‐ ble lipid contents are considerably lower compared with the HPH based formulations.

**Figure 2.** Partitioning effects on drug during the hot homogenization technique production of SLNs. *Left:* Partitioning of drug from the lipid phase to the water phase at increased temperature. *Right:* Re-partitioning of the drug to the lipid phase during cooling of the produced O/W nanoemulsion. Source: Muller RH et al. Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. European Journal of Pharmaceutics and Biopharmaceu‐ tics 50: (2000) 161-177.

#### **4.1. Spray drying method**

processing [31]. SLNs can be prepared by the rapid expansion of supercritical carbon dioxide

This method is based on the dilution of microemulsions that are two-phase systems composed of an inner and outer phase (e.g. o/w microemulsions) [32]. They are made by stirring an optical‐ ly transparent mixture at 65-70°C, which typically composed of a low melting fatty acid (e.g. stearic acid), an emulsifier (e.g. polysorbate 20), co-emulsifiers (e.g. butanol) and water. The hot microemulsion is dispersed in cold water (2-3°C) with stirring. SLN dispersion can be used as granulation fluid for transferring into solid product (tablets, pellets) by granulation process, but in case of low particle content, excess water must first be removed. High-temperature gradients facilitate rapid lipid crystallization and prevent aggregation. Due to the dilution step, achieva‐

solutions (RESS) method, where carbon dioxide (99.99%) is a good choice as solvent.

ble lipid contents are considerably lower compared with the HPH based formulations.

**Figure 2.** Partitioning effects on drug during the hot homogenization technique production of SLNs. *Left:* Partitioning of drug from the lipid phase to the water phase at increased temperature. *Right:* Re-partitioning of the drug to the lipid phase during cooling of the produced O/W nanoemulsion. Source: Muller RH et al. Solid lipid nanoparticles (SLN) for controlled drug delivery - a review of the state of the art. European Journal of Pharmaceutics and Biopharmaceu‐

**4. Microemulsion method**

58 Novel Gene Therapy Approaches

tics 50: (2000) 161-177.

Spray drying is an alternative procedure to lyophilization in the transformation of an aqueous SLN dispersion into a solid drug product. This method results in particle aggregation due to high temperature, shear forces and partial melting of the particle. The use of lipid with melting point >70°C for spray drying is recommended [33]. Best results are obtained with an SLN concentration of 1% in a solution of trehalose in water or 20% trehalose in ethanol-water mixtures (10/90 v/v).

#### **4.2. Double emulsion method**

For the preparation of hydrophilic loaded SLN, double emulsion method, a novel approach based on solvent emulsification-evaporation can be employed. Here, the drug is encapsulated with a stabilizer to prevent drug partitioning to external water phase during solvent evapo‐ ration in the external water phase of w/o/w double emulsion [34]

## **5. SLNs cellular uptake, pharmacokinetics and bio-distribution**

Research on cellular uptake mechanisms has repeatedly demonstrated that endocytosis is the preferred route of internalization of non-viral gene vectors via a number of distinct endocytic processes. The most studied mechanisms include macropinocytosis, circular dorsal ruffles, clathrin-mediated endocytosis and several clathrin-independent endocytic pathways [35]. Endocytic uptake mechanisms are highly dependent on cell type and on the nature of gene vectors [36]. Clathrin-mediated processes are limited to particles un‐ der 200 nm in size, whereas caveolae-dependent uptake prevails for particles between 200 and 500 nm [37]. The prevalent pathway for the cell internalization of PEI polyplex‐ es is however, clathrin-dependent [38].

Apart from overcoming cellular barriers of uptake, an **anticancer** drug must be specifi‐ cally targeted to the tumor in order to maximize its therapeutic effect, and therefore biodistribution studies are critical to assess the safety of a nanomedicine. However, since most groups work on healthy instead of tumor-bearing animals, it is difficult to confirm whether SLNs can lead to increased tumor drug concentrations by way of en‐ hanced permeability and retention [9]. Recently, Zhang *et al.* (2010) evaluated antitu‐ mor efficacy of docetaxel-loaded **solid lipid nanoparticles** (DSN) in a murine ovarian **cancer** model [39]. In this study, SLN biodistribution from RES more toward the circu‐ lation system was observed. Moreover, SLNs in comparison to the free drug demon‐ strated more potent *in vivo* anti-ovarian **cancer** activity with improved pharmacokinetics. In contrast, paclitaxel loaded in pegylated **solid lipid** nanoparticles were mainly taken up by the RES after intravenous administration in rats, showing 8 fold and 3-fold higher levels in liver and spleen, respectively, 8 h after administration compared to paclitaxel in Taxol® [40]. Moreover, paclitaxel levels in kidney, heart and lung were indistinguishable between the two formulations. The difference in biodistri‐ bution of SLNs reported in literature may be due to several factors including varia‐ tions in size, surface functionalization and composition.

**SLN Composition Characterization Indication Drug/Gene Reference**

Poloxamer 188 and Tween 80 Size, Zeta potential Breast Cancer Emodin [54]

Size, Zeta Potential Ovarian Cancer

Size, Zeta Potential, Differential Scanning Calorimetry

microscopy

Size, Zeta Potential, Transmission Electron Microscopy

Zeta potential and Gel

Size, Zeta Potential

Size, Zeta Potential Prostate Cancer Plasmid DNA [s51]

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Size, Zeta Potential Breast Cancer Tryptanthrin [55]

retardation Epithelial Cancer Paclitaxel and siRNA [58]

Brain Cancer (glioblastomas)

Stearic acid, Glyceryl behenate Size, Zeta Potential Skin Cancer Doxorubicin [60]

**Table 1.** Solid Lipid Nanoparticles loaded with DNA/ Drug as anti-cancer delivery systems in various cancers.

Breast Cancer

Lung Cancer Plasmid DNA [52]

Lung Cancer Phospho-Sulindac [53]

Doxorubicin and Mitomycin -C

Doxorubicin and mixedbackbone GCS antisense oligonucleotides (MBO-asGCS)

c-Met siRNA [59]

[56]

[57]

Stearic acid, DOTAP, Pluronic F68

dioleoylphosphatidylethanolami

Stearic acid, Lecithin and PS Transmission electron

Tricaprin as a core, 3beta[Ncarbamoyl] cholesterol (DC-Chol), DOPE and Tween 80

Precirol, Compritol, soybean Phosphatidylcholine, Tween 80

Myristic acid, Stearic acid, Palmitic acid, lauric acid, poly(ethylene glycol)-100 stearate (PEG100SA),

block copolymer)

Stearyl alcohol and cetyltrimethylammonium bromide (CTAB), Ceramide VI,

polysorbate 60

2000-distearoyl

Chol

phosphatidylethanolamine (mPEG-DSPE), glyceryl trioleate.

Cholesteryl oleate, glyceryl trioleate, DOPE, Chol , and DC-

1,2-Dioleoyl-sn-glycero-3 ethylphosphocholine 1,2 diphytanoyl-sn-glycero-3 phosphatidylethanolamine (DPhPE), 3â[N-(N′,N′ dimethylaminoethane) carbamoyl] cholesterol (DC-Chol), 1,2-Dioleoyl-sn-glycero-3 ethylphosphocholine (EDOPC), and methoxypolyethylene glycol

poly(ethylene glycol)-40-stearate (PEG40SA), Hydrolyzed polymer of epoxidized soybean oil, Pluronic F68 (PF68) (non-ionic

and

ne (DOPE)

The biodistribution of an anticancer drug delivered by SLN may be further man‐ oeuvred by route of injection to achieve the desired therapeutic goal. Harivardhan Reddy *et al.* (2005) compared the biodistribution of free 99 mTechnetium-labeled etopo‐ side and radio-labeled etoposide loaded SLNs in Dalton's lymphoma tumor-bearing mice [41]. They showed that administration via the subcutaneous route resulted in high tumor uptake of etoposide and etoposide loaded tripalmitin nanoparticles and was the preferred route as compared to intravenous or intraperitoneal administration. However, elevated tumor drug concentrations were also found with intravenously ad‐ ministered etoposide loaded SLN in comparison to the free drug, (approximately 67% increase 1 h post-injection, 30% increase 24 h post-injection). In yet another study by Zara *et al.* (2002) duodenal administration of idarubicin-loaded SLN led to higher bioa‐ vailability than intravenously administered SLNs [42]. Also, idarubicin and its main metabolite, idarubicinol, were detected in the brain after IDA-SLN administration, indi‐ cating that the SLNs were able to pass the blood-brain barrier; an attractive attribute in the tratement of brain tumors. Thus, the route of administration of SLN formulation is a key consideration in the design of animal or clinical anti-cancer drug delivery studies.

## **6. SLNs as anti-cancer gene/drug delivery vectors: Challenges and successes**

Solid lipid nanoparticles have rapidly established themselves during the past decade as stable, reliable and easy to produce vectors. SLN advantages over other existing trans‐ fection vectors include safety, good storage stability, possibility of lyophilization and a high degree of flexibility in design and optimization [25]. Cationic SLNs can efficiently bind DNA directly *via* ionic interaction and mediate gene transfection. However, as with all non-viral vectors, many cellular obstacles have to be overcome to achieve sat‐ isfactory levels of transfection activity: i) binding to the cell surface; ii) cellular inter‐ nalization; iii) escape from the endolysosomal compartment; and iv) translocation through the nuclear envelope. In order to surmount these barriers, cationic SLNs are designed as multifunctional "smart" carriers for efficient gene expression [43]. Compo‐ nents such as chitosan [44] and surface functionalization moieties e.g. poly(styrene-4 sodium sulfonate) (PSS) and poly(L-lysine hydrochloride) (PLL) [45], folate–chitosan and cholesterol derivative (CHETA) [46], cetyltrimethyl ammonium bromide (CTAB) [47] and a phyto-ceramide [48] and TAT peptides [49], may each individually assist in overcoming the barriers of efficient transfection. In addition, protamine a cationic small protein rich in arginine exerts both DNA condensation activity and proton sponge ef‐ fect facilitating endosomal escape as well as assisting nanovectors to enter the nucleus owing to its nuclear localization signal (NLS) [50]. Table 1 lists some of the successful SLN formulations evaluated as anti-cancer agents in various cancers.


bution of SLNs reported in literature may be due to several factors including varia‐

The biodistribution of an anticancer drug delivered by SLN may be further man‐ oeuvred by route of injection to achieve the desired therapeutic goal. Harivardhan Reddy *et al.* (2005) compared the biodistribution of free 99 mTechnetium-labeled etopo‐ side and radio-labeled etoposide loaded SLNs in Dalton's lymphoma tumor-bearing mice [41]. They showed that administration via the subcutaneous route resulted in high tumor uptake of etoposide and etoposide loaded tripalmitin nanoparticles and was the preferred route as compared to intravenous or intraperitoneal administration. However, elevated tumor drug concentrations were also found with intravenously ad‐ ministered etoposide loaded SLN in comparison to the free drug, (approximately 67% increase 1 h post-injection, 30% increase 24 h post-injection). In yet another study by Zara *et al.* (2002) duodenal administration of idarubicin-loaded SLN led to higher bioa‐ vailability than intravenously administered SLNs [42]. Also, idarubicin and its main metabolite, idarubicinol, were detected in the brain after IDA-SLN administration, indi‐ cating that the SLNs were able to pass the blood-brain barrier; an attractive attribute in the tratement of brain tumors. Thus, the route of administration of SLN formulation is a key consideration in the design of animal or clinical anti-cancer drug delivery

**6. SLNs as anti-cancer gene/drug delivery vectors: Challenges and**

SLN formulations evaluated as anti-cancer agents in various cancers.

Solid lipid nanoparticles have rapidly established themselves during the past decade as stable, reliable and easy to produce vectors. SLN advantages over other existing trans‐ fection vectors include safety, good storage stability, possibility of lyophilization and a high degree of flexibility in design and optimization [25]. Cationic SLNs can efficiently bind DNA directly *via* ionic interaction and mediate gene transfection. However, as with all non-viral vectors, many cellular obstacles have to be overcome to achieve sat‐ isfactory levels of transfection activity: i) binding to the cell surface; ii) cellular inter‐ nalization; iii) escape from the endolysosomal compartment; and iv) translocation through the nuclear envelope. In order to surmount these barriers, cationic SLNs are designed as multifunctional "smart" carriers for efficient gene expression [43]. Compo‐ nents such as chitosan [44] and surface functionalization moieties e.g. poly(styrene-4 sodium sulfonate) (PSS) and poly(L-lysine hydrochloride) (PLL) [45], folate–chitosan and cholesterol derivative (CHETA) [46], cetyltrimethyl ammonium bromide (CTAB) [47] and a phyto-ceramide [48] and TAT peptides [49], may each individually assist in overcoming the barriers of efficient transfection. In addition, protamine a cationic small protein rich in arginine exerts both DNA condensation activity and proton sponge ef‐ fect facilitating endosomal escape as well as assisting nanovectors to enter the nucleus owing to its nuclear localization signal (NLS) [50]. Table 1 lists some of the successful

tions in size, surface functionalization and composition.

studies.

60 Novel Gene Therapy Approaches

**successes**

**Table 1.** Solid Lipid Nanoparticles loaded with DNA/ Drug as anti-cancer delivery systems in various cancers.

Nanovectors offer the potential to both detect and treat cancer at a very early stage, thereby maximizing survival rates. The NCI (National Cancer Institute) Alliance for Nanotechnology in Cancer provides up-to-date information in nano-cancer research and its promise for cancer diagnosis and treatment (http://nano.cancer.gov/). Using siRNA molecules loaded in nano‐ vectors, early proof-of-principle experiments in various tumor cells suggest that RNA silencing may have great potential as a strategy for treating cancer. However, siRNA therapeutics are hindered by poor intracellular uptake, limited blood stability and undesirable non-specific immune stimulation [61]. An interesting strategy used to target the vector employs threeamino-acid peptide, arginine-glycine-aspartic acid (known by its amino acid code RGD) that binds to integrins, which in turn are involved in angiogenesis, tumor cell growth, metastasis, and inflammation. Intravenous administration into tumor-bearing mice of nanoparticles combined with a dual strategy of siRNA inhibiting vascular endothelial growth factor receptor-2 and RGD peptide ligand attached at the distal end of the polyethylene glycol [40], conferred selective tumor uptake, and inhibition of both tumor angiogenesis and growth rate, achieving both tissue and gene selectivity [62]. In February 2012, Calando Pharmaceuticals, in Pasadena, Canada, and the National Cancer Institute (NCI) entered into a collaborative development program for a nanoparticle-based siRNA therapeutic aimed at treating neuro‐ blastoma, the most common extracranial solid tumor in children less than five years of age. Previous attempts to develop targeted nanoparticles were unsuccessful due to the inherent difficulties of designing and scaling up a particle capable of targeting, long-circulating via immune-response evasion and controlled drug release. Very recently, Hrkach *et al.* (2012) reported the preclinical development and clinical translation of a docetaxel nanoparticle with prostate-specific membrane antigen, a clinically validated tumor antigen expressed on prostate cancer cells and on the neovasculature of most non-prostate solid tumors including breast, head, lung, neck, prostate and stomach [63]. This targeted nanoparticle-based compound called "BIND-014" is currently the first one to enter clinical trial, although with small number of only 17 patients. Patients with advanced or metastatic cancer receive an injection of the nanodrug once every three weeks and are showing signals of efficacy even at relatively low doses. This initial but positive result shows promise and the potential impact of nanomedicines as a paradigm shift in the treatment of cancer.

good combinations of two-tailed cationic lipids and matrix lipids. Hence, structural or compositional design changes of nanovectors may influence the outcome in relation with cell physiology, cell internalization pathways and transfection efficiency. The above results

Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems

http://dx.doi.org/10.5772/54781

63

Under optimised conditions SLNs can be designed to incorporate lipophilic or hydrophilic drugs and seem to fulfil the requirements for an optimum particulate carrier system. Stability studies were performed on SLNs loaded with all-*trans* retinoic acid (ATRA), another com‐ pound that is sensitive to light, heat and oxidants, and quickly degrades into less active products such as isotretinoin and all-*trans*-4-oxo [66]. After 3 months of storage at 4 °C, more than 90% of the ATRA drug molecules in SLN remained chemically intact. This can be compared to approximately 50% drug degradation when stored at the same temperature in the form of methanol solution or 1% polysorbate-80 solution for only 1 month. Hence, SLNs are useful for the protection of anticancer compounds that are sensitive to light, and probably heat and oxidants as well. In a study conducted by our group, modulatory effects of encap‐ sulated and free forms of sesamol (anti-oxidant and anti-cancer compound) were evaluated by the topical delivery systems in a skin cancer mice model. Both free sesamol and SLN dispersion were applied as gels (using 1% w/v of Carbopol 934P®) on the skin of mice. Encapsulated or nanosesamol was found to safely exert chemopreventive effects by decreasing the lipid peroxidation levels and increasing the anti-oxidant levels, thereby decreasing the development and promotion of skin tumors. Immunofluorescence studies of pro- and antiapoptotic markers, bcl-2 and bax protein expression revealed higher expression of antiapoptotic protein, bcl-2, in the tissue sections of tumor bearing mice in comparison to their control counterparts and groups which received sesamol treatment, reinforcing the role of bcl-2 in skin carcinogenesis. Higher expression of bax was also observed in sesamol treated animals as compared to the tumor bearing mice. Up-regulation of bax in the control and sesamol treated groups suggests that it follows the intrinsic pathway of apoptosis (unpublished results).

Ongoing work by our group compared neutraceutical curcumin-loaded SLNs to the free form as a chemopreventive topical delivery system in 7,12-dimethylbenz [*a*]anthracene (DMBA) induced skin cancer model mice. In order to understand the molecular events underlying nanocurcumin-mediated chemoprevention, protein expression of various biomolecules e.g. anti and pro inflammatory cytokines (Il-4 and Il-1β) were analyzed by Western immunoblot‐ ting and immunoflourscence. For cancer induction, male Balb/c mice were subcutaneously injected with 30 mg/Kg body weight of DMBA (in olive oil) once a week for three weeks. DMBA skin cancer induced mice were topically applied free and encapsulated curcumin (50mg/Kg b.w) as a chemopreventive agent from one week before DMBA injection to the experiment's end (18 weeks). We found that free and nanocurcumin treatment of DMBA treated mice reduced the levels of malondialdehyde, a by-product of lipid degradation (Figure 3). Antiox‐ idant analysis revealed increased levels of enzymes (SOD, Catalase, Reduced Glutathione, Total Glutathione) in encapsulated nanocurcumin treated group as compared to free curcumin group (Figure 4-7). Immunofluorscence studies and western blot analysis of Il-4 and Il-1β suggest enhanced anti-inflammatory potential of encapsulated curcumin in comparison to mice treated with free curcumin. Mice bearing skin tumors showed increased expression of

support the use of SLNs to serve as nano/microcarriers for anti-cancer gene therapies.

Very recently, Vighi E *et al.* (2012) developed a multicomponent cationic SLN as a pDNA delivery vehicle. The formulations were prepared using stearic acid as the main component in the lipid phase, stearylamine, the main component in the aqueous phase, as cationic agent and protamine as transfection promoter along with the phosphatidylcholine (SLN–PC), cholesterol (SLN–Chol) or both (SLN–PC–Chol). Transfection results on various cell lines in this study revealed the best transfection for SLN–PC–Chol on COS-1 cells (African green monkey kidney cell line) [64]. However, lower transfection levels than poly [62] were observed on HepG2 cells (human hepatocellular liver carcinoma cell line), regardless of the SLN composition. Using COS-1 monkey kidney fibroblast-like cells, SLNs and liposomes formu‐ lated from the same cationic lipids, demonstrated equipotent *in vitro* transfection efficiencies [65]. This study suggests that only the lipid composition in the tested lipid-based formulations affected transfection efficiencies. The intrinsic toxicity that is common in cationic gene delivery vehicles may also be minimized, while maintaining high transfection efficiency, by selecting good combinations of two-tailed cationic lipids and matrix lipids. Hence, structural or compositional design changes of nanovectors may influence the outcome in relation with cell physiology, cell internalization pathways and transfection efficiency. The above results support the use of SLNs to serve as nano/microcarriers for anti-cancer gene therapies.

Nanovectors offer the potential to both detect and treat cancer at a very early stage, thereby maximizing survival rates. The NCI (National Cancer Institute) Alliance for Nanotechnology in Cancer provides up-to-date information in nano-cancer research and its promise for cancer diagnosis and treatment (http://nano.cancer.gov/). Using siRNA molecules loaded in nano‐ vectors, early proof-of-principle experiments in various tumor cells suggest that RNA silencing may have great potential as a strategy for treating cancer. However, siRNA therapeutics are hindered by poor intracellular uptake, limited blood stability and undesirable non-specific immune stimulation [61]. An interesting strategy used to target the vector employs threeamino-acid peptide, arginine-glycine-aspartic acid (known by its amino acid code RGD) that binds to integrins, which in turn are involved in angiogenesis, tumor cell growth, metastasis, and inflammation. Intravenous administration into tumor-bearing mice of nanoparticles combined with a dual strategy of siRNA inhibiting vascular endothelial growth factor receptor-2 and RGD peptide ligand attached at the distal end of the polyethylene glycol [40], conferred selective tumor uptake, and inhibition of both tumor angiogenesis and growth rate, achieving both tissue and gene selectivity [62]. In February 2012, Calando Pharmaceuticals, in Pasadena, Canada, and the National Cancer Institute (NCI) entered into a collaborative development program for a nanoparticle-based siRNA therapeutic aimed at treating neuro‐ blastoma, the most common extracranial solid tumor in children less than five years of age. Previous attempts to develop targeted nanoparticles were unsuccessful due to the inherent difficulties of designing and scaling up a particle capable of targeting, long-circulating via immune-response evasion and controlled drug release. Very recently, Hrkach *et al.* (2012) reported the preclinical development and clinical translation of a docetaxel nanoparticle with prostate-specific membrane antigen, a clinically validated tumor antigen expressed on prostate cancer cells and on the neovasculature of most non-prostate solid tumors including breast, head, lung, neck, prostate and stomach [63]. This targeted nanoparticle-based compound called "BIND-014" is currently the first one to enter clinical trial, although with small number of only 17 patients. Patients with advanced or metastatic cancer receive an injection of the nanodrug once every three weeks and are showing signals of efficacy even at relatively low doses. This initial but positive result shows promise and the potential impact of nanomedicines as a

Very recently, Vighi E *et al.* (2012) developed a multicomponent cationic SLN as a pDNA delivery vehicle. The formulations were prepared using stearic acid as the main component in the lipid phase, stearylamine, the main component in the aqueous phase, as cationic agent and protamine as transfection promoter along with the phosphatidylcholine (SLN–PC), cholesterol (SLN–Chol) or both (SLN–PC–Chol). Transfection results on various cell lines in this study revealed the best transfection for SLN–PC–Chol on COS-1 cells (African green monkey kidney cell line) [64]. However, lower transfection levels than poly [62] were observed on HepG2 cells (human hepatocellular liver carcinoma cell line), regardless of the SLN composition. Using COS-1 monkey kidney fibroblast-like cells, SLNs and liposomes formu‐ lated from the same cationic lipids, demonstrated equipotent *in vitro* transfection efficiencies [65]. This study suggests that only the lipid composition in the tested lipid-based formulations affected transfection efficiencies. The intrinsic toxicity that is common in cationic gene delivery vehicles may also be minimized, while maintaining high transfection efficiency, by selecting

paradigm shift in the treatment of cancer.

62 Novel Gene Therapy Approaches

Under optimised conditions SLNs can be designed to incorporate lipophilic or hydrophilic drugs and seem to fulfil the requirements for an optimum particulate carrier system. Stability studies were performed on SLNs loaded with all-*trans* retinoic acid (ATRA), another com‐ pound that is sensitive to light, heat and oxidants, and quickly degrades into less active products such as isotretinoin and all-*trans*-4-oxo [66]. After 3 months of storage at 4 °C, more than 90% of the ATRA drug molecules in SLN remained chemically intact. This can be compared to approximately 50% drug degradation when stored at the same temperature in the form of methanol solution or 1% polysorbate-80 solution for only 1 month. Hence, SLNs are useful for the protection of anticancer compounds that are sensitive to light, and probably heat and oxidants as well. In a study conducted by our group, modulatory effects of encap‐ sulated and free forms of sesamol (anti-oxidant and anti-cancer compound) were evaluated by the topical delivery systems in a skin cancer mice model. Both free sesamol and SLN dispersion were applied as gels (using 1% w/v of Carbopol 934P®) on the skin of mice. Encapsulated or nanosesamol was found to safely exert chemopreventive effects by decreasing the lipid peroxidation levels and increasing the anti-oxidant levels, thereby decreasing the development and promotion of skin tumors. Immunofluorescence studies of pro- and antiapoptotic markers, bcl-2 and bax protein expression revealed higher expression of antiapoptotic protein, bcl-2, in the tissue sections of tumor bearing mice in comparison to their control counterparts and groups which received sesamol treatment, reinforcing the role of bcl-2 in skin carcinogenesis. Higher expression of bax was also observed in sesamol treated animals as compared to the tumor bearing mice. Up-regulation of bax in the control and sesamol treated groups suggests that it follows the intrinsic pathway of apoptosis (unpublished results).

Ongoing work by our group compared neutraceutical curcumin-loaded SLNs to the free form as a chemopreventive topical delivery system in 7,12-dimethylbenz [*a*]anthracene (DMBA) induced skin cancer model mice. In order to understand the molecular events underlying nanocurcumin-mediated chemoprevention, protein expression of various biomolecules e.g. anti and pro inflammatory cytokines (Il-4 and Il-1β) were analyzed by Western immunoblot‐ ting and immunoflourscence. For cancer induction, male Balb/c mice were subcutaneously injected with 30 mg/Kg body weight of DMBA (in olive oil) once a week for three weeks. DMBA skin cancer induced mice were topically applied free and encapsulated curcumin (50mg/Kg b.w) as a chemopreventive agent from one week before DMBA injection to the experiment's end (18 weeks). We found that free and nanocurcumin treatment of DMBA treated mice reduced the levels of malondialdehyde, a by-product of lipid degradation (Figure 3). Antiox‐ idant analysis revealed increased levels of enzymes (SOD, Catalase, Reduced Glutathione, Total Glutathione) in encapsulated nanocurcumin treated group as compared to free curcumin group (Figure 4-7). Immunofluorscence studies and western blot analysis of Il-4 and Il-1β suggest enhanced anti-inflammatory potential of encapsulated curcumin in comparison to mice treated with free curcumin. Mice bearing skin tumors showed increased expression of pro-inflammatory interleukins when compared to the control, which was decreased on treatment with curcumin (Figure 8). Furthermore, the immunoflourscence assay of antiinflammatory interleukin (IL-4) showed a far greater increase in IL-4 expression by topical treatment with encapsulated curcumin as compared to the free curcumin in mice bearing skin tumors (Figure 9).

## **7. Conclusion**

Solid Lipid Nanoparticles serve as efficient and safe DNA/ drug loaded nanosystems in both the imaging and treatment of cancer. Traditional drug delivery systems are often hindered by their low bioavailabilty, low solubility, toxicity and rapid clearance. In future, clinicians and researchers will be able to "**tune and time**" the amount of DNA/Drug delivery by controlling the release at specific location thereby minimizing their toxicity and side- effects.

0

20

40

nmoles/mg protein

60

80

a

nmoles/mg protein

Curcumin + DMBA (CGD).

cumin + DMBA (CGD).

C D FCC FCD CGC CGD

:DMBA vs FCD and FCC p≤0.001<sup>b</sup> :DMBA vs CGD and CGC p≤0.001<sup>c</sup>

C D FCC FCD CGC CGD

:DMBA vs FCD and FCC p≤0.001<sup>b</sup> :DMBA vs CGD and CGC p≤0.001<sup>c</sup>

Total Glutathione

Groups

The value are presented as Mean ± SEM., N=8-10 Statistical significance: Control vs DMBA p≤0.001<sup>a</sup>

**Figure 5.** Effect of encapsulated and free curcumin on total glutathione in control and experimental groups. Control (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Cur‐

<sup>b</sup> b c <sup>c</sup>

Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems

http://dx.doi.org/10.5772/54781

65

Groups

The value are presented as Mean ± SEM., N=8-10 Statistical significanc: Control vs DMBA p≤0.001<sup>a</sup>

**Figure 4.** Effect of encapsulated and free curcumin on reduced glutathione in control and experimental groups. Con‐ trol (C), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated


**Figure 3.** Effect of encapsulated and free curcumin on lipid peroxidation (LPO) in control and experimental groups. Control (C), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulat‐ ed Curcumin + DMBA (CGD).

pro-inflammatory interleukins when compared to the control, which was decreased on treatment with curcumin (Figure 8). Furthermore, the immunoflourscence assay of antiinflammatory interleukin (IL-4) showed a far greater increase in IL-4 expression by topical treatment with encapsulated curcumin as compared to the free curcumin in mice bearing skin

Solid Lipid Nanoparticles serve as efficient and safe DNA/ drug loaded nanosystems in both the imaging and treatment of cancer. Traditional drug delivery systems are often hindered by their low bioavailabilty, low solubility, toxicity and rapid clearance. In future, clinicians and researchers will be able to "**tune and time**" the amount of DNA/Drug delivery by controlling

C D FCC FCD CGC CGD

:DMBA vs FCD and FCC p≤0.001<sup>b</sup> :DMBA vs CGD and CGC p≤0.001<sup>c</sup>

<sup>a</sup> LPO

b

c

c

b

The value are presented as Mean ± SEM., N=8-10 Statistical significance : Control vs DMBA p≤0.001<sup>a</sup>

**Figure 3.** Effect of encapsulated and free curcumin on lipid peroxidation (LPO) in control and experimental groups. Control (C), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulat‐

Groups

the release at specific location thereby minimizing their toxicity and side- effects.

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08

nmoles/mg protein

ed Curcumin + DMBA (CGD).

tumors (Figure 9).

64 Novel Gene Therapy Approaches

**7. Conclusion**


**Figure 4.** Effect of encapsulated and free curcumin on reduced glutathione in control and experimental groups. Con‐ trol (C), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Curcumin + DMBA (CGD).


**Figure 5.** Effect of encapsulated and free curcumin on total glutathione in control and experimental groups. Control (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Cur‐ cumin + DMBA (CGD).


**Figure 6.** Effect of encapsulated and free curcumin on superoxide dismutase in control and experimental groups. Con‐ trol (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Curcumin + DMBA (CGD).

The value are presented as Mean± SEM., N=8-10 Statistical significance: Control vs DMBA p≤0.05<sup>a</sup>


24

*Free curcumin + DMBA Enc\* curcumin control Enc\* curcumin + DMBA*

**Figure 9.** Photomicrographs (20X) showing expression of IL-4 in paraffin sections by immunofluoroscence after 18

*Control DMBA Free curcumin control*

26

*Control DMBA Free curcumin control*

Solid Lipid Nanoparticles: Tuneable Anti-Cancer Gene/Drug Delivery Systems

http://dx.doi.org/10.5772/54781

67

*Free curcumin + DMBA Enc\* curcumin control Enc\* curcumin + DMBA*

**Figure 8.** Photomicrographs (20X) showing expression of IL-1Beta in paraffin sections by immunofluoroscence after

*Immunofluorscence Expression of IL‐1β*

*Immunofluorscence expression of IL‐4*

18 weeks of treatment.

weeks of treatment.

**Figure 7.** Effect of encapsulated and free curcumin on Catalase in control and experimental groups. Control (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Curcumin + DMBA (CGD).

#### *Immunofluorscence Expression of IL‐1β*

*Control DMBA Free curcumin control*

0

5

IU/mg protein

10

15

IU/mg protein

Curcumin + DMBA (CGD).

66 Novel Gene Therapy Approaches

DMBA (CGD).

C D FCC FCD CGC CGD

The value are presented as Mean± SEM., N=8-10 Statistical significance: Control vs DMBA p≤0.05<sup>a</sup>

**Figure 6.** Effect of encapsulated and free curcumin on superoxide dismutase in control and experimental groups. Con‐ trol (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin + DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated

Catalase

C D FCC FCD CGC CGD

Groups

:DMBA (D) vs FCD and FCC p≤0.05<sup>b</sup> :DMBA (D) vs CGD and CGC p≤0.01<sup>c</sup>

The value are presented as Mean± SEM., N=8-10

Statistical significance: Control vs DMBA p≤0.05<sup>a</sup>

**Figure 7.** Effect of encapsulated and free curcumin on Catalase in control and experimental groups. Control (C ), DMBA (D), Free Curcumin (FCC), Free Curcumin DMBA (FCD), Encapsulated Curcumin (CGC), Encapsulated Curcumin +

SOD

Groups

:DMBA vs FCD and FCC p≤0.01<sup>b</sup> :DMBA vs CGD and CGC p≤0.01<sup>c</sup>

**Figure 8.** Photomicrographs (20X) showing expression of IL-1Beta in paraffin sections by immunofluoroscence after 18 weeks of treatment.

*Free curcumin + DMBA Enc\* curcumin control Enc\* curcumin + DMBA*

**Figure 9.** Photomicrographs (20X) showing expression of IL-4 in paraffin sections by immunofluoroscence after 18 weeks of treatment.

24

26

## **Author details**

Tranum Kaur1 and Roderick Slavcev2\*

\*Address all correspondence to: slavcev@uwaterloo.ca

1 Department of Biophysics, Panjab University, Chandigarh, India

2 School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada

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[12] Miele, E, Spinelli, G. P, Miele, E, Tomao, F, & Tomao, S. Albumin-bound formulation of paclitaxel (Abraxane ABI-007) in the treatment of breast cancer. Int J Nanomedicine (2009). Epub;%2009 Apr;%20.:99-105., 4, 99-105.

**Author details**

68 Novel Gene Therapy Approaches

and Roderick Slavcev2\*

\*Address all correspondence to: slavcev@uwaterloo.ca

Carrier Syst (2011). , 28(2), 101-64.

1 Department of Biophysics, Panjab University, Chandigarh, India

2 School of Pharmacy, University of Waterloo, Kitchener, Ontario, Canada

doi:IJN.S30726. Epub;%2012 Sep 14.:4943-51., 7, 4943-51.

applications. Adv Drug Deliv Rev (2012). Sep;(12):10.

Therapy. Anticancer Agents Med Chem (2012). Feb.

systems. J Control Release (2001). Jun;73(2-3):137-72.

Adv Drug Deliv Rev (2012). Sep;(12):10.

cine drug targeting. Methods Mol Biol (2010). , 624, 25-37.

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[4] Pearson, R. M, Sunoqrot, S, Hsu, H. J, Bae, J. W, & Hong, S. Dendritic nanoparticles: the

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**Chapter 4**

**Extracellular and Intracellular Barriers**

**to Non-Viral Gene Transfer**

Lynn F. Gottfried and David A. Dean

http://dx.doi.org/10.5772/54699

**1. Introduction**

Additional information is available at the end of the chapter

barriers in order to improve the efficacy of these vectors.

**2. Extracellular barriers**

Non-viral gene therapy vectors are highly desirable tools for the introduction of DNA into cells; they have better safety profiles than viral methods of delivery and are more amenable to repeated administration. Non-viral vectors include naked DNA, cationic lipid-DNA com‐ plexes (lipoplex), polymer-DNA complexes (polyplex) or combinations of lipids and polymers. Successful gene delivery depends upon the ability of the vector of choice to target a specific cell type, enter the cell and obtain sufficient levels of gene expression. This is not a simple task since there are several barriers encountered by both viral and non-viral vectors that make this process difficult. First, the vector must have a method by which to target a specific cell type, while also avoiding extracellular insults including nucleases and the immune system. Next, once the vector has reached its particular target, it must traverse the plasma membrane and/or escape the endosome, and pass through the dense cytoskeletal network en route to the nucleus. The nuclear envelope presents a final barrier, since DNA must enter the nucleus in order to be transcribed. While viruses have evolved mechanisms to enter target cells, deliver their genetic material and continue to propagate, non-viral systems lack these innate mecha‐ nisms. Consequently, there has been much work aimed at characterizing and overcoming these

Regardless of the method by which a non-viral vector is administered *in vivo* (e.g., by inhalation, intramuscular injection, gavage, intravascular injection, etc), it will unavoidably come into con‐ tact with the extracellular environment. Within the extracellular milieu, multiple factors exist

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

© 2013 Gottfried and Dean; 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

## **Chapter 4**

## **Extracellular and Intracellular Barriers to Non-Viral Gene Transfer**

Lynn F. Gottfried and David A. Dean

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54699

## **1. Introduction**

Non-viral gene therapy vectors are highly desirable tools for the introduction of DNA into cells; they have better safety profiles than viral methods of delivery and are more amenable to repeated administration. Non-viral vectors include naked DNA, cationic lipid-DNA com‐ plexes (lipoplex), polymer-DNA complexes (polyplex) or combinations of lipids and polymers. Successful gene delivery depends upon the ability of the vector of choice to target a specific cell type, enter the cell and obtain sufficient levels of gene expression. This is not a simple task since there are several barriers encountered by both viral and non-viral vectors that make this process difficult. First, the vector must have a method by which to target a specific cell type, while also avoiding extracellular insults including nucleases and the immune system. Next, once the vector has reached its particular target, it must traverse the plasma membrane and/or escape the endosome, and pass through the dense cytoskeletal network en route to the nucleus. The nuclear envelope presents a final barrier, since DNA must enter the nucleus in order to be transcribed. While viruses have evolved mechanisms to enter target cells, deliver their genetic material and continue to propagate, non-viral systems lack these innate mecha‐ nisms. Consequently, there has been much work aimed at characterizing and overcoming these barriers in order to improve the efficacy of these vectors.

## **2. Extracellular barriers**

Regardless of the method by which a non-viral vector is administered *in vivo* (e.g., by inhalation, intramuscular injection, gavage, intravascular injection, etc), it will unavoidably come into con‐ tact with the extracellular environment. Within the extracellular milieu, multiple factors exist

© 2013 Gottfried and Dean; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

which can result in rapid clearance and/or degradation of the vector before it ever reaches its targeted organ. Intravenously delivered naked DNA has been shown to have a very short halflife within serum, on the range of 1.2 to 21 minutes depending upon the topoform of the DNA [1]. This is believed to be the result of both endo and exonuclease activity in the plasma. Similar degradation has been observed in plasmid DNA delivered intramuscularly [2]. Strategies to protect DNA from nuclease activity include the use of cationic lipids, such as DOTAP:DOPE, to encapsulate the plasmid and shield it from the enzymatic environment outside the cell [1]. In addition, the use of PEGylated lipids and polymers has been demonstrated to enhance the sta‐ bility of complexes in serum, greatly lengthening the vectors' half-life [3].

anti-PEG IgM has the potential to interfere with repeated administration of the vector [12-17]. Thus, much care and consideration must be put into the selection of the proper non-viral vector

Extracellular and Intracellular Barriers to Non-Viral Gene Transfer

http://dx.doi.org/10.5772/54699

77

Once the delivery vehicle has reached a cell, it then encounters a second significant barrier: the plasma membrane. Successful transfection relies on the ability of the vector to enter the cell of interest. In the absence of a delivery vehicle, naked DNA does not efficiently associate with the plasma membrane due to the negative charge density on both the DNA and the cell surface. De‐ livery vehicles help circumvent this problem through the use of polycations to neutralize the negative charge of the DNA, thereby increasing association with the plasma membrane. This non-specific electrostatic association is mediated largely by heparin sulfate proteoglycans on

While most chemical transfection methods require endocytosis for transport across the plasma membrane into the cytoplasm, more recently, several short peptides have been reported to facilitate cell entry in an endocytosis-independent manner [18-20]. These have been termed cell-penetrating peptides (CPPs) and include peptides from proteins such as Tat, antennape‐ dia, and penetratin as well as polyarginine peptides. Depending upon the CPP used, different internalization mechanisms are used to access the cytoplasm including direct transduction through the lipid bilayer as well as energy-dependent macropinocytosis [21]. Regardless of the mechanism for cell entry, these peptides may prove very useful in delivery of small DNAs

Physical methods of gene delivery, such as the gene gun, electroporation (electric fields), sonoporation (ultrasound) and hydrodynamic (high pressure) delivery can also facilitate delivery of DNA across the plasma membrane. The gene gun utilizes metal particles coated with plasmid DNA that are accelerated and bombard a tissue of interest [22]. This technique, however is limited by the superficial penetration of the DNA into the tissue and is therefore most successful for percutaneous delivery of DNA. Electroporation is a more versatile technique that has been used on a variety of tissues with success. During this process, a series of electrical pulses are delivered that result in destabilization of the cell membrane. Transient pores are then created which allow the passage of plasmid DNA into the cell. Blood vessels, skin, muscle, heart, liver, and lung have all been successfully transfected with electroporation [23-27]. Sonoporation has seen most success in soft tissues and it's use deep within the body is a major potential strength. This technique uses ultrasound to enhance cell permeability through acoustic cavitation and subsequent gene transfer through passive diffusion of DNA across pores in the cell membranes. A number of in vivo studies have demonstrated successful gene transfer in skeletal muscle, cardiac muscle, kidney, carotid artery, pancreas and liver of mice and rats [28-37]. Finally, hydrodynamic gene delivery is a highly efficient method for gene transfer to highly perfused organs such as the liver or muscle in peripheral limbs. A large volume of DNA injected into the tail vein of a mouse has been shown to result in transient

the cell surface that trigger endocytosis of the delivery vehicle and entry into the cell.

for specific disease therapeutics.

**3. The plasma membrane**

and synthetic RNAs.

DNA that does evade nucleases, however, also comes into contact with proteins and cells within the extracellular environment. Serum contains a variety of proteins which have the ability to bind to non-viral vectors and, as a result, inhibit the biological activity of the vector or sequester it for degradation and/or removal. For example, negatively charged serum proteins form connections between cationic liposomes, which results in the aggregration of the delivery vehicles. When this happens, the vectors are quickly removed from circulation by the reticuloendothelial system. Some of the key blood proteins that have been identified to associate with non-viral vectors include albumin, complement, immunoglobulins, fibronectin, apolipoproteins, C-reactive protein, and b2-glycoprotein I [4]. PEGylation of both lipolexes and polyplexes as well as the use of cholesterol as a helper lipid have shown promise in the prevention of this type of aggregation [3].

It is important to note that DNA delivery vehicles also come into contact with blood cells. These cells, which include erythrocytes, leukocytes, macrophages, and platelets, have a negative surface charge, thereby allowing for electrostatic interactions to occur between cells and cationic vectors. In particular, the interaction of lipoplexes with erythrocytes has been shown to be a significant factor in *in vivo* gene delivery, as binding occurs within minutes of *in vivo* intravenous gene transfer [5]. The vector is then able to directly associate (in the case of DOTMA/cholesterol complexes) and/or fuse (in the case of DOTMA/DOPE complexes) with erythrocytes, thereby decreasing transfection efficiency and encouraging removal of the delivery vehicle by way of the liver and spleen [5]. (Since erythrocytes have no nuclei, entry of vectors into these cells represents a dead end.) In the lung, the alveolar macrophage is regarded as a major barrier to both viral and non-viral delivery since this professional phagocytic cell "eats" up delivery agents before they can transfect any other cell type [6].

Another extracellular barrier to consider is activation of the immune system. While immune activation has been most associated with viral gene delivery, some non-viral methods have been shown to induce an immune response. For example, intravenously injected cationic lipoplexes can induce an inflammatory response involving the release of TNFα and IFNγ into the serum [7]. This is believed to be a result of unmethylated CpG motifs on the plasmid DNA and the subsequent recognition by Toll-like receptors [8, 9]. Thus, the removal of these CpG motifs is important for successful gene delivery [10]. The cationic polymer, PEI, has also been shown to activate the immune system through complement and activation of both a Th1 and Th2 response [11] Finally, as previously discussed, PEGylation of non-viral vectors is a common technique used for avoiding some extracellular barriers, however the production of anti-PEG IgM has the potential to interfere with repeated administration of the vector [12-17]. Thus, much care and consideration must be put into the selection of the proper non-viral vector for specific disease therapeutics.

## **3. The plasma membrane**

which can result in rapid clearance and/or degradation of the vector before it ever reaches its targeted organ. Intravenously delivered naked DNA has been shown to have a very short halflife within serum, on the range of 1.2 to 21 minutes depending upon the topoform of the DNA [1]. This is believed to be the result of both endo and exonuclease activity in the plasma. Similar degradation has been observed in plasmid DNA delivered intramuscularly [2]. Strategies to protect DNA from nuclease activity include the use of cationic lipids, such as DOTAP:DOPE, to encapsulate the plasmid and shield it from the enzymatic environment outside the cell [1]. In addition, the use of PEGylated lipids and polymers has been demonstrated to enhance the sta‐

DNA that does evade nucleases, however, also comes into contact with proteins and cells within the extracellular environment. Serum contains a variety of proteins which have the ability to bind to non-viral vectors and, as a result, inhibit the biological activity of the vector or sequester it for degradation and/or removal. For example, negatively charged serum proteins form connections between cationic liposomes, which results in the aggregration of the delivery vehicles. When this happens, the vectors are quickly removed from circulation by the reticuloendothelial system. Some of the key blood proteins that have been identified to associate with non-viral vectors include albumin, complement, immunoglobulins, fibronectin, apolipoproteins, C-reactive protein, and b2-glycoprotein I [4]. PEGylation of both lipolexes and polyplexes as well as the use of cholesterol as a helper lipid have shown promise in the

It is important to note that DNA delivery vehicles also come into contact with blood cells. These cells, which include erythrocytes, leukocytes, macrophages, and platelets, have a negative surface charge, thereby allowing for electrostatic interactions to occur between cells and cationic vectors. In particular, the interaction of lipoplexes with erythrocytes has been shown to be a significant factor in *in vivo* gene delivery, as binding occurs within minutes of *in vivo* intravenous gene transfer [5]. The vector is then able to directly associate (in the case of DOTMA/cholesterol complexes) and/or fuse (in the case of DOTMA/DOPE complexes) with erythrocytes, thereby decreasing transfection efficiency and encouraging removal of the delivery vehicle by way of the liver and spleen [5]. (Since erythrocytes have no nuclei, entry of vectors into these cells represents a dead end.) In the lung, the alveolar macrophage is regarded as a major barrier to both viral and non-viral delivery since this professional phagocytic cell "eats" up delivery agents before they can transfect any other cell type [6].

Another extracellular barrier to consider is activation of the immune system. While immune activation has been most associated with viral gene delivery, some non-viral methods have been shown to induce an immune response. For example, intravenously injected cationic lipoplexes can induce an inflammatory response involving the release of TNFα and IFNγ into the serum [7]. This is believed to be a result of unmethylated CpG motifs on the plasmid DNA and the subsequent recognition by Toll-like receptors [8, 9]. Thus, the removal of these CpG motifs is important for successful gene delivery [10]. The cationic polymer, PEI, has also been shown to activate the immune system through complement and activation of both a Th1 and Th2 response [11] Finally, as previously discussed, PEGylation of non-viral vectors is a common technique used for avoiding some extracellular barriers, however the production of

bility of complexes in serum, greatly lengthening the vectors' half-life [3].

prevention of this type of aggregation [3].

76 Novel Gene Therapy Approaches

Once the delivery vehicle has reached a cell, it then encounters a second significant barrier: the plasma membrane. Successful transfection relies on the ability of the vector to enter the cell of interest. In the absence of a delivery vehicle, naked DNA does not efficiently associate with the plasma membrane due to the negative charge density on both the DNA and the cell surface. De‐ livery vehicles help circumvent this problem through the use of polycations to neutralize the negative charge of the DNA, thereby increasing association with the plasma membrane. This non-specific electrostatic association is mediated largely by heparin sulfate proteoglycans on the cell surface that trigger endocytosis of the delivery vehicle and entry into the cell.

While most chemical transfection methods require endocytosis for transport across the plasma membrane into the cytoplasm, more recently, several short peptides have been reported to facilitate cell entry in an endocytosis-independent manner [18-20]. These have been termed cell-penetrating peptides (CPPs) and include peptides from proteins such as Tat, antennape‐ dia, and penetratin as well as polyarginine peptides. Depending upon the CPP used, different internalization mechanisms are used to access the cytoplasm including direct transduction through the lipid bilayer as well as energy-dependent macropinocytosis [21]. Regardless of the mechanism for cell entry, these peptides may prove very useful in delivery of small DNAs and synthetic RNAs.

Physical methods of gene delivery, such as the gene gun, electroporation (electric fields), sonoporation (ultrasound) and hydrodynamic (high pressure) delivery can also facilitate delivery of DNA across the plasma membrane. The gene gun utilizes metal particles coated with plasmid DNA that are accelerated and bombard a tissue of interest [22]. This technique, however is limited by the superficial penetration of the DNA into the tissue and is therefore most successful for percutaneous delivery of DNA. Electroporation is a more versatile technique that has been used on a variety of tissues with success. During this process, a series of electrical pulses are delivered that result in destabilization of the cell membrane. Transient pores are then created which allow the passage of plasmid DNA into the cell. Blood vessels, skin, muscle, heart, liver, and lung have all been successfully transfected with electroporation [23-27]. Sonoporation has seen most success in soft tissues and it's use deep within the body is a major potential strength. This technique uses ultrasound to enhance cell permeability through acoustic cavitation and subsequent gene transfer through passive diffusion of DNA across pores in the cell membranes. A number of in vivo studies have demonstrated successful gene transfer in skeletal muscle, cardiac muscle, kidney, carotid artery, pancreas and liver of mice and rats [28-37]. Finally, hydrodynamic gene delivery is a highly efficient method for gene transfer to highly perfused organs such as the liver or muscle in peripheral limbs. A large volume of DNA injected into the tail vein of a mouse has been shown to result in transient membrane changes in hepatocytes, resulting in direct transfer of DNA into the cytoplasm [38, 39]. Like electroporation and sonoporation, pores within the plasma membrane are thought to be formed allowing entry of plasmids, in this case by the rapid change in hydrostatic pressure [40]. Modifications of this technique using balloon catheters in larger animals has suggested potential for ultimate success in humans as well [41-43].

of buffering capacity increases the amount of time before passage of DNA to lysosomes, which

Extracellular and Intracellular Barriers to Non-Viral Gene Transfer

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Finally, many fusogenic and pore-forming peptides have been discovered and incorporat‐ ed into gene delivery vehicles. The influenza-derived peptides GALA and KALA under‐ go pH-dependent conformational changes that result in disruption of endosomal membranes [55-58]. Several bacteria derived and animal derived peptides with membrane disruptive properties have also been developed and appear to increase endosomal escape

Once DNA has successfully been released into the cytoplasm, it must then traffic to the nucleus in order for gene expression to occur. This step represents another significant barrier to gene delivery. First, the cytoplasm contains nucleases that will degrade free DNA. Studies have demonstrated that plasmid DNA is degraded in the cytoplasm of HeLa and COS cells with a half-life of 50 to 90 minutes [60]. This poses a big problem for delivery of naked DNA and DNA-lipid complexes that are believed to dissociate prior to nuclear entry. Additionally, the cytoplasm itself poses a diffusional barrier as well. The cytoplasm is a viscous environment crowded with molecules, which results in decreased mobility of macromolecules [61-63]. Thus, if DNA is released from an endosome at a distant site from the nucleus, the DNA cannot simply diffuse toward its desired location. This has been demonstrated in the case of liposome transfections where some DNA is left free in the cytoplasm and never reaches the nucleus [64, 65]. Although it has been shown that lipoplex-containing endosomes themselves traffic toward the nucleus and the interior of the cell, there is still quite a lot of distance for the free DNA following endosomal release to move before it reaches the nucleus. We and others have shown that DNA in the cytoplasm utilizes the microtubule network and the molecular motor, dynein [66-68] for its trafficking to the nucleus. Since DNA does not directly bind to dynein, the mechanism of this interaction was investigated and was found to involve a multiprotein complex that bridges the DNA and dynein. Our laboratory has shown that transcription factors are key proteins in this complex, and they are involved in the movement of DNA along microtubules [69]. Furthermore, the velocity of plasmid DNA movement can be increased through addition of specific transcription factor binding sites in the plasmid, such as CREB [69] or by inducing acetylation of the microtubules themselves [70]. The acetylation status is largely controlled by histone deacetylase 6 (HDAC6), and studies have shown that modulation of this enzyme can increase the efficiency of gene transfer [70]. Thus, despite the fact that the cytoplasm poses as a significant barrier to gene transfer, many techniques for overcoming this

DNA that has successfully navigated all of the barriers previously discussed finally comes into contact with the nuclear envelope. However, depending upon cell type, DNA dose and detection method, only 1 to 10% of transfected plasmid can be found within the nucleus [71, 72]. This suggests that an overwhelming proportion of DNA that enters the cytoplasm will

therefore increases the likelihood of the DNA getting transferred to the cytoplasm.

and transfection efficiency [59].

problem are being revealed.

**5. Nuclear import**

While all of these approaches facilitate endocytosis or direct entry into the cytoplasm of any cell type, one of the goals of gene therapy is often cell-specific targeting. Thus, the development of delivery vehicles that only interact with specific cell types is highly desirable. In most cases, cell-specificity is acheived by specific interactions between ligands on the vector and receptors on the cell surface. For example, as many tumor cells overexpress receptors for nutrients, such as folate and transferrin, this phentoype has been exploited for the development of DNA and drug delivery vehicles carrying either folic acid or transferrin as ligands. Recently, a transfer‐ rin-PEG-PE conjugated cationic lipid carrier and a PEG-transferrin-PEI nanocomplex were developed, which exhibited increased transfection efficiency in vitro as well as in vivo [44, 45]. Similar results have been obtained using folate linked nanoparticles as well [46]. Delivery of DNA to specific cell types has also been achieved through the use of glycosylated carriers, specifically cationic liposomes [47]. Mannosylated and galactose conjugated liposomes have demonstrated efficacy as delivery agents for macrophages and hepatocytes [48, 49].

## **4. Vector release and cytoplasmic trafficking**

Gene delivery vectors that utilize endocytosis to access the cytoplasm are then moved through the endocytic compartment. Thus, another major barrier to successful gene deliv‐ ery is the release of the DNA from the endosome before it is degraded at the lysosomal level. There are several mechanisms employed to increase the likelihood of endosomal escape including membrane fusion, the proton-sponge effect and incorporation of fuso‐ genic and pore-forming peptides.

Lipoplexes are able to escape the endosome through fusion of the liposome with the en‐ dosomal membrane. In particular, inclusion of dioleoylphosphatidylethanolamine (DOPE) has been shown to enhance endosomal escape due to its ability to transition from bilayer to inverted hexagonal structures [50, 51]. The instability of this type of structure increas‐ es fusion with endosomes and subsequently releases the DNA [52]. This is not common across all lipids as a similar phospholipid, dioleoylphosphatidylcholine (DOPC), does not exhibit similar activity [53, 54]. Therefore, complex structure plays an important role in enhancement of endosomal escape.

In contrast to lipoplexes, cationic polymers such as PEI achieve endosomal escape through a 'proton sponge' mechanism. PEI possesses a very high buffering capacity due to the presence of amino nitrogen at every third atom, which can be protonated within the acidifying endosomes. Consequently, an accumulation of protons causes an influx of chloride ions thereby resulting in osmotic swelling and lysis of the endosome. Therefore, use of cationic polymers with this type of buffering capacity increases the amount of time before passage of DNA to lysosomes, which therefore increases the likelihood of the DNA getting transferred to the cytoplasm.

Finally, many fusogenic and pore-forming peptides have been discovered and incorporat‐ ed into gene delivery vehicles. The influenza-derived peptides GALA and KALA under‐ go pH-dependent conformational changes that result in disruption of endosomal membranes [55-58]. Several bacteria derived and animal derived peptides with membrane disruptive properties have also been developed and appear to increase endosomal escape and transfection efficiency [59].

Once DNA has successfully been released into the cytoplasm, it must then traffic to the nucleus in order for gene expression to occur. This step represents another significant barrier to gene delivery. First, the cytoplasm contains nucleases that will degrade free DNA. Studies have demonstrated that plasmid DNA is degraded in the cytoplasm of HeLa and COS cells with a half-life of 50 to 90 minutes [60]. This poses a big problem for delivery of naked DNA and DNA-lipid complexes that are believed to dissociate prior to nuclear entry. Additionally, the cytoplasm itself poses a diffusional barrier as well. The cytoplasm is a viscous environment crowded with molecules, which results in decreased mobility of macromolecules [61-63]. Thus, if DNA is released from an endosome at a distant site from the nucleus, the DNA cannot simply diffuse toward its desired location. This has been demonstrated in the case of liposome transfections where some DNA is left free in the cytoplasm and never reaches the nucleus [64, 65]. Although it has been shown that lipoplex-containing endosomes themselves traffic toward the nucleus and the interior of the cell, there is still quite a lot of distance for the free DNA following endosomal release to move before it reaches the nucleus. We and others have shown that DNA in the cytoplasm utilizes the microtubule network and the molecular motor, dynein [66-68] for its trafficking to the nucleus. Since DNA does not directly bind to dynein, the mechanism of this interaction was investigated and was found to involve a multiprotein complex that bridges the DNA and dynein. Our laboratory has shown that transcription factors are key proteins in this complex, and they are involved in the movement of DNA along microtubules [69]. Furthermore, the velocity of plasmid DNA movement can be increased through addition of specific transcription factor binding sites in the plasmid, such as CREB [69] or by inducing acetylation of the microtubules themselves [70]. The acetylation status is largely controlled by histone deacetylase 6 (HDAC6), and studies have shown that modulation of this enzyme can increase the efficiency of gene transfer [70]. Thus, despite the fact that the cytoplasm poses as a significant barrier to gene transfer, many techniques for overcoming this problem are being revealed.

## **5. Nuclear import**

membrane changes in hepatocytes, resulting in direct transfer of DNA into the cytoplasm [38, 39]. Like electroporation and sonoporation, pores within the plasma membrane are thought to be formed allowing entry of plasmids, in this case by the rapid change in hydrostatic pressure [40]. Modifications of this technique using balloon catheters in larger animals has

While all of these approaches facilitate endocytosis or direct entry into the cytoplasm of any cell type, one of the goals of gene therapy is often cell-specific targeting. Thus, the development of delivery vehicles that only interact with specific cell types is highly desirable. In most cases, cell-specificity is acheived by specific interactions between ligands on the vector and receptors on the cell surface. For example, as many tumor cells overexpress receptors for nutrients, such as folate and transferrin, this phentoype has been exploited for the development of DNA and drug delivery vehicles carrying either folic acid or transferrin as ligands. Recently, a transfer‐ rin-PEG-PE conjugated cationic lipid carrier and a PEG-transferrin-PEI nanocomplex were developed, which exhibited increased transfection efficiency in vitro as well as in vivo [44, 45]. Similar results have been obtained using folate linked nanoparticles as well [46]. Delivery of DNA to specific cell types has also been achieved through the use of glycosylated carriers, specifically cationic liposomes [47]. Mannosylated and galactose conjugated liposomes have

demonstrated efficacy as delivery agents for macrophages and hepatocytes [48, 49].

Gene delivery vectors that utilize endocytosis to access the cytoplasm are then moved through the endocytic compartment. Thus, another major barrier to successful gene deliv‐ ery is the release of the DNA from the endosome before it is degraded at the lysosomal level. There are several mechanisms employed to increase the likelihood of endosomal escape including membrane fusion, the proton-sponge effect and incorporation of fuso‐

Lipoplexes are able to escape the endosome through fusion of the liposome with the en‐ dosomal membrane. In particular, inclusion of dioleoylphosphatidylethanolamine (DOPE) has been shown to enhance endosomal escape due to its ability to transition from bilayer to inverted hexagonal structures [50, 51]. The instability of this type of structure increas‐ es fusion with endosomes and subsequently releases the DNA [52]. This is not common across all lipids as a similar phospholipid, dioleoylphosphatidylcholine (DOPC), does not exhibit similar activity [53, 54]. Therefore, complex structure plays an important role in

In contrast to lipoplexes, cationic polymers such as PEI achieve endosomal escape through a 'proton sponge' mechanism. PEI possesses a very high buffering capacity due to the presence of amino nitrogen at every third atom, which can be protonated within the acidifying endosomes. Consequently, an accumulation of protons causes an influx of chloride ions thereby resulting in osmotic swelling and lysis of the endosome. Therefore, use of cationic polymers with this type

suggested potential for ultimate success in humans as well [41-43].

**4. Vector release and cytoplasmic trafficking**

genic and pore-forming peptides.

78 Novel Gene Therapy Approaches

enhancement of endosomal escape.

DNA that has successfully navigated all of the barriers previously discussed finally comes into contact with the nuclear envelope. However, depending upon cell type, DNA dose and detection method, only 1 to 10% of transfected plasmid can be found within the nucleus [71, 72]. This suggests that an overwhelming proportion of DNA that enters the cytoplasm will never successfully enter the nucleus. It has been appreciated for over 30 years that the nuclear envelope is a major barrier to DNA delivery [73]. Our laboratory has shown in microinjection experiments using non-dividing cells that 30 to 100 times more plasmid must be injected into the cytoplasm of a cell to equal levels of gene expression of plasmid injected directly into the nucleus [74]. While it is true that DNA delivery to the nucleus is greater within dividing cells, even breakdown of the nuclear envelope does not completely eliminate nuclear import as a barrier to transfer [75]. Therefore, studies aimed at understanding how DNA is imported into the nucleus as well as development of strategies to improve this process have been key to enhancing the efficiency of non-viral gene delivery.

to the importin proteins for nuclear import to occur [87]. However, in vivo studies using DNA with conjugated NLS peptides have demonstrated increased gene expression in muscle as well as increased immune response against the expressed antigen [88]. Also, more recently, analysis of a bipartite NLS construct as a non-viral gene carrier has revealed the potential success of this type of method over traditional monopartite peptides [89]. Due to the varied success of NLS peptides at promotion of nuclear import, it is still unclear if this will be a promising

Extracellular and Intracellular Barriers to Non-Viral Gene Transfer

http://dx.doi.org/10.5772/54699

81

As an alternative to NLS peptides, some work has tested direct conjugation of importins to plasmid DNA. The importin-β-binding domain of importin-α was covalently coupled to plasmid DNA, but this also failed to enhance nuclear import [90]. In a separate study, a plasmid DNA/importin-β conjugate was made via binding of biotinylated plasmid DNA and recombi‐ nant streptavidin-importin-β chimeric protein. While this did enhance nuclear import, gene

To maximize non-viral gene delivery, levels of expression must be improved. Unfortunately, many extracellular and intracellular barriers (including the extracellular environment, immune scavengers, the cell membrane, endosomal escape, the cytoskeletal network and the nuclear membrane) preclude efficient gene transfer. In this review, we have focused on these barriers and various means to overcome them. The goal of all gene therapy approaches is to target enough DNA to the nuclei of cells to obtain sufficient expression for a therapeutic effect. By characterizing and understanding these barriers, we can overcome our relative inability to target substantial amounts of DNA to the nucleus and increase transfection efficiency and

Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA

dation in rat plasma. AAPS PharmSci. (1999). E9.

[1] Houk, B. E, Hochhaus, G, & Hughes, J. A. Kinetic modeling of plasmid DNA degra‐

[2] Mumper, R. J, Duguid, J. G, Anwer, K, Barron, M. K, Nitta, H, & Rolland, A. P. Polyvinyl derivatives as novel interactive polymers for controlled gene delivery to muscle. Pharm

expression was very low due to the highly modified plasmid [91].

approach for gene therapy.

**6. Conclusion**

ultimately gene therapy.

Lynn F. Gottfried and David A. Dean

Res. (1996). May;, 13(5), 701-9.

**Author details**

**References**

A number of studies have shown that the vast majority of transfected DNA enters the nucleus during mitosis when the nuclear envelope has broken down [76, 77]. While plasmids can enter the nucleus in the absence of cell division, the process is slow and highly inefficient, resulting in very low levels of nuclear entry. However, our laboratory has shown that the delivery of plasmid DNA into the nucleus can be greatly increased by the addition of specific DNA sequences. We have demonstrated that plasmids containing only 72bp of the SV40 enhancer are able to target the nucleus of non-dividing cells within a few hours [78]. This sequence, termed a DNA nuclear targeting sequence (DTS), functions to enhance nuclear import in all cell types tested. It is the presence of ubiquitously expressed transcription factor binding sites within the SV40 DTS that mediate this effect. Since these transcription factors contain nuclear localization signals (NLS) to allow their targeting to the nucleus, nuclear import of the DNA is controlled by the interaction of these proteins with the NLS-receptors importin-α and importin-β, that then transport cargo through the nuclear pore complex (NPC). Thus, when NLS-containing transcription factors bind to the SV40 DTS on a plasmid, the plasmid utilizes this system to enter the nucleus [79, 80]. We have shown that these DTS sequences act not only in microinjected cells, but in transfected cells as well to increase DNA nuclear uptake and gene expression as well as in tissues in living animals [81-83].

We have also identified several cell-specific DTSs, in which nuclear import of a plasmid is regu‐ lated by the presence of cell-specific transcription factor binding sites within the DTS. DTSs spe‐ cific for smooth muscle cells, osteoblasts, endothelial cells, alveolar epithelial type I cells and alveolar epithelial type II cells have been identified and large studies are underway to screen hundreds of DNA sequences for the potential to act as cell-specific DTSs [83-86]. In all of these cases, the cell-specific DTS contains binding sites for cell specific transcription factors that are expressed in unique cell types. Thus, if the plasmid is delivered to a cell that expresses those transcription factors, it will be transported into the nucleus and gene expression will ensue; if, however, the plasmid enters any other cell type that does not express the specific transcription factor, the DNA will remain in the cytoplasm until cell division or until it is degraded by cyto‐ plasmic nucleases. Again, as for the SV40 DTS, these cell-specific DTSs work in cultured cells and in animal tissues to increase gene expression in a cell-restricted manner [83, 86].

A number of other methods for enhancing nuclear import have been studied, all of which center around exploiting the cells protein nuclear import machinery. These approaches include complexing plasmid DNA with NLS peptides, nuclear proteins or small molecule ligands. The success of NLS peptides has been variable, likely due to the fact that the NLS must be visible to the importin proteins for nuclear import to occur [87]. However, in vivo studies using DNA with conjugated NLS peptides have demonstrated increased gene expression in muscle as well as increased immune response against the expressed antigen [88]. Also, more recently, analysis of a bipartite NLS construct as a non-viral gene carrier has revealed the potential success of this type of method over traditional monopartite peptides [89]. Due to the varied success of NLS peptides at promotion of nuclear import, it is still unclear if this will be a promising approach for gene therapy.

As an alternative to NLS peptides, some work has tested direct conjugation of importins to plasmid DNA. The importin-β-binding domain of importin-α was covalently coupled to plasmid DNA, but this also failed to enhance nuclear import [90]. In a separate study, a plasmid DNA/importin-β conjugate was made via binding of biotinylated plasmid DNA and recombi‐ nant streptavidin-importin-β chimeric protein. While this did enhance nuclear import, gene expression was very low due to the highly modified plasmid [91].

## **6. Conclusion**

never successfully enter the nucleus. It has been appreciated for over 30 years that the nuclear envelope is a major barrier to DNA delivery [73]. Our laboratory has shown in microinjection experiments using non-dividing cells that 30 to 100 times more plasmid must be injected into the cytoplasm of a cell to equal levels of gene expression of plasmid injected directly into the nucleus [74]. While it is true that DNA delivery to the nucleus is greater within dividing cells, even breakdown of the nuclear envelope does not completely eliminate nuclear import as a barrier to transfer [75]. Therefore, studies aimed at understanding how DNA is imported into the nucleus as well as development of strategies to improve this process have been key to

A number of studies have shown that the vast majority of transfected DNA enters the nucleus during mitosis when the nuclear envelope has broken down [76, 77]. While plasmids can enter the nucleus in the absence of cell division, the process is slow and highly inefficient, resulting in very low levels of nuclear entry. However, our laboratory has shown that the delivery of plasmid DNA into the nucleus can be greatly increased by the addition of specific DNA sequences. We have demonstrated that plasmids containing only 72bp of the SV40 enhancer are able to target the nucleus of non-dividing cells within a few hours [78]. This sequence, termed a DNA nuclear targeting sequence (DTS), functions to enhance nuclear import in all cell types tested. It is the presence of ubiquitously expressed transcription factor binding sites within the SV40 DTS that mediate this effect. Since these transcription factors contain nuclear localization signals (NLS) to allow their targeting to the nucleus, nuclear import of the DNA is controlled by the interaction of these proteins with the NLS-receptors importin-α and importin-β, that then transport cargo through the nuclear pore complex (NPC). Thus, when NLS-containing transcription factors bind to the SV40 DTS on a plasmid, the plasmid utilizes this system to enter the nucleus [79, 80]. We have shown that these DTS sequences act not only in microinjected cells, but in transfected cells as well to increase DNA nuclear uptake and gene

We have also identified several cell-specific DTSs, in which nuclear import of a plasmid is regu‐ lated by the presence of cell-specific transcription factor binding sites within the DTS. DTSs spe‐ cific for smooth muscle cells, osteoblasts, endothelial cells, alveolar epithelial type I cells and alveolar epithelial type II cells have been identified and large studies are underway to screen hundreds of DNA sequences for the potential to act as cell-specific DTSs [83-86]. In all of these cases, the cell-specific DTS contains binding sites for cell specific transcription factors that are expressed in unique cell types. Thus, if the plasmid is delivered to a cell that expresses those transcription factors, it will be transported into the nucleus and gene expression will ensue; if, however, the plasmid enters any other cell type that does not express the specific transcription factor, the DNA will remain in the cytoplasm until cell division or until it is degraded by cyto‐ plasmic nucleases. Again, as for the SV40 DTS, these cell-specific DTSs work in cultured cells

and in animal tissues to increase gene expression in a cell-restricted manner [83, 86].

A number of other methods for enhancing nuclear import have been studied, all of which center around exploiting the cells protein nuclear import machinery. These approaches include complexing plasmid DNA with NLS peptides, nuclear proteins or small molecule ligands. The success of NLS peptides has been variable, likely due to the fact that the NLS must be visible

enhancing the efficiency of non-viral gene delivery.

80 Novel Gene Therapy Approaches

expression as well as in tissues in living animals [81-83].

To maximize non-viral gene delivery, levels of expression must be improved. Unfortunately, many extracellular and intracellular barriers (including the extracellular environment, immune scavengers, the cell membrane, endosomal escape, the cytoskeletal network and the nuclear membrane) preclude efficient gene transfer. In this review, we have focused on these barriers and various means to overcome them. The goal of all gene therapy approaches is to target enough DNA to the nuclei of cells to obtain sufficient expression for a therapeutic effect. By characterizing and understanding these barriers, we can overcome our relative inability to target substantial amounts of DNA to the nucleus and increase transfection efficiency and ultimately gene therapy.

## **Author details**

Lynn F. Gottfried and David A. Dean

Department of Pediatrics, University of Rochester Medical Center, Rochester, NY, USA

## **References**


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Extracellular and Intracellular Barriers to Non-Viral Gene Transfer

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**Section 2**

**Gene Therpay Using Viral Vectors**


**Gene Therpay Using Viral Vectors**

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**Chapter 5**

**Viral Vectors for Vaccine Development**

Recombinant vectors can be used to deliver antigens and to stimulate immune responses in humans. Viral vectors possess various intrinsic properties which may lend to advantages and disadvantages for usage for a given therapeutic application [reviewed by Larocca and Schlom] [1]. The safety and flexibility of recombinant viral vectors have lead to their usage in gene therapy, virotherapy, and vaccine applications. In this chapter, we will discuss the utility and importance of recombinant vectors as vaccine agents. This chapter will highlight some of the uses of recombinant viral vectors for therapeutic vaccines; and will mostly focus on the application of a range of recombinant viral vectors for prophylactic vaccines against infectious agents. More specifically, this chapter will focus in depth on the use of recombinant adenovirus

Viruses can be used as gene delivery tools for a variety of diseases and conditions [1]. Most viruses are naturally immunogenic and can be engineered to express genes that modulate the immune system or express tumor antigen transgenes. Human Ad vectors have been widely used as vehicles for gene therapy [2]. Replication-defective Ads were the first vectors to be evaluated for *in vivo* gene transfer in a wide variety of preclinical models. For instance, Stratford-Perricaudet, and group reported efficient, long-term *in vivo* gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant Ad vector [3]. This study focused on the transfer of the report gene, *β*-*galactosidase*; however, studies similar to this lead to the delivery of therapeutic genes by means of recombinant vectors. Routinely, viral vectors such as human Ad vectors have been engineered to express

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

© 2013 Matthews 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Qiana L. Matthews, Linlin Gu,

http://dx.doi.org/10.5772/54700

**1. Introduction**

Alexandre Krendelchtchikov and Zan C. Li

Additional information is available at the end of the chapter

(Ad) for vaccine development against infectious agents.

**2. Gene therapy vectors and oncolytic vectors**

## **Viral Vectors for Vaccine Development**

Qiana L. Matthews, Linlin Gu, Alexandre Krendelchtchikov and Zan C. Li

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54700

## **1. Introduction**

Recombinant vectors can be used to deliver antigens and to stimulate immune responses in humans. Viral vectors possess various intrinsic properties which may lend to advantages and disadvantages for usage for a given therapeutic application [reviewed by Larocca and Schlom] [1]. The safety and flexibility of recombinant viral vectors have lead to their usage in gene therapy, virotherapy, and vaccine applications. In this chapter, we will discuss the utility and importance of recombinant vectors as vaccine agents. This chapter will highlight some of the uses of recombinant viral vectors for therapeutic vaccines; and will mostly focus on the application of a range of recombinant viral vectors for prophylactic vaccines against infectious agents. More specifically, this chapter will focus in depth on the use of recombinant adenovirus (Ad) for vaccine development against infectious agents.

## **2. Gene therapy vectors and oncolytic vectors**

Viruses can be used as gene delivery tools for a variety of diseases and conditions [1]. Most viruses are naturally immunogenic and can be engineered to express genes that modulate the immune system or express tumor antigen transgenes. Human Ad vectors have been widely used as vehicles for gene therapy [2]. Replication-defective Ads were the first vectors to be evaluated for *in vivo* gene transfer in a wide variety of preclinical models. For instance, Stratford-Perricaudet, and group reported efficient, long-term *in vivo* gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant Ad vector [3]. This study focused on the transfer of the report gene, *β*-*galactosidase*; however, studies similar to this lead to the delivery of therapeutic genes by means of recombinant vectors. Routinely, viral vectors such as human Ad vectors have been engineered to express

© 2013 Matthews 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

[4–6] or display [7–9] herpes simplex virus type 1 thymidine kinase which kills proliferating tumor cells in the presence of the prodrug gancyclovir. This strategy is commonly referred to as cancer gene therapy. Concerning non-cancerous diseases, viral vectors have been utilized to deliver genes to a multitude of cells types ranging from dental cells [10], islets cells [11], and many other cell types. In these instances, viral vectors transduce cells to deliver genes which may lead to an overexpression or knock down of protein, leading to a corrective phenomenon or destruction of damaged cells.

cancer [29]. Therapeutic strategies related to DCs, cancer vaccination, and oncolytic vectors

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 93

Each viral vector has its own distinct advantage and disadvantage. The most extensively studied viral vectors are from the poxviridae family. They include derivatives of vaccinia virus from the orthopoxvirus genera, and members of the avipoxvirus genera, such as fowlpox and canarypox (ALVAC). Poxviruses have a long and successful track record related to vaccination. In particular, vaccinia virus was used to vaccinate over one billion people against smallpox, leading to the eradication of the disease in 1978. Poxviruses are double stranded DNA viruses with a linear genome. Poxviruses have the ability to accept large inserts of foreign DNA, and therefore can accommodate multiple genes. Vaccinia virus has a genome of ~190 kilobases (kb), which encodes for ~250 genes [32]. Fowlpox viruses have a ~260 kb to 309 kb genome with approximately 260 recognized genes. Attenuated canarypox strain ALVAC has an approxi‐

Viral replication and transcription of the poxvirus genome is limited to the cytoplasm of the host cell. This extranuclear replication eliminates the risk of insertional mutagenesis, the random insertion of viral genetic sequences into host cell genomic DNA [34]. Vaccinia virus infects mammalian cells, and expresses recombinant genes for about 7 days before the infected cell are eliminated by the immune system [21]. Avipoxviruses infect mammalian cells and express their transgenes for 14 to 21 days [35,36]. Despite the attractive features of poxviruses, replication competent viruses like vaccinia should not be administered to severely immuno‐ compromised patients. To circumvent this problem, an attenuated vaccinia virus called modified vaccinia virus Ankara (MVA) was developed for high-risk individuals. MVA was developed by over 500 serial passages of a smallpox vaccine from Ankara, Turkey, in chicken embryo fibroblasts (CEF). This technique resulted in over 15% loss of the vaccinia virus genome [37]. MVA can infect mammalian cells and express transgenes, but it cannot produce infectious viral particles. Similarly, fowlpox and canarypox, which are pathogenic in some avian species, are unable to productively infect humans because they cannot complete their life cycle and form infectious particles [38]. As a result, mammalian poxviruses generate a stronger immune response compared to avipoxviruses. Unfortunately, MVA and vaccinia virus vectors can only be given once or twice to vaccinia immune or vaccinia naïve patients due to the development of host neutralizing antibodies against these vectors [39]. Neutralizing antibodies (NAbs) are not developed against the avipoxvirus vectors, allowing them to be given several times to patients as booster vaccinations [40]. Similarly, alphaviruses, like avipox viruses, are also desirable vectors because infected hosts do not develop neutralizing antibodies to the vectors,

In 2010, adenovirus-based vectors accounted for 23.9% of gene-therapy clinical trials [41]. The broad utility of these vectors is derived from several key characteristics: (a) the recombinant viral genome is readily manipulated; (b) replication-defective Ads can be propagated in

are summarized in the following reviews [17,29–31].

**3. Viral vectors for vaccine development**

mate 330 kb genome with ~320 putative genes [33].

allowing for multiple administrations.

Viruses can also be used as oncolytic agents. Oncolytic viruses which have been identified or engineered belong to several viral families. They include herpes simplex viruses, adenovirus, retroviruses, paramyxoviruses, and poxviruses [12]. These viruses can be categorized into four major groups on the basis of their oncolytic restriction: (1) mutation/deletion derived viruses, (2) transcriptionally targeted oncolytic viruses, (3) transductionally targeted oncolytic vectors, and (4) "naturally smart" viruses [13]. Oncolytic viruses for cancer exploit the difference of the molecular makeup between the tumor cells and their normal counterparts; they also utilize recombinant DNA technology to engineer viral vectors to selectively replicate in the tumor cells and destroy them. Conditionally replicative Ads (CRAds) [14,15], measles virus [16,17], herpes simplex virus [18], and vesicular stomatitis virus [reviewed in [1]], have been shown to preferentially infect and propagate in tumor cells. It has been demonstrated that these vectors not only have direct cytopathic effect on tumor cells but in addition, these oncolytic vectors are likely to enhance immune-mediated killing of tumor cells likely through the release of tumor antigens. Tumor antigens have been demonstrated to be more immunogenic when delivered as transgenes in a viral vector, compared with employing tumor antigens used as a peptide or protein vaccine [19,20].

However, some oncolytic vectors are unlikely to be used as cancer vaccines due to the short duration of transgene expression in infected cells given the onset of lysis; this might limit their ability to elicit a robust immune response against the transgene. Many types of recombinant vectors can infect antigen presenting cells (APC), specifically dendritic cells (DCs). Once engineered recombinant vectors infect APCs and then express antigens or transgenes which are then presented to the immune system [21–26]. However, some oncolytic vectors have a limited tropism for DCs. Ads or CRAds do not infect DCs well due to the fact that DCs possess limited expression of the primary Ad5 docking receptor, Cosxackie-Adenovirus Receptor (CAR). Naturally, DCs are virtually resistant to Ad5 infection, presenting a challenge for effective transduction of DCs by Ad [27]. Direct *in vivo* administration of untargeted Ad5 may result in cytopathic effects due to ectopic gene transfer to CAR expressing bystander cells rather than DCs. Moreover, additional antigen presentation by these transduced non-proces‐ sional APCs may lead to suboptimal T cell activation, or even tolerance induction [28]. Despite these caveats, DCs are key orchestrators of the adaptive immune system. DCs have an exceptional ability to capture, process, and present antigens to activate naïve T cells. DCs have the ability to regulate the nature of the T cell response by providing appropriate co-stimulatory signals that dictate immunogenic or tolerogenic T cell stimulation. These unique features make targeted manipulation of DCs an attractive approach for modulating immune response against cancer [29]. Therapeutic strategies related to DCs, cancer vaccination, and oncolytic vectors are summarized in the following reviews [17,29–31].

## **3. Viral vectors for vaccine development**

[4–6] or display [7–9] herpes simplex virus type 1 thymidine kinase which kills proliferating tumor cells in the presence of the prodrug gancyclovir. This strategy is commonly referred to as cancer gene therapy. Concerning non-cancerous diseases, viral vectors have been utilized to deliver genes to a multitude of cells types ranging from dental cells [10], islets cells [11], and many other cell types. In these instances, viral vectors transduce cells to deliver genes which may lead to an overexpression or knock down of protein, leading to a corrective phenomenon

Viruses can also be used as oncolytic agents. Oncolytic viruses which have been identified or engineered belong to several viral families. They include herpes simplex viruses, adenovirus, retroviruses, paramyxoviruses, and poxviruses [12]. These viruses can be categorized into four major groups on the basis of their oncolytic restriction: (1) mutation/deletion derived viruses, (2) transcriptionally targeted oncolytic viruses, (3) transductionally targeted oncolytic vectors, and (4) "naturally smart" viruses [13]. Oncolytic viruses for cancer exploit the difference of the molecular makeup between the tumor cells and their normal counterparts; they also utilize recombinant DNA technology to engineer viral vectors to selectively replicate in the tumor cells and destroy them. Conditionally replicative Ads (CRAds) [14,15], measles virus [16,17], herpes simplex virus [18], and vesicular stomatitis virus [reviewed in [1]], have been shown to preferentially infect and propagate in tumor cells. It has been demonstrated that these vectors not only have direct cytopathic effect on tumor cells but in addition, these oncolytic vectors are likely to enhance immune-mediated killing of tumor cells likely through the release of tumor antigens. Tumor antigens have been demonstrated to be more immunogenic when delivered as transgenes in a viral vector, compared with employing tumor antigens used as a

However, some oncolytic vectors are unlikely to be used as cancer vaccines due to the short duration of transgene expression in infected cells given the onset of lysis; this might limit their ability to elicit a robust immune response against the transgene. Many types of recombinant vectors can infect antigen presenting cells (APC), specifically dendritic cells (DCs). Once engineered recombinant vectors infect APCs and then express antigens or transgenes which are then presented to the immune system [21–26]. However, some oncolytic vectors have a limited tropism for DCs. Ads or CRAds do not infect DCs well due to the fact that DCs possess limited expression of the primary Ad5 docking receptor, Cosxackie-Adenovirus Receptor (CAR). Naturally, DCs are virtually resistant to Ad5 infection, presenting a challenge for effective transduction of DCs by Ad [27]. Direct *in vivo* administration of untargeted Ad5 may result in cytopathic effects due to ectopic gene transfer to CAR expressing bystander cells rather than DCs. Moreover, additional antigen presentation by these transduced non-proces‐ sional APCs may lead to suboptimal T cell activation, or even tolerance induction [28]. Despite these caveats, DCs are key orchestrators of the adaptive immune system. DCs have an exceptional ability to capture, process, and present antigens to activate naïve T cells. DCs have the ability to regulate the nature of the T cell response by providing appropriate co-stimulatory signals that dictate immunogenic or tolerogenic T cell stimulation. These unique features make targeted manipulation of DCs an attractive approach for modulating immune response against

or destruction of damaged cells.

92 Novel Gene Therapy Approaches

peptide or protein vaccine [19,20].

Each viral vector has its own distinct advantage and disadvantage. The most extensively studied viral vectors are from the poxviridae family. They include derivatives of vaccinia virus from the orthopoxvirus genera, and members of the avipoxvirus genera, such as fowlpox and canarypox (ALVAC). Poxviruses have a long and successful track record related to vaccination. In particular, vaccinia virus was used to vaccinate over one billion people against smallpox, leading to the eradication of the disease in 1978. Poxviruses are double stranded DNA viruses with a linear genome. Poxviruses have the ability to accept large inserts of foreign DNA, and therefore can accommodate multiple genes. Vaccinia virus has a genome of ~190 kilobases (kb), which encodes for ~250 genes [32]. Fowlpox viruses have a ~260 kb to 309 kb genome with approximately 260 recognized genes. Attenuated canarypox strain ALVAC has an approxi‐ mate 330 kb genome with ~320 putative genes [33].

Viral replication and transcription of the poxvirus genome is limited to the cytoplasm of the host cell. This extranuclear replication eliminates the risk of insertional mutagenesis, the random insertion of viral genetic sequences into host cell genomic DNA [34]. Vaccinia virus infects mammalian cells, and expresses recombinant genes for about 7 days before the infected cell are eliminated by the immune system [21]. Avipoxviruses infect mammalian cells and express their transgenes for 14 to 21 days [35,36]. Despite the attractive features of poxviruses, replication competent viruses like vaccinia should not be administered to severely immuno‐ compromised patients. To circumvent this problem, an attenuated vaccinia virus called modified vaccinia virus Ankara (MVA) was developed for high-risk individuals. MVA was developed by over 500 serial passages of a smallpox vaccine from Ankara, Turkey, in chicken embryo fibroblasts (CEF). This technique resulted in over 15% loss of the vaccinia virus genome [37]. MVA can infect mammalian cells and express transgenes, but it cannot produce infectious viral particles. Similarly, fowlpox and canarypox, which are pathogenic in some avian species, are unable to productively infect humans because they cannot complete their life cycle and form infectious particles [38]. As a result, mammalian poxviruses generate a stronger immune response compared to avipoxviruses. Unfortunately, MVA and vaccinia virus vectors can only be given once or twice to vaccinia immune or vaccinia naïve patients due to the development of host neutralizing antibodies against these vectors [39]. Neutralizing antibodies (NAbs) are not developed against the avipoxvirus vectors, allowing them to be given several times to patients as booster vaccinations [40]. Similarly, alphaviruses, like avipox viruses, are also desirable vectors because infected hosts do not develop neutralizing antibodies to the vectors, allowing for multiple administrations.

In 2010, adenovirus-based vectors accounted for 23.9% of gene-therapy clinical trials [41]. The broad utility of these vectors is derived from several key characteristics: (a) the recombinant viral genome is readily manipulated; (b) replication-defective Ads can be propagated in complementing cell lines; (c) Ads infect a broad range of target cells, [42,43] and (d) Ads can achieve high levels of *in viv*o gene transfer with concomitantly high levels of transgene expression [44]. Adenovirus is a non-enveloped double stranded DNA virus. The 36 kb genome can accommodate cDNA sequences of up to 7.5 kb. Replication of the adenovirus occurs in the nucleus but remains extrachromosomal, minimizing the risk associated with insertional mutagenesis. The majority of Ad vectors are replication- incompetent because of a deletion of the viral gene, E1. This limits the vectors' pathogenicity, while still allowing for humoral and cellular responses to the transgene. Most Ad vectors are E3-deleted [E1-, E3-], for the potential to have increased cloning capacity. However, retainment of the E3 gene-encoding regions within an [E1-, E3+] Ad vector would given an optimal effect related to vector characteristics. There has been some speculation that E3 gene promoters are dependent primarily upon the trans-activation capabilities of the E1 gene products. There have been various studies where the E3 region (or selected genes from E3) is re-introduced into the Ad vector under appropriate control of E1 independent promoters. These studies have shown some improvement in small animal models, including reduced humoral and CD8 T cell responses to the vector, and/or long-term transgene expression [45–47]. Oncolytic vectors have, in some cases, the E1 regions intact and, therefore, could potentially benefit from expression of these immune evasion proteins [48]. Most importantly, Ad vectors can be easily manipulated in the laboratory setting, which allows researchers to easily modify these vectors. This includes retargeting the viral tropism to infect DCs which are usually resistant to Ad infection. These properties have also led to Ads being used as molecular vaccine agents.

the "antigen capsid-incorporation" strategy, vector chimeras, covalent modifications (i.e. such as polyethylene glycol, PEGylation) [66–68], and Gutless (helper-dependent) Ad vectors. Gutless vectors are devoid of the majority of viral genes. Therefore, they avoid cellular immunity to Ad viral genes and diminish liver toxicity, thus promoting long-term transgene

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 95

**5. Antigen capsid-incorporation strategies for vaccination schemes**

base, and protein IX (pIX), similar to the illustration depicted in Figure 1 [82,83].

tide or antigen incorporation [84,88–94].

Fiber, penton base, and pIX have been used for antigen capsid-incorporation strategies [84]. However, the major capsid protein hexon has been involved in the majority of anti‐ gen capsid-incorporation strategies. Hexon is the most plentiful of the capsid's structural proteins, accounting for 63% of the total protein mass [85,86]. Current hexon sequence analysis from different species revealed that, in addition to the conserved regions, there were 9 discrete hypervariable regions (HVRs). The HVRs of hexon contain serotype-spe‐ cific epitopes [85,87]. The loops at the top of the HVRs are the most amenable to modifi‐ cation by genetic engineering. Some research groups have shown that short heterologous peptides can be incorporated within the HVRs of the hexon without affecting the viri‐ on's stability or function. Of note, a subset of these modifiable loops were exposed on the surface of the capsid [88,89]. HVRs1, 2, and 5 have been utilized respectively for pep‐

The "antigen capsid-incorporation" or "capsid-display of antigen" strategies are currently being used to circumvent drawbacks associated with conventional transgene expression of antigens by viral vectors. Initially, the "capsid-display" strategy had been developed and utilized to present ligands [8,71,72], imaging motifs [7–9,73–75], and more recently immunomodulatory inhibitors and/or activators ligands [76–78]. More recently, the cap‐ sid-display of antigen strategy has been used to present antigens for vaccination applica‐ tions. The antigen capsid-incorporation strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The human rhinovirus has been uti‐ lized for HIV vaccination in the context of the antigen capsid-incorporation strategy. Re‐ searchers have constructed human rhinovirus: HIV chimeras to stimulate immunity against HIV-1 [79]. As well, researchers have designed combinatorial libraries of human rhinovirus capsid incorporated HIV-1 glycoprotein 41 (gp41) epitope, eliciting antibodies whose activity can mimic the NAb effect [80]. Commercial and clinical development of Ad-based HIV vaccines has progressed faster than the development of vector systems such as human rhinovirus because the tremendous flexibility of Ad generally exceeds that of current rhinovirus systems. For instance, since human rhinovirus is a relatively small RNA virus, the human rhinovirus platform can only display 60 copies of a single HIV-1 epitope [79]. In contrast, the Ad vector capsid platform could allow incorporation of HIV-1 epitopes into 4 structurally distinct domains including hexon [81], fiber, penton

expression [67,69,70].

#### **4. Adenoviral vectors as vaccine agents**

Traditionally, Ad vaccination embodies the concept that the vector uses the host-cell machi‐ nery to express antigens that are encoded as transgenes within the viral vector, specifically within the E1 and/or the E3 regions. Cellular and humoral immune responses are generated against these antigens for a vaccine effect. Several preclinical successes have used this approach in animal model systems. In one example, an Ad serotype 5 (Ad5) vector encoding Ebola surface glycoprotein generated neutralizing antibodies and protected monkeys after a single administration of Ebola [49]. In murine models protection against malaria has also been observed using Ad vectors that express the circumsporozite antigen in *Plasmodium yoelii* [50– 52] or *Plasmodium berghei [53].* Ad vectors are currently being used in clinical trials for vaccine development against tuberculosis [54], HIV [55–57], and malaria [58–60]. Ad5-based HIV and malaria vaccines were well tolerated and induced antigen-specific CD4+ T cell, CD8+ T cell, and antibody responses in volunteers [55,59,60].

However, in some instances, these conventional Ad-based vaccines have yielded suboptimal clinical results. These suboptimal results are attributed, in part, to preexisting Ad5 immunity. It is estimated that 50% to 90% of the adult population has preexisting immunity (PEI) to Ad serotype 2 (Ad2) or Ad5 [61–65]; and this Ad PEI can limit efficacy of Ad based vaccinations due to Ad clearance by the immune system. In this regard, innovative strategies have been developed to circumvent drawbacks associated with Ad5 PEI, some of these strategies include the "antigen capsid-incorporation" strategy, vector chimeras, covalent modifications (i.e. such as polyethylene glycol, PEGylation) [66–68], and Gutless (helper-dependent) Ad vectors. Gutless vectors are devoid of the majority of viral genes. Therefore, they avoid cellular immunity to Ad viral genes and diminish liver toxicity, thus promoting long-term transgene expression [67,69,70].

## **5. Antigen capsid-incorporation strategies for vaccination schemes**

complementing cell lines; (c) Ads infect a broad range of target cells, [42,43] and (d) Ads can achieve high levels of *in viv*o gene transfer with concomitantly high levels of transgene expression [44]. Adenovirus is a non-enveloped double stranded DNA virus. The 36 kb genome can accommodate cDNA sequences of up to 7.5 kb. Replication of the adenovirus occurs in the nucleus but remains extrachromosomal, minimizing the risk associated with insertional mutagenesis. The majority of Ad vectors are replication- incompetent because of a deletion of the viral gene, E1. This limits the vectors' pathogenicity, while still allowing for humoral and cellular responses to the transgene. Most Ad vectors are E3-deleted [E1-, E3-], for the potential to have increased cloning capacity. However, retainment of the E3 gene-encoding regions within an [E1-, E3+] Ad vector would given an optimal effect related to vector characteristics. There has been some speculation that E3 gene promoters are dependent primarily upon the trans-activation capabilities of the E1 gene products. There have been various studies where the E3 region (or selected genes from E3) is re-introduced into the Ad vector under appropriate control of E1 independent promoters. These studies have shown some improvement in small animal models, including reduced humoral and CD8 T cell responses to the vector, and/or long-term transgene expression [45–47]. Oncolytic vectors have, in some cases, the E1 regions intact and, therefore, could potentially benefit from expression of these immune evasion proteins [48]. Most importantly, Ad vectors can be easily manipulated in the laboratory setting, which allows researchers to easily modify these vectors. This includes retargeting the viral tropism to infect DCs which are usually resistant to Ad infection. These

properties have also led to Ads being used as molecular vaccine agents.

Traditionally, Ad vaccination embodies the concept that the vector uses the host-cell machi‐ nery to express antigens that are encoded as transgenes within the viral vector, specifically within the E1 and/or the E3 regions. Cellular and humoral immune responses are generated against these antigens for a vaccine effect. Several preclinical successes have used this approach in animal model systems. In one example, an Ad serotype 5 (Ad5) vector encoding Ebola surface glycoprotein generated neutralizing antibodies and protected monkeys after a single administration of Ebola [49]. In murine models protection against malaria has also been observed using Ad vectors that express the circumsporozite antigen in *Plasmodium yoelii* [50– 52] or *Plasmodium berghei [53].* Ad vectors are currently being used in clinical trials for vaccine development against tuberculosis [54], HIV [55–57], and malaria [58–60]. Ad5-based HIV and malaria vaccines were well tolerated and induced antigen-specific CD4+ T cell, CD8+ T cell,

However, in some instances, these conventional Ad-based vaccines have yielded suboptimal clinical results. These suboptimal results are attributed, in part, to preexisting Ad5 immunity. It is estimated that 50% to 90% of the adult population has preexisting immunity (PEI) to Ad serotype 2 (Ad2) or Ad5 [61–65]; and this Ad PEI can limit efficacy of Ad based vaccinations due to Ad clearance by the immune system. In this regard, innovative strategies have been developed to circumvent drawbacks associated with Ad5 PEI, some of these strategies include

**4. Adenoviral vectors as vaccine agents**

94 Novel Gene Therapy Approaches

and antibody responses in volunteers [55,59,60].

The "antigen capsid-incorporation" or "capsid-display of antigen" strategies are currently being used to circumvent drawbacks associated with conventional transgene expression of antigens by viral vectors. Initially, the "capsid-display" strategy had been developed and utilized to present ligands [8,71,72], imaging motifs [7–9,73–75], and more recently immunomodulatory inhibitors and/or activators ligands [76–78]. More recently, the cap‐ sid-display of antigen strategy has been used to present antigens for vaccination applica‐ tions. The antigen capsid-incorporation strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. The human rhinovirus has been uti‐ lized for HIV vaccination in the context of the antigen capsid-incorporation strategy. Re‐ searchers have constructed human rhinovirus: HIV chimeras to stimulate immunity against HIV-1 [79]. As well, researchers have designed combinatorial libraries of human rhinovirus capsid incorporated HIV-1 glycoprotein 41 (gp41) epitope, eliciting antibodies whose activity can mimic the NAb effect [80]. Commercial and clinical development of Ad-based HIV vaccines has progressed faster than the development of vector systems such as human rhinovirus because the tremendous flexibility of Ad generally exceeds that of current rhinovirus systems. For instance, since human rhinovirus is a relatively small RNA virus, the human rhinovirus platform can only display 60 copies of a single HIV-1 epitope [79]. In contrast, the Ad vector capsid platform could allow incorporation of HIV-1 epitopes into 4 structurally distinct domains including hexon [81], fiber, penton base, and protein IX (pIX), similar to the illustration depicted in Figure 1 [82,83].

Fiber, penton base, and pIX have been used for antigen capsid-incorporation strategies [84]. However, the major capsid protein hexon has been involved in the majority of anti‐ gen capsid-incorporation strategies. Hexon is the most plentiful of the capsid's structural proteins, accounting for 63% of the total protein mass [85,86]. Current hexon sequence analysis from different species revealed that, in addition to the conserved regions, there were 9 discrete hypervariable regions (HVRs). The HVRs of hexon contain serotype-spe‐ cific epitopes [85,87]. The loops at the top of the HVRs are the most amenable to modifi‐ cation by genetic engineering. Some research groups have shown that short heterologous peptides can be incorporated within the HVRs of the hexon without affecting the viri‐ on's stability or function. Of note, a subset of these modifiable loops were exposed on the surface of the capsid [88,89]. HVRs1, 2, and 5 have been utilized respectively for pep‐ tide or antigen incorporation [84,88–94].

nized the VP1 capsid of the polio virus [89]. More recently, similar studies have been performed by other research groups. For example, Worgall and colleagues used the antigen capsidincorporation strategy to vaccinate against *Pseudomonas aeruginosa* (*pseudomonas*), a Gramnegative bacterium that causes respiratory tract infections in individuals who are immunocompromised or who have cystic fibrosis [102]. Because *pseudomonas* is an extracel‐ lular pathogen, anti-*pseudomonas* humoral immunity should be sufficient to provide protective immunity. Therefore, *pseudomonas* can be a candidate agent for vaccine development. Several immunogenic peptides have been identified in the outer membrane protein F (OprF) of *pseudomonas*, including the immunodominant 14-mer peptide Epi8. This study characterizes genetic incorporations of a neutralizing epitope from the *pseudomonas* Epi8 into Ad5 HVR5 (AdZ.Epi8) [90]. BALB/c mice immunized with the capsid-modified vectors showed an increase in antibody response consisting of both anti-*pseudomonas* IgG1 and IgG2a subtypes. In addition, mice immunized with the vector containing the OprF epitope were subjected to pulmonary challenge with *pseudomonas*, 60% to 80% of them survived. This group also performed additional studies where they attempted DC targeting in combination with the

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 97

To expand on knowledge gained from previous antigen capsid-incorporation studies, our group set out to create novel vaccine vectors that would yield optimal vaccine efficacy by maximizing the size of antigens which could be incorporated within the capsid protein, hexon. Our 2008 manuscript evaluated the use of Ad5 HVR2 or HVR5 vectors containing identical antigenic epitopes in either region. To compare the capacities and flexibility of Ad5 HVR2 to those of HVR5, we genetically incorporated identical epitopes of increasing size within HVR2 or HVR5 of the Ad5 hexon. The epitopes ranged in size from 33-83 amino acids. Stable vectors were produced with incorporations of 33 amino acids plus a 12 amino acid linker at HVR2 or HVR5. In addition, stable vectors were produced with incorporations up to 53 amino acids plus a 12 amino acid linker in HVR5. With respect to the selected antigens, HVR5 was more permissive, allowing an epitope incorporation of 65 amino acids. Whole virus enzyme-linked immunosorbent assay (ELISA) analysis revealed that the model antigens were virion surfaceexposed, and *in vivo* immunization with these vectors elicited antigen-specific immune

In our most recent published study we evaluated the antigen capsid-incorporation strategy further by using novel vectors that were constructed to provide cellular and humoral HIV immunity [104]. Our study was the first of its kind to genetically incorporate an HIV antigen within the Ad5 hexon's HVR2, alone or in combination with the genomic/E1 incorporation of the HIV Gag gene (Ad5/HVR2-MPER-L15(Gag)). In this study, we incorporated a 24 amino acid epitope of HIV within Ad5 HVR2. The HIV region selected was the membrane proximal ectodomain region (MPER) derived from HIV gp41. When the MPER epitope was incorporated within HVR2 in combination with transgene incorporation, we observed growth kinetics and thermostability changes similar to those observed in other studies after using antigen capsidincorporated vectors [7,105], indicating that incorporation of the MPER epitope within HVR2 was not substantially detrimental to vector characteristics [9,105]. In this study we demon‐ strated for the first time that a disease-specific antigen could be incorporated within HVR2 of

antigen capsid-incorporation strategy [103].

responses [93].

**Figure 1. Antigen Capsid-Incorporation within Adenovirus Structural Proteins.** Adenoviral capsid protein consists of: Hexon (II), Penton Base (III), Fiber (IV), and protein IX (pIX). Antigenic epitopes can be incorporated into these cap‐ sid structural proteins to induce antigen-specific immune responses. For example, this figure depicts the incorporation of HIV antigens from the variable region 2 (CSFNITTT), glycoprotein 41(KWAS) and glycoprotein 120 (INCTRP). This figure is adapted from Nemerow et al., 2009. Virology 384 (2009) 380–388, copyright Elsevier.

One drawback associated with conventional transgene expression of antigen is the inability of Ad-based vectors to produce a potent humoral immune response against certain antigens (as seen in the case of some malaria antigens) [95]. The antigen capsid-incorporation strategy may circumvent this drawback because this strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. By incorporating antigens directly into the capsid proteins, the capsid-incorporated antigen is processed through the exogenous pathway leading to strong humoral response, similar to the response generated by native Ad capsid proteins. Incorporating immunogenic peptides into the Ad capsid offers potential advantages. This strategy may allow vectors to circumvent Ad5 PEI allowing a more robust immune response to either the antigen presented on the vector capsid or the antigen that is expressed as a transgene. Additionally, because anti-Ad capsid responses are augmented by administering the vector repeatedly, immune responses against antigenic epitopes that are part of the Ad capsid should be increased by this approach as well, thus allowing boosting of the response [96–98]. This strategy may also allow for cross-priming [99,100] and activation of CD8+ T cells by means of incorporating T cell helper epitopes into the Ad capsid proteins [90]. Therefore, this antigen capsid-incorporation approach offers feasible opportunities to create Ad-based vaccine vector strategies that circumvent the major limitations associated with traditional Ad-based vaccine vectors.

Preclinically, incorporating antigens into viral capsid structures has been used as a vaccination approach for several diseases [84,90,91,93,94,101]. In 1994, Crompton and colleagues used this strategy for the first time in the context of Ad [101]. Crompton's group genetically incorporated an 8 amino acid sequence of the VP1 capsid protein of poliovirus type 3 into 2 regions of the adenovirus serotype 2 hexon. One of the chimeric vectors produced was able to grow well in tissue culture, and antiserum raised against the Ad with the polio antigen specifically recog‐ nized the VP1 capsid of the polio virus [89]. More recently, similar studies have been performed by other research groups. For example, Worgall and colleagues used the antigen capsidincorporation strategy to vaccinate against *Pseudomonas aeruginosa* (*pseudomonas*), a Gramnegative bacterium that causes respiratory tract infections in individuals who are immunocompromised or who have cystic fibrosis [102]. Because *pseudomonas* is an extracel‐ lular pathogen, anti-*pseudomonas* humoral immunity should be sufficient to provide protective immunity. Therefore, *pseudomonas* can be a candidate agent for vaccine development. Several immunogenic peptides have been identified in the outer membrane protein F (OprF) of *pseudomonas*, including the immunodominant 14-mer peptide Epi8. This study characterizes genetic incorporations of a neutralizing epitope from the *pseudomonas* Epi8 into Ad5 HVR5 (AdZ.Epi8) [90]. BALB/c mice immunized with the capsid-modified vectors showed an increase in antibody response consisting of both anti-*pseudomonas* IgG1 and IgG2a subtypes. In addition, mice immunized with the vector containing the OprF epitope were subjected to pulmonary challenge with *pseudomonas*, 60% to 80% of them survived. This group also performed additional studies where they attempted DC targeting in combination with the antigen capsid-incorporation strategy [103].

**Figure 1. Antigen Capsid-Incorporation within Adenovirus Structural Proteins.** Adenoviral capsid protein consists of: Hexon (II), Penton Base (III), Fiber (IV), and protein IX (pIX). Antigenic epitopes can be incorporated into these cap‐ sid structural proteins to induce antigen-specific immune responses. For example, this figure depicts the incorporation of HIV antigens from the variable region 2 (CSFNITTT), glycoprotein 41(KWAS) and glycoprotein 120 (INCTRP). This

One drawback associated with conventional transgene expression of antigen is the inability of Ad-based vectors to produce a potent humoral immune response against certain antigens (as seen in the case of some malaria antigens) [95]. The antigen capsid-incorporation strategy may circumvent this drawback because this strategy embodies the incorporation of antigenic peptides within the capsid structure of viral vectors. By incorporating antigens directly into the capsid proteins, the capsid-incorporated antigen is processed through the exogenous pathway leading to strong humoral response, similar to the response generated by native Ad capsid proteins. Incorporating immunogenic peptides into the Ad capsid offers potential advantages. This strategy may allow vectors to circumvent Ad5 PEI allowing a more robust immune response to either the antigen presented on the vector capsid or the antigen that is expressed as a transgene. Additionally, because anti-Ad capsid responses are augmented by administering the vector repeatedly, immune responses against antigenic epitopes that are part of the Ad capsid should be increased by this approach as well, thus allowing boosting of the response [96–98]. This strategy may also allow for cross-priming [99,100] and activation of CD8+ T cells by means of incorporating T cell helper epitopes into the Ad capsid proteins [90]. Therefore, this antigen capsid-incorporation approach offers feasible opportunities to create Ad-based vaccine vector strategies that circumvent the major limitations associated with

Preclinically, incorporating antigens into viral capsid structures has been used as a vaccination approach for several diseases [84,90,91,93,94,101]. In 1994, Crompton and colleagues used this strategy for the first time in the context of Ad [101]. Crompton's group genetically incorporated an 8 amino acid sequence of the VP1 capsid protein of poliovirus type 3 into 2 regions of the adenovirus serotype 2 hexon. One of the chimeric vectors produced was able to grow well in tissue culture, and antiserum raised against the Ad with the polio antigen specifically recog‐

figure is adapted from Nemerow et al., 2009. Virology 384 (2009) 380–388, copyright Elsevier.

traditional Ad-based vaccine vectors.

96 Novel Gene Therapy Approaches

To expand on knowledge gained from previous antigen capsid-incorporation studies, our group set out to create novel vaccine vectors that would yield optimal vaccine efficacy by maximizing the size of antigens which could be incorporated within the capsid protein, hexon. Our 2008 manuscript evaluated the use of Ad5 HVR2 or HVR5 vectors containing identical antigenic epitopes in either region. To compare the capacities and flexibility of Ad5 HVR2 to those of HVR5, we genetically incorporated identical epitopes of increasing size within HVR2 or HVR5 of the Ad5 hexon. The epitopes ranged in size from 33-83 amino acids. Stable vectors were produced with incorporations of 33 amino acids plus a 12 amino acid linker at HVR2 or HVR5. In addition, stable vectors were produced with incorporations up to 53 amino acids plus a 12 amino acid linker in HVR5. With respect to the selected antigens, HVR5 was more permissive, allowing an epitope incorporation of 65 amino acids. Whole virus enzyme-linked immunosorbent assay (ELISA) analysis revealed that the model antigens were virion surfaceexposed, and *in vivo* immunization with these vectors elicited antigen-specific immune responses [93].

In our most recent published study we evaluated the antigen capsid-incorporation strategy further by using novel vectors that were constructed to provide cellular and humoral HIV immunity [104]. Our study was the first of its kind to genetically incorporate an HIV antigen within the Ad5 hexon's HVR2, alone or in combination with the genomic/E1 incorporation of the HIV Gag gene (Ad5/HVR2-MPER-L15(Gag)). In this study, we incorporated a 24 amino acid epitope of HIV within Ad5 HVR2. The HIV region selected was the membrane proximal ectodomain region (MPER) derived from HIV gp41. When the MPER epitope was incorporated within HVR2 in combination with transgene incorporation, we observed growth kinetics and thermostability changes similar to those observed in other studies after using antigen capsidincorporated vectors [7,105], indicating that incorporation of the MPER epitope within HVR2 was not substantially detrimental to vector characteristics [9,105]. In this study we demon‐ strated for the first time that a disease-specific antigen could be incorporated within HVR2 of Ad5. Also, we demonstrated that the MPER epitope is surface exposed within HVR2. Most importantly, we observed a humoral anti-HIV response in mice immunized with the hexonmodified vector. Immunization with the MPER-modified vector allows boosting when compared to that of immunization with AdCMVGag vector, possibly because the Ad5/HVR2- MPER-L15(Gag) vector elicits less of an anti-Ad5 immune response. It is plausible that the MPER epitope which is incorporated within this vector reduces the immunogenicity of the Ad5 vector. This finding is notable because HVR2 has not been fully explored for its potential use in antigen capsid-incorporation strategies.

two or more distinct organisms. In this regard, our current unpublished data herein demon‐ strates for the first time ever that multiple antigens can be incorporated in combination at two sites within the major capsid protein, hexon (Figures 3, 4 and 5). In order to create a multivalent vaccine vector, we created vectors that display antigens within HVR1 and HVR2 or HVR1 and HVR5. Our unpublished findings focus on the generation of proof-ofconcept vectors that can ultimately result in the development of multivalent vaccine vectors displaying dual antigens within the hexon of one Ad virion particle. These novel vectors uti‐ lize HVR1 as an incorporation site for a seven amino acid region (ELDKWAS) (called KWAS in this chapter) derived from HIV gp41; in combination with a six Histidine (His6) incorpo‐ ration within HVR2 or HVR5. After these vectors were rescued they were designated as Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6. In order to de‐ termine the quality of these vectors, we determined the viral particle (VP)/infectious particle (IP) ratios for the hexon-modified vectors. We compared these parameters to unmodified Ad5. Importantly, we observed similar VP/IP ratios for Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6 as compared to Ad5 (Figure 3). These values are

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 99

**Figure 2. Adenovirus Presenting Capsid-Incorporated HIV Antigen Elicits an HIV Humoral Immune Response in C57BL/6 Mice.** C57BL/6 mice (n=8) were primed and boosted with 1 x 1010 VP of Ad vectors. Post-prime and postboost sera was collected at various time points for ELISA binding assays. 10 μM of purified MPER (EKNEKELLELDK‐ WASLWNWFDITN) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera and the binding was detected with HRP conjugated

secondary antibody. OD absorbance at 450 nm represents MPER antibody levels in sera.

similar to what we observed in our previous 2008 study [93].

While many studies have examined the efficacy of targeting genetic vector based vaccines to DCs to enhance cellular immune responses, our group will examine a novel question. How does DC targeting affect the vector capsid antigenicity with respect to focusing humoral immune responses to finite amounts of capsid-incorporated HIV antigen? Specifically, we are interested in evaluating how DC targeting impacts the quality and potency of humoral responses generated from our capsid-incorporated antigen approach. As previously men‐ tioned, in our 2010 manuscript, we illustrated that immunizations with Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15ΔE1 yielded MPER-specific humoral responses in BALB/ c mice [104]. However, we eventually plan to use the antigen-capsid incorporation system in combination with DCs activation. The C57BL/6 mouse model will allow us to better evaluate the antigen capsid-incorporation strategy in combination with DC targeting. Our initial data illustrates (data not shown) that there are substantially more DCs available for targeting in C57BL/6 mice as compared to BALB/c mice. Therefore, it was necessary to evaluate our antigen capsid-incorporation strategy in C57BL/6 mice. In brief, C57BL/6 mice were immunized with Ad5/HVR2-MPER-L15(G/L) (green fluorescent protein/ luciferase) and Ad5/HVR2-MPER-L15(Gag), respectively. 17 days later these mice were boosted in a similar manner with the same vectors. This data indicates that there is MPER-specific humoral response produced after immunizations with both vectors in C57BL/6 mice (Figure 2). In summary, we observed a similar outcome with our antigen capsid-incorporated vectors in C57BL/6 mice; therefore, we can continue with our DC targeted antigen capsid-incorporated studies. These experiments are likely to be very informative because DCs represent a unique junction for intervention by antigen-specific vaccination strategies.

With the vast diversity of many bacterial pathogens and viral pathogens, such as HIV, the need remains for vaccine vectors that yield a broad immune response. Successful HIV vacci‐ nation remains a tremendous challenge because HIV-1 vaccine strategies must contend with the enormous global sequence diversity of HIV-1. To attempt to overcome this obstacle, mo‐ saic vectors and Ad vectors schemes that utilize "heterologous inserts" in prime-boost regi‐ mens have been developed in order to increase the breadth and depth of cellular immune responses in nonhuman primate models [106,107]. These vectors have shown promise; how‐ ever, these constructs focused primarily on cellular immunity. It is likely that the most suc‐ cessful prophylactic HIV-1 vaccine will elicit a broad and robust cellular and humoral response. In order to create vectors that could provide a varied humoral response we gener‐ ated multivalent proof-of-concept vectors. Our definition of a multivalent vector is, a vector that has the ability to vaccinate against several strains of an organism or vaccinate against two or more distinct organisms. In this regard, our current unpublished data herein demon‐ strates for the first time ever that multiple antigens can be incorporated in combination at two sites within the major capsid protein, hexon (Figures 3, 4 and 5). In order to create a multivalent vaccine vector, we created vectors that display antigens within HVR1 and HVR2 or HVR1 and HVR5. Our unpublished findings focus on the generation of proof-ofconcept vectors that can ultimately result in the development of multivalent vaccine vectors displaying dual antigens within the hexon of one Ad virion particle. These novel vectors uti‐ lize HVR1 as an incorporation site for a seven amino acid region (ELDKWAS) (called KWAS in this chapter) derived from HIV gp41; in combination with a six Histidine (His6) incorpo‐ ration within HVR2 or HVR5. After these vectors were rescued they were designated as Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6. In order to de‐ termine the quality of these vectors, we determined the viral particle (VP)/infectious particle (IP) ratios for the hexon-modified vectors. We compared these parameters to unmodified Ad5. Importantly, we observed similar VP/IP ratios for Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6 as compared to Ad5 (Figure 3). These values are similar to what we observed in our previous 2008 study [93].

Ad5. Also, we demonstrated that the MPER epitope is surface exposed within HVR2. Most importantly, we observed a humoral anti-HIV response in mice immunized with the hexonmodified vector. Immunization with the MPER-modified vector allows boosting when compared to that of immunization with AdCMVGag vector, possibly because the Ad5/HVR2- MPER-L15(Gag) vector elicits less of an anti-Ad5 immune response. It is plausible that the MPER epitope which is incorporated within this vector reduces the immunogenicity of the Ad5 vector. This finding is notable because HVR2 has not been fully explored for its potential

While many studies have examined the efficacy of targeting genetic vector based vaccines to DCs to enhance cellular immune responses, our group will examine a novel question. How does DC targeting affect the vector capsid antigenicity with respect to focusing humoral immune responses to finite amounts of capsid-incorporated HIV antigen? Specifically, we are interested in evaluating how DC targeting impacts the quality and potency of humoral responses generated from our capsid-incorporated antigen approach. As previously men‐ tioned, in our 2010 manuscript, we illustrated that immunizations with Ad5/HVR2-MPER-L15(Gag) and Ad5/HVR2-MPER-L15ΔE1 yielded MPER-specific humoral responses in BALB/ c mice [104]. However, we eventually plan to use the antigen-capsid incorporation system in combination with DCs activation. The C57BL/6 mouse model will allow us to better evaluate the antigen capsid-incorporation strategy in combination with DC targeting. Our initial data illustrates (data not shown) that there are substantially more DCs available for targeting in C57BL/6 mice as compared to BALB/c mice. Therefore, it was necessary to evaluate our antigen capsid-incorporation strategy in C57BL/6 mice. In brief, C57BL/6 mice were immunized with Ad5/HVR2-MPER-L15(G/L) (green fluorescent protein/ luciferase) and Ad5/HVR2-MPER-L15(Gag), respectively. 17 days later these mice were boosted in a similar manner with the same vectors. This data indicates that there is MPER-specific humoral response produced after immunizations with both vectors in C57BL/6 mice (Figure 2). In summary, we observed a similar outcome with our antigen capsid-incorporated vectors in C57BL/6 mice; therefore, we can continue with our DC targeted antigen capsid-incorporated studies. These experiments are likely to be very informative because DCs represent a unique junction for intervention by

With the vast diversity of many bacterial pathogens and viral pathogens, such as HIV, the need remains for vaccine vectors that yield a broad immune response. Successful HIV vacci‐ nation remains a tremendous challenge because HIV-1 vaccine strategies must contend with the enormous global sequence diversity of HIV-1. To attempt to overcome this obstacle, mo‐ saic vectors and Ad vectors schemes that utilize "heterologous inserts" in prime-boost regi‐ mens have been developed in order to increase the breadth and depth of cellular immune responses in nonhuman primate models [106,107]. These vectors have shown promise; how‐ ever, these constructs focused primarily on cellular immunity. It is likely that the most suc‐ cessful prophylactic HIV-1 vaccine will elicit a broad and robust cellular and humoral response. In order to create vectors that could provide a varied humoral response we gener‐ ated multivalent proof-of-concept vectors. Our definition of a multivalent vector is, a vector that has the ability to vaccinate against several strains of an organism or vaccinate against

use in antigen capsid-incorporation strategies.

98 Novel Gene Therapy Approaches

antigen-specific vaccination strategies.

**Figure 2. Adenovirus Presenting Capsid-Incorporated HIV Antigen Elicits an HIV Humoral Immune Response in C57BL/6 Mice.** C57BL/6 mice (n=8) were primed and boosted with 1 x 1010 VP of Ad vectors. Post-prime and postboost sera was collected at various time points for ELISA binding assays. 10 μM of purified MPER (EKNEKELLELDK‐ WASLWNWFDITN) antigenic peptide was bound to ELISA plates. Residual unbound peptide was washed from the plates. The plates were then incubated with immunized mice sera and the binding was detected with HRP conjugated secondary antibody. OD absorbance at 450 nm represents MPER antibody levels in sera.


**Figure 3. Virological Characterization of Multivalent Vaccine Vectors Displaying Dual Antigens.**

After the successful rescue of the multivalent vectors we next sought to verify expression of genetic incorporations at the protein level by Western blot analysis. In order to determine if the dual hexon-modified vectors were presenting His6 tag within HVR2 or HVR5, the vectors were subjected to Western blot analysis. The His6 tag was detected as a 117 kilodalton (kDa) protein band associated with Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6. Figure 4, lanes 2 and 3, respectively. The size of the 117 kDa band corresponds to the expected size of the Ad5 hexon protein with His6 peptide genetically incorporated into the HVR2 or HVR5 region. As expected, there was no His6 protein detected on Ad5 wild type particles (Figure 4, lane 1). Similar results were observed when these vectors were analyzed in order to verify expression of KWAS incorporations within the HVR1 locale of our dual hexon-modified vectors (Figure 5, lanes 2 and 3, respectively).

**Figure 5. Western Blotting Confirms the Presence of KWAS on Multivalent Vaccine Vectors Displaying Dual An‐ tigens.** Western blotting confirmed the presence of KWAS incorporation within the dual modified vectors. In this as‐ say, 1 x 1010 VP of Ad5 (lane 1), Ad5/H5-HVR1-KWAS-HVR2-His6 (lane 2), and Ad5/H5-HVR1-KWAS-HVR5-His6 (lane 3) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinyli‐ dene fluoride membrane then blotted with anti-gp41 antibody (NIH AIDS Reagent Program). The arrow indicates

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 101

The size of the 117 kDa band corresponds to the expected size of the Ad5 hexon protein with KWAS peptide genetically incorporated into the HVR1 region. There was a slight KWAS protein detected on Ad5 wild type particles, we attribute this to a cross reactive sequence within the Ad vector (Figure 5, lane 1). Most importantly, Figures 4 and 5 illustrate that KWAS and His6 proteins were incorporated at comparable levels within HVR1 and HVR2 or HVR5. We also performed a series of ELISA assays to verify that the KWAS and His6 motifs were surface accessible on the hexon double-modified virions. These results indicated that the His6 epitope was properly exposed on the virion surfaces when incorporated within HVR2 or HVR5 (data not shown). In addition, the HIV-specific ELISA also illustrated that that the HIV motif was accessible on the virion surface within the HVR1 region. Our results showed significant binding of the anti-HIV antibody to the Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5- HVR1-KWAS-HVR5-His6, whereas no binding was seen in response to Ad5 control (data not shown). Currently, we are in the process of testing these vectors *in vivo*. Our initial findings lead us to believe that these vectors can have tremendous impact for multivalent vaccine

**6. Chimeric serotype Ad vectors and rare serotype vectors for vaccine usage**

In the near future, it is possible that viral vector-based vaccination will become a common clinical intervention; therefore, it has become increasingly necessary to design vectors that can overcome Ad5 pre-existing immunity [108,109]. In order to overcome Ad5 pre-existing immunity rare and non-human Ad serotypes have been used. Chimeric Ad vectors consist of

KWAS/gp41 protein genetically incorporated into the hexon.

development.

**Figure 4. Western Blotting Confirms the Presence of His6 on Multivalent Vaccine Vectors Displaying Dual Anti‐ gens.** Western blotting confirmed the presence of His6 incorporation within the dual modified vectors. In this assay, 1 x 1010 VP of Ad5 (lane 1), Ad5/H5-HVR1-KWAS-HVR2-His6 (lane 2), and Ad5/H5-HVR1-KWAS-HVR5-His6 (lane 3) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinylidene fluo‐ ride membrane then blotted with anti-His antibody. The arrow indicates the His tag is genetically incorporated into the hexon protein.

**Figure 3. Virological Characterization of Multivalent Vaccine Vectors Displaying Dual Antigens.**

hexon-modified vectors (Figure 5, lanes 2 and 3, respectively).

the hexon protein.

100 Novel Gene Therapy Approaches

After the successful rescue of the multivalent vectors we next sought to verify expression of genetic incorporations at the protein level by Western blot analysis. In order to determine if the dual hexon-modified vectors were presenting His6 tag within HVR2 or HVR5, the vectors were subjected to Western blot analysis. The His6 tag was detected as a 117 kilodalton (kDa) protein band associated with Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5-HVR1-KWAS-HVR5-His6. Figure 4, lanes 2 and 3, respectively. The size of the 117 kDa band corresponds to the expected size of the Ad5 hexon protein with His6 peptide genetically incorporated into the HVR2 or HVR5 region. As expected, there was no His6 protein detected on Ad5 wild type particles (Figure 4, lane 1). Similar results were observed when these vectors were analyzed in order to verify expression of KWAS incorporations within the HVR1 locale of our dual

**Figure 4. Western Blotting Confirms the Presence of His6 on Multivalent Vaccine Vectors Displaying Dual Anti‐ gens.** Western blotting confirmed the presence of His6 incorporation within the dual modified vectors. In this assay, 1 x 1010 VP of Ad5 (lane 1), Ad5/H5-HVR1-KWAS-HVR2-His6 (lane 2), and Ad5/H5-HVR1-KWAS-HVR5-His6 (lane 3) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinylidene fluo‐ ride membrane then blotted with anti-His antibody. The arrow indicates the His tag is genetically incorporated into

**Figure 5. Western Blotting Confirms the Presence of KWAS on Multivalent Vaccine Vectors Displaying Dual An‐ tigens.** Western blotting confirmed the presence of KWAS incorporation within the dual modified vectors. In this as‐ say, 1 x 1010 VP of Ad5 (lane 1), Ad5/H5-HVR1-KWAS-HVR2-His6 (lane 2), and Ad5/H5-HVR1-KWAS-HVR5-His6 (lane 3) were separated on 4 to 15% polyacrylamide gradient SDS-PAGE gel. The proteins were transferred to polyvinyli‐ dene fluoride membrane then blotted with anti-gp41 antibody (NIH AIDS Reagent Program). The arrow indicates KWAS/gp41 protein genetically incorporated into the hexon.

The size of the 117 kDa band corresponds to the expected size of the Ad5 hexon protein with KWAS peptide genetically incorporated into the HVR1 region. There was a slight KWAS protein detected on Ad5 wild type particles, we attribute this to a cross reactive sequence within the Ad vector (Figure 5, lane 1). Most importantly, Figures 4 and 5 illustrate that KWAS and His6 proteins were incorporated at comparable levels within HVR1 and HVR2 or HVR5.

We also performed a series of ELISA assays to verify that the KWAS and His6 motifs were surface accessible on the hexon double-modified virions. These results indicated that the His6 epitope was properly exposed on the virion surfaces when incorporated within HVR2 or HVR5 (data not shown). In addition, the HIV-specific ELISA also illustrated that that the HIV motif was accessible on the virion surface within the HVR1 region. Our results showed significant binding of the anti-HIV antibody to the Ad5/H5-HVR1-KWAS-HVR2-His6 and Ad5/H5- HVR1-KWAS-HVR5-His6, whereas no binding was seen in response to Ad5 control (data not shown). Currently, we are in the process of testing these vectors *in vivo*. Our initial findings lead us to believe that these vectors can have tremendous impact for multivalent vaccine development.

### **6. Chimeric serotype Ad vectors and rare serotype vectors for vaccine usage**

In the near future, it is possible that viral vector-based vaccination will become a common clinical intervention; therefore, it has become increasingly necessary to design vectors that can overcome Ad5 pre-existing immunity [108,109]. In order to overcome Ad5 pre-existing immunity rare and non-human Ad serotypes have been used. Chimeric Ad vectors consist of either a sub-portion of the Ad5 vector genome that is replaced with genomic portions of another alternative serotype, thus creating "chimeric" Ad vectors, or, in a more drastic approach, the entire Ad vector genome is composed of proteins solely derived from alternative Ad serotypes [27,109–114]. Ad hexon and fiber have been the proteins manipulated genetically in chimeric strategies, primarily because these proteins are known to be the target of vector neutralizing antibodies [115–118]. Several chimeric fiber and hexon strategies have been endeavored [109]. Specifically, NAbs generated against hexon HVRs account for 80-90% of the Anti-Ad NAb response. These antibodies appear to be most critical for vector clearance, therefore, diminishing therapeutic efficacy of the vaccine vector administered [119]. The importance of the seven HVRs as NAbs epitopes remains unclear as it relates to Ad5 and other serotypes [120]. Therefore, exact mapping of the NAb epitopes in these HVRs, maybe necessary to obtain improved chimeric Ad5-based vectors [121].

studies which are currently underway. Recent studies suggested that Ad5 responses maybe focused on one specific HVR, such as HVR1 or HVR5 [81,124]. Bradley and group attempted to answer some of these issues; in their study they characterized the contribution of the individual hexon HVRs as Ad5 NAb epitopes. They constructed chimeric Ad5 vectors in which subsets of Ad5 HVRs were exchanged for Ad48 HVRs. These partial HVR-chimeric vectors were evaluated by NAb assays and immunogenicity studies with and without baseline Ad5 immunity. Through a series of complex and thorough experiments they demonstrated that Ad5-specific NAbs are targeted against several of the HVRs. This data suggest that it is necessary to replace all HVRs to optimize evasion of Anti-Ad5 immunity [125]. Along those same lines, another group evaluated Ad5-based vectors where the hexon HVRs are replaced with that of the HVRs of rare serotypes, Ad43 and Ad34. Ad43 and 34 are group D and B viruses, each of these have low prevalence of neutralizing antibodies in humans. They demonstrated that these hexon-modified Ad vectors are not neutralized efficiently by Ad5 neutralizing antibodies *in vitro* using sera from mice, rabbits, and human volunteers. This research yielded significant findings related to malaria antigen expression, in combination with hexon-modified vectors. Their data also demonstrates that hexon-modified vectors can be highly immunogenic in the presence of Ad5 pre-existing immunity. The authors comment that these hexon-modified vectors may have useful applications in places such as sub-Saharan

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 103

Africa where there is high prevalence of pre-existing Ad5 immunity [126].

Liver sequestration of Ad5-based vectors is another major drawback that hinders Ad5-based therapies. Previous studies illustrate that human coagulation factor X (FX) binds Ad5 hexon through an interaction between HVRs and the FX Gla domain leading to liver infection after systemic delivery [127,128]. The binding affinities for FX vary among vector serotypes, and may explain differences in heptaocyte transduction *in vivo* previously observed between serotypes. Although, some differences in binding affinities were noted in this report, overall, Ad2 and Ad5 bound factor X with the highest affinity, however, weak or no binding was detected with Ad9, Ad35, Ad48, and Ad51. This interaction has been observed in multiple human adenovirus serotypes and shows diversity and affinity. The domains and amino acid sequences in the HVRs are integral for high-affinity interaction with FX, however, several aspects of this binding and mechanism remain unclear [121]. In recent studies, Yu and colleagues evaluated the role of chimeric hexon HVRs with FX binding and affinity. In this study they constructed and expressed several chimeric HVR proteins and demonstrated that the native proteins were oligomers and had consistent structure and function with that of the virus. Their data demonstrated that HVR5 and HVR7 had only a fraction of hexon activity to NAbs compared to a group of HVRs, 1 through 7. In addition, they demonstrated a differential high-affinity interaction of the HVR proteins with FX and indicated that the HVRs had similar binding activity with corresponding Ad vector serotypes. This study highlighted some properties of chimeric HVR proteins and exposed the influences on the structure and function of hexon proteins and Ad vectors resulting from the incorporations of these HVRs [129].

The use of vectors derived completely from alternative human serotypes (including Ad26 and Ad35) have also shown great promise, in particular, in terms of ability to deliver transgenes [110,113,130,131]. The development of vectors based on Ads which normally infect nonhuman

One of the first reports of Ad5-based chimeric vectors generated was performed in 1998. This was done by replacing Ad serotype 5 hexon gene with sequences from Ad2 [122]. This study was the launching point for other chimeric vectors, such as experiments performed in 2002 by Wu and group. They constructed a chimeric adenoviral vector, Ad5/H3, by replacing the Ad5 hex‐ on gene with the hexon gene of serotype Ad3. The chimeric vector was successfully rescued in 293 cells. Ad5/H3 had a significantly lower growth profile as compared to Ad5/H5. Indicating that the Ad3 hexon could encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficiency of Ad5H3 in HeLa cells was also lower than that of Ad5/H5. They also tested the host neutralization responses against the two vectors after immunizing C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the im‐ munized mice did not cross-neutralize each other in the context of *invitro* infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of vectors also did not prevent sub‐ sequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5 [117]. Along these same lines, another research group constructed a chimeric Ad vector, Ad3/H7. This construction was generated by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimer‐ ic vectors were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. In addition, this group tested the host neutralization responses against the vectors using BALB/c mice. Up to 97% of the NAbs produced by mice that were infected with these vectors were specific for the hexon protein *in vi‐ tro*. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other vector *in vivo*. In contrast, preimmunization of mice with either Ad3 or Ad3/H7 did not remarkably prevent subsequent infection of the other vector [123].

Roberts et. al, previously demonstrated that replacing seven of the HVRs in Ad5 with that of rare serotype, Ad48, resulted in a chimeric vector, Ad5HVR48 (1-7). Ad5HVR48 (1-7) was able to evade the majority of Ad5 pre-existing immunity in preclinical studies in mice and rhesus monkeys, [112] Ad5 chimeric vectors in which all seven HVRs were exchanged induced the same level of anti-antigen immune responses in mice with Ad5 PEI as in naïve mice. However, replacing only one HVR provided little improvement over non-chimeric Ad5 vectors. Since the role of the seven individual HVRs as NAb epitopes remains unclear, there are several studies which are currently underway. Recent studies suggested that Ad5 responses maybe focused on one specific HVR, such as HVR1 or HVR5 [81,124]. Bradley and group attempted to answer some of these issues; in their study they characterized the contribution of the individual hexon HVRs as Ad5 NAb epitopes. They constructed chimeric Ad5 vectors in which subsets of Ad5 HVRs were exchanged for Ad48 HVRs. These partial HVR-chimeric vectors were evaluated by NAb assays and immunogenicity studies with and without baseline Ad5 immunity. Through a series of complex and thorough experiments they demonstrated that Ad5-specific NAbs are targeted against several of the HVRs. This data suggest that it is necessary to replace all HVRs to optimize evasion of Anti-Ad5 immunity [125]. Along those same lines, another group evaluated Ad5-based vectors where the hexon HVRs are replaced with that of the HVRs of rare serotypes, Ad43 and Ad34. Ad43 and 34 are group D and B viruses, each of these have low prevalence of neutralizing antibodies in humans. They demonstrated that these hexon-modified Ad vectors are not neutralized efficiently by Ad5 neutralizing antibodies *in vitro* using sera from mice, rabbits, and human volunteers. This research yielded significant findings related to malaria antigen expression, in combination with hexon-modified vectors. Their data also demonstrates that hexon-modified vectors can be highly immunogenic in the presence of Ad5 pre-existing immunity. The authors comment that these hexon-modified vectors may have useful applications in places such as sub-Saharan Africa where there is high prevalence of pre-existing Ad5 immunity [126].

either a sub-portion of the Ad5 vector genome that is replaced with genomic portions of another alternative serotype, thus creating "chimeric" Ad vectors, or, in a more drastic approach, the entire Ad vector genome is composed of proteins solely derived from alternative Ad serotypes [27,109–114]. Ad hexon and fiber have been the proteins manipulated genetically in chimeric strategies, primarily because these proteins are known to be the target of vector neutralizing antibodies [115–118]. Several chimeric fiber and hexon strategies have been endeavored [109]. Specifically, NAbs generated against hexon HVRs account for 80-90% of the Anti-Ad NAb response. These antibodies appear to be most critical for vector clearance, therefore, diminishing therapeutic efficacy of the vaccine vector administered [119]. The importance of the seven HVRs as NAbs epitopes remains unclear as it relates to Ad5 and other serotypes [120]. Therefore, exact mapping of the NAb epitopes in these HVRs, maybe necessary

One of the first reports of Ad5-based chimeric vectors generated was performed in 1998. This was done by replacing Ad serotype 5 hexon gene with sequences from Ad2 [122]. This study was the launching point for other chimeric vectors, such as experiments performed in 2002 by Wu and group. They constructed a chimeric adenoviral vector, Ad5/H3, by replacing the Ad5 hex‐ on gene with the hexon gene of serotype Ad3. The chimeric vector was successfully rescued in 293 cells. Ad5/H3 had a significantly lower growth profile as compared to Ad5/H5. Indicating that the Ad3 hexon could encapsidate the Ad5 genome, but with less efficiency than the Ad5 hexon. The gene transfer efficiency of Ad5H3 in HeLa cells was also lower than that of Ad5/H5. They also tested the host neutralization responses against the two vectors after immunizing C57BL/6 mice. The neutralizing antibodies against Ad5/H3 and Ad5/H5 generated by the im‐ munized mice did not cross-neutralize each other in the context of *invitro* infection of HeLa cells. Preimmunization of C57BL/6 mice with one of the two types of vectors also did not prevent sub‐ sequent infection of the other type. These data suggest that replacing the Ad5 hexon with the Ad3 hexon can circumvent the host neutralization response to Ad5 [117]. Along these same lines, another research group constructed a chimeric Ad vector, Ad3/H7. This construction was generated by replacing the Ad3 hexon gene (H3) with the hexon gene (H7) of Ad7. The chimer‐ ic vectors were successfully rescued in HEp-2 cells, and the Ad7 hexon was able to encapsidate the Ad3 genome, and functioned as efficiently as the Ad3 hexon. In addition, this group tested the host neutralization responses against the vectors using BALB/c mice. Up to 97% of the NAbs produced by mice that were infected with these vectors were specific for the hexon protein *in vi‐ tro*. Preimmunization of mice with one of Ad7 and Ad3/H7 significantly prevented subsequent intranasal infection of the other vector *in vivo*. In contrast, preimmunization of mice with either Ad3 or Ad3/H7 did not remarkably prevent subsequent infection of the other vector [123].

Roberts et. al, previously demonstrated that replacing seven of the HVRs in Ad5 with that of rare serotype, Ad48, resulted in a chimeric vector, Ad5HVR48 (1-7). Ad5HVR48 (1-7) was able to evade the majority of Ad5 pre-existing immunity in preclinical studies in mice and rhesus monkeys, [112] Ad5 chimeric vectors in which all seven HVRs were exchanged induced the same level of anti-antigen immune responses in mice with Ad5 PEI as in naïve mice. However, replacing only one HVR provided little improvement over non-chimeric Ad5 vectors. Since the role of the seven individual HVRs as NAb epitopes remains unclear, there are several

to obtain improved chimeric Ad5-based vectors [121].

102 Novel Gene Therapy Approaches

Liver sequestration of Ad5-based vectors is another major drawback that hinders Ad5-based therapies. Previous studies illustrate that human coagulation factor X (FX) binds Ad5 hexon through an interaction between HVRs and the FX Gla domain leading to liver infection after systemic delivery [127,128]. The binding affinities for FX vary among vector serotypes, and may explain differences in heptaocyte transduction *in vivo* previously observed between serotypes. Although, some differences in binding affinities were noted in this report, overall, Ad2 and Ad5 bound factor X with the highest affinity, however, weak or no binding was detected with Ad9, Ad35, Ad48, and Ad51. This interaction has been observed in multiple human adenovirus serotypes and shows diversity and affinity. The domains and amino acid sequences in the HVRs are integral for high-affinity interaction with FX, however, several aspects of this binding and mechanism remain unclear [121]. In recent studies, Yu and colleagues evaluated the role of chimeric hexon HVRs with FX binding and affinity. In this study they constructed and expressed several chimeric HVR proteins and demonstrated that the native proteins were oligomers and had consistent structure and function with that of the virus. Their data demonstrated that HVR5 and HVR7 had only a fraction of hexon activity to NAbs compared to a group of HVRs, 1 through 7. In addition, they demonstrated a differential high-affinity interaction of the HVR proteins with FX and indicated that the HVRs had similar binding activity with corresponding Ad vector serotypes. This study highlighted some properties of chimeric HVR proteins and exposed the influences on the structure and function of hexon proteins and Ad vectors resulting from the incorporations of these HVRs [129].

The use of vectors derived completely from alternative human serotypes (including Ad26 and Ad35) have also shown great promise, in particular, in terms of ability to deliver transgenes [110,113,130,131]. The development of vectors based on Ads which normally infect nonhuman species have also shown a great deal of promise. These nonhuman Ad vectors have been developed from multiple species, including, bovine, canine, chimpanzee and porcine [67]. Vectors derived from chimpanzee Ads C1 or C8 (AdC), have been recently developed, initially these vectors gained popularity since it was demonstrated that human sera does not signifi‐ cantly neutralize AdC vectors [132]. Importantly, unlike some other serotypes, the E1-deleted version of AdC7 is easily propagated [133]. An AdC7 vector expressing the SARS-coronavirus antigen elicited higher T-and B-cell responses than an Ad5 vector in mice with Ad5 PEI [134]. Importantly, a single injection of AdC7 encoding the Ebola glycoprotein provided protection from a lethal challenge, unlike the corresponding Ad5 vector [133].

**7. Conclusion**

Recombinant viral vectors have been utilized as therapeutic agents to prevent or cure disease because of their tremendous capacity to deliver antigens and to stimulate immune responses in humans. Viral vectors are generally more immunogenic than antigen administered adjuvant [19,145]. In addition, it is easier to generate recombinant vectors as compared to tumor cell or DC-based vaccines. Tumor cell or DC-based vaccines can be complex to acquire. They are often time consuming to produce and costly because they are customized treatments. Whereas, on the other hand, recombinant vectors are thought to be "off the shelf" treatments because they are relatively easy to produce, purify, and store. One major advantage of utilizing viral vectors for vaccination in multi-center clinical trials is the relatively low cost of vector production. However, the paramount factor to overcome when using viral vectors for gene therapy, virotherapy, and vaccine applications is the development of host-induced neutralizing

Viral Vectors for Vaccine Development http://dx.doi.org/10.5772/54700 105

The past few years have bought forward exciting technical advances, along with critical structure/function analyses of viral vectors which have allowed for better understanding of the interaction of recombinant vector and host immune systems. It has become increasingly more obvious that there are many factors which must be evaluated to optimize each specific vaccine. In order to achieve optimal therapeutic outcomes when treating patients with vector PEI, careful consideration must be given to determine prime-boost schemes, epitope-capsid incorporation (monovalent versus polyvalent), transgene selection (homologous versus

This work was supported by a grant from the National Institutes of Health: 5R01AI089337-02 (Matthews). I would like to thank Dr. Phoebe L. Stewart for allowing me to reproduce her figure (Nemerow et al. Virology 2009, 384, 380-388), copyright Elsevier. I would also like to thank Dr. Glenn C. Rowe for his thoughtful insight and review of this chapter. We would also like to thank the NIH AIDS Reagent Program, for providing HIV-1 gp41 monoclonal antibody

, Linlin Gu, Alexandre Krendelchtchikov and Zan C. Li

Department of Medicine, Division of Infectious Diseases, The University of Alabama, Bir‐

antibodies to the vector itself which limits continued usage.

heterologous), vector dosing, and serotype selection.

**Acknowledgements**

(2F5), Cat # 1475.

**Author details**

Qiana L. Matthews\*

mingham, AL, USA

It is essential to note that several Ad epitopes recognized by T cells are conserved among a broad range of human and nonhuman primate-derived Ads, making it possible that the T cells in patient with Ad5 PEI will also recognize vectors derived from these viruses [135–138]. Bovine Ads have been examined, since NAbs to bovine Ad3 (BAd3) have not been reported in humans. In a mouse model, a single immunization of BAd3 encoding the hemagglutination antigen of H5N1 influenza induced greater levels of cellular immunity than Ad5 vectors, and this was not diminished by Ad5 PEI [139]. It is important to note that, mice which had Ad5 PEI and received a prime-boost regimen of BAd3-Ad5 vectors encoding HA were fully protected from lethal influenza virus challenge. However, those receiving a homologous Ad5- Ad5 regimen were not. Therefore, Ad vectors that normally infect nonhuman species may induce responses and offer protection comparable or superior to Ad5, while maintaining protection in the presence of Ad5 PEI. The use of alternative serotype Ads allow for improved induction of immune responses to vector re-administration in host that have Ad5 PEI [110,113,140]. As a result of these earlier studies, alternative serotypes vectors have been tested in patient populations for HIV vaccine development [141]. In addition, human clinical trials utilizing Ad26 as a HIV vaccine agent have been initiated.

There are benefits to using alternative serotype vectors, however, the use of alternative serotypes vectors can also have limitations as well as potential side effects for human use. One limitation of alternative serotype usage is that, some alternative serotypes do not afford the same benefits of Ad5 because they are unable to induce high levels of transgene expression and are less amenable to large scale purification [108]. Humans have evolved with previous exposure to human Ad vectors, and have not been exposed to Ads derived from other species. Consequently, it may be predicted that the human innate immune system may react to the capsid proteins of alternative serotype Ads in a way that is different from that of human Ad vectors. It is also possible that the human immune system may have a response which is more robust when challenged with alternative serotypes as compared with human serotypes Ads. Recently, it has been demonstrated that the innate immune response to capsid proteins of alternative serotypes Ads have not only been shown to be significantly more robust as compared to Ad5, but in some cases toxic in animal models [110,142–144]. Alternative serotype vectors have different tropism than Ad5; therefore the biodistribution of these vectors could be quite different than that of Ad5-based vectors. Ad5 vectors have been proven to be safe in humans and animals over the last decade and the knowledge gained from this experimentation must be applied and tested as it relates to alternative serotype vectors.

## **7. Conclusion**

species have also shown a great deal of promise. These nonhuman Ad vectors have been developed from multiple species, including, bovine, canine, chimpanzee and porcine [67]. Vectors derived from chimpanzee Ads C1 or C8 (AdC), have been recently developed, initially these vectors gained popularity since it was demonstrated that human sera does not signifi‐ cantly neutralize AdC vectors [132]. Importantly, unlike some other serotypes, the E1-deleted version of AdC7 is easily propagated [133]. An AdC7 vector expressing the SARS-coronavirus antigen elicited higher T-and B-cell responses than an Ad5 vector in mice with Ad5 PEI [134]. Importantly, a single injection of AdC7 encoding the Ebola glycoprotein provided protection

It is essential to note that several Ad epitopes recognized by T cells are conserved among a broad range of human and nonhuman primate-derived Ads, making it possible that the T cells in patient with Ad5 PEI will also recognize vectors derived from these viruses [135–138]. Bovine Ads have been examined, since NAbs to bovine Ad3 (BAd3) have not been reported in humans. In a mouse model, a single immunization of BAd3 encoding the hemagglutination antigen of H5N1 influenza induced greater levels of cellular immunity than Ad5 vectors, and this was not diminished by Ad5 PEI [139]. It is important to note that, mice which had Ad5 PEI and received a prime-boost regimen of BAd3-Ad5 vectors encoding HA were fully protected from lethal influenza virus challenge. However, those receiving a homologous Ad5- Ad5 regimen were not. Therefore, Ad vectors that normally infect nonhuman species may induce responses and offer protection comparable or superior to Ad5, while maintaining protection in the presence of Ad5 PEI. The use of alternative serotype Ads allow for improved induction of immune responses to vector re-administration in host that have Ad5 PEI [110,113,140]. As a result of these earlier studies, alternative serotypes vectors have been tested in patient populations for HIV vaccine development [141]. In addition, human clinical trials

There are benefits to using alternative serotype vectors, however, the use of alternative serotypes vectors can also have limitations as well as potential side effects for human use. One limitation of alternative serotype usage is that, some alternative serotypes do not afford the same benefits of Ad5 because they are unable to induce high levels of transgene expression and are less amenable to large scale purification [108]. Humans have evolved with previous exposure to human Ad vectors, and have not been exposed to Ads derived from other species. Consequently, it may be predicted that the human innate immune system may react to the capsid proteins of alternative serotype Ads in a way that is different from that of human Ad vectors. It is also possible that the human immune system may have a response which is more robust when challenged with alternative serotypes as compared with human serotypes Ads. Recently, it has been demonstrated that the innate immune response to capsid proteins of alternative serotypes Ads have not only been shown to be significantly more robust as compared to Ad5, but in some cases toxic in animal models [110,142–144]. Alternative serotype vectors have different tropism than Ad5; therefore the biodistribution of these vectors could be quite different than that of Ad5-based vectors. Ad5 vectors have been proven to be safe in humans and animals over the last decade and the knowledge gained from this experimentation

from a lethal challenge, unlike the corresponding Ad5 vector [133].

104 Novel Gene Therapy Approaches

utilizing Ad26 as a HIV vaccine agent have been initiated.

must be applied and tested as it relates to alternative serotype vectors.

Recombinant viral vectors have been utilized as therapeutic agents to prevent or cure disease because of their tremendous capacity to deliver antigens and to stimulate immune responses in humans. Viral vectors are generally more immunogenic than antigen administered adjuvant [19,145]. In addition, it is easier to generate recombinant vectors as compared to tumor cell or DC-based vaccines. Tumor cell or DC-based vaccines can be complex to acquire. They are often time consuming to produce and costly because they are customized treatments. Whereas, on the other hand, recombinant vectors are thought to be "off the shelf" treatments because they are relatively easy to produce, purify, and store. One major advantage of utilizing viral vectors for vaccination in multi-center clinical trials is the relatively low cost of vector production. However, the paramount factor to overcome when using viral vectors for gene therapy, virotherapy, and vaccine applications is the development of host-induced neutralizing antibodies to the vector itself which limits continued usage.

The past few years have bought forward exciting technical advances, along with critical structure/function analyses of viral vectors which have allowed for better understanding of the interaction of recombinant vector and host immune systems. It has become increasingly more obvious that there are many factors which must be evaluated to optimize each specific vaccine. In order to achieve optimal therapeutic outcomes when treating patients with vector PEI, careful consideration must be given to determine prime-boost schemes, epitope-capsid incorporation (monovalent versus polyvalent), transgene selection (homologous versus heterologous), vector dosing, and serotype selection.

## **Acknowledgements**

This work was supported by a grant from the National Institutes of Health: 5R01AI089337-02 (Matthews). I would like to thank Dr. Phoebe L. Stewart for allowing me to reproduce her figure (Nemerow et al. Virology 2009, 384, 380-388), copyright Elsevier. I would also like to thank Dr. Glenn C. Rowe for his thoughtful insight and review of this chapter. We would also like to thank the NIH AIDS Reagent Program, for providing HIV-1 gp41 monoclonal antibody (2F5), Cat # 1475.

## **Author details**

Qiana L. Matthews\* , Linlin Gu, Alexandre Krendelchtchikov and Zan C. Li

Department of Medicine, Division of Infectious Diseases, The University of Alabama, Bir‐ mingham, AL, USA

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Kibuuka H, Robb ML, Michael NL, Anzala O, Amornkul PN, Gilmour J, Hural J, Buchbinder SP, Seaman MS, Dolin R, Baden LR, Carville A, Mansfield KG, Pau MG, Goudsmit J (2011) International seroepidemiology of adenovirus serotypes 5, 26, 35, and 48 in pediatric and adult populations. Vaccine 29: 5203-5209.

**Chapter 6**

**Development of Muscle-Directed**

**Systemic Cancer Gene Therapy**

Additional information is available at the end of the chapter

(AAV)-based systemic anti-angiogenic cancer gene therapy.

**2. Muscle-directed systemic cancer gene therapy**

In recent years, one of the main focuses of the effort to improve cancer treatment and patient prognosis has been gene therapy. Furthermore, the complex nature of cancer has meant that a variety of therapeutic strategies have been developed along two main avenues: local gene therapies and systemic gene therapies (Table 1). The strategies for local cancer gene therapy include suppression of an oncogene, activation of a tumor suppressor gene, and introduction of a suicide gene into cancer cells (McCormick 2001). Unfortunately, delivering a therapeutic gene to every individual cancer cell in a patient with metastatic cancer has so far proven to be an insurmountable task. It has not been possible to treat all cancerous lesions, which can in‐ clude undetectable ones such as individual cancer cells and micrometastases. In addition, it is difficult to selectively target cancer cells without affecting normal cells. On the other hand, systemic cancer gene therapy such as immunotherapy appears more promising for both in‐ hibiting tumor growth and preventing metastasis. This chapter will summarize the utility of viral vector-mediated systemic cancer gene therapy in the treatment of human malignancies, focusing on the powerful and promising approach of recombinant adeno-associated virus

Because a neovasculature is essential for tumor growth and metastasis (Folkman 1971), inhib‐ iting the development of the tumor vasculature using anti-angiogenic agents has emerged as an attractive new strategy for the treatment of cancer (Kerbel & Folkman 2002). For many types

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

© 2013 Miyake and Shimada; 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

Koichi Miyake and Takashi Shimada

http://dx.doi.org/10.5772/54779

**1. Introduction**

**2.1. Why gene therapy?**


**Chapter 6**

## **Development of Muscle-Directed Systemic Cancer Gene Therapy**

Koichi Miyake and Takashi Shimada

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54779

## **1. Introduction**

Kibuuka H, Robb ML, Michael NL, Anzala O, Amornkul PN, Gilmour J, Hural J, Buchbinder SP, Seaman MS, Dolin R, Baden LR, Carville A, Mansfield KG, Pau MG, Goudsmit J (2011) International seroepidemiology of adenovirus serotypes 5, 26, 35,

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[144] Hensley SE, Cun AS, Giles-Davis W, Li Y, Xiang Z, Lasaro MO, Williams BR, Silverman RH, Ertl HC (2007) Type I interferon inhibits antibody responses induced by a chim‐

[145] Kantor J, Irvine K, Abrams S, Kaufman H, DiPietro J, Schlom J (1992) Antitumor activity and immune responses induced by a recombinant carcinoembryonic antigen-vaccinia

and 48 in pediatric and adult populations. Vaccine 29: 5203-5209.

interactions with the complement system. Virology 374: 453-467.

complement dependent. Gene Ther 15: 885-901.

118 Novel Gene Therapy Approaches

panzee adenovirus vector. Mol Ther 15: 393-403.

virus vaccine. J Natl Cancer Inst 84: 1084-1091.

In recent years, one of the main focuses of the effort to improve cancer treatment and patient prognosis has been gene therapy. Furthermore, the complex nature of cancer has meant that a variety of therapeutic strategies have been developed along two main avenues: local gene therapies and systemic gene therapies (Table 1). The strategies for local cancer gene therapy include suppression of an oncogene, activation of a tumor suppressor gene, and introduction of a suicide gene into cancer cells (McCormick 2001). Unfortunately, delivering a therapeutic gene to every individual cancer cell in a patient with metastatic cancer has so far proven to be an insurmountable task. It has not been possible to treat all cancerous lesions, which can in‐ clude undetectable ones such as individual cancer cells and micrometastases. In addition, it is difficult to selectively target cancer cells without affecting normal cells. On the other hand, systemic cancer gene therapy such as immunotherapy appears more promising for both in‐ hibiting tumor growth and preventing metastasis. This chapter will summarize the utility of viral vector-mediated systemic cancer gene therapy in the treatment of human malignancies, focusing on the powerful and promising approach of recombinant adeno-associated virus (AAV)-based systemic anti-angiogenic cancer gene therapy.

## **2. Muscle-directed systemic cancer gene therapy**

#### **2.1. Why gene therapy?**

Because a neovasculature is essential for tumor growth and metastasis (Folkman 1971), inhib‐ iting the development of the tumor vasculature using anti-angiogenic agents has emerged as an attractive new strategy for the treatment of cancer (Kerbel & Folkman 2002). For many types

© 2013 Miyake and Shimada; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

of cancer, targeted biological therapies that selectively interfere with tumor angiogenesis have the potential to improve survival among patients, and a number of angiogenesis inhibitors are currently being tested in clinical trials (Shojaei 2012). In addition, preclinical studies using purified anti-angiogenic factors have indicated the ability of anti-angiogenic compounds to minimize the size of established tumors. However, although they are capable of significantly inhibiting tumor cell growth in animal models, the clinical efficacy of administering purified anti-angiogenic factors would likely be limited by the peptides' short half-life. On the other hand, synthesis and secretion of anti-angiogenic factors following gene transfer may overcome that limitation. If so, gene therapy could play an important role in this field, as anti-angiogenic factors need to be delivered for long periods to control the progression of tumors.

**2.3. Why muscle directed?**

Noro et al. 2004; Yan et al. 2005).

essary for anti-angiogenic therapy.

the effect.

**2.5. A concept of muscle-directed systemic cancer gene therapy**

**Figure 1.** A concept of muscle-directed systemic cancer gene therapy

**2.4. Why an AAV vector?**

To express therapeutic proteins, skeletal muscles are considered an attractive target for gene delivery because they are large, have a good capacity for protein synthesis and are easily ac‐ cessible for intramuscular injection. In addition, muscle fibers are capable of expressing and secreting biologically active gene products that they do not normally synthesize (Arruda et al. 2001). Thus, direct injection of viral vectors into muscles has been widely used for both the treatment of muscular disorders and for expression of therapeutic proteins used for the treat‐ ment of metabolic disease, genetic bleeding disorders and malignant diseases (Liu et al. 2004;

Development of Muscle-Directed Systemic Cancer Gene Therapy

http://dx.doi.org/10.5772/54779

121

The potential of anti-angiogenic gene therapy in cancer is currently being evaluated using viral and non-viral vectors. The development of an effective gene delivery system is absolutely critical to the effectiveness and safety of gene therapy. Among the several gene transfer strat‐ egies being considered at present, the AAV vector appears the most promising, in view of its lack of pathogenicity, wide tropisms and long-term transgene expression *in vivo*. Gene therapy studies using different serotypes of recombinant AAVs as delivery vehicles have demonstrated that AAVs are an effective modality for cancer gene therapy that meet the requirements nec‐

Figure 1 illustrates the concept of muscle-directed systemic cancer gene therapy. First, an AAV vector encoding anti-angiogenic agents is injected into a muscle. After a single injection, se‐ creted anti-angiogenic agents are circulating throughout the entire body. These circulating factors suppress both the primary tumor and undetectable metastatic lesions through inhibi‐ tion of tumor angiogenesis. If the gene therapy alone is not sufficient to suppress all the tumors, other therapies such as radiation, chemotherapy or immunotherapy can be added to enhance


**Table 1.** Strategy of cancer gene therapy

#### **2.2. Why anti-angiogenic?**

Anti-angiogenic gene therapy has several advantageous features (Table 2). As mentioned above, this strategy can potentially suppress both the main tumor and small metastatic tumors. Moreover, since angiogenesis is essential for the development of all tumors, this strategy could be applied to a wide variety of cancers. In contrast to genetic therapies targeting tumor cells directly, anti-angiogenic gene therapy does not require the targeting and transduction of can‐ cer cells. Instead, systemic levels of anti-angiogenic factors may be achieved by targeting nontumor cells, which provide a stable platform for transgene expression and subsequent secretion of the translated proteins. Finally, cancers do not develop resistance to anti-angiogenic therapy, and the patients experience no side effects or side effects that are much milder than those associated with conventional anti-cancer therapies.

5. No or mild side-effect

**Table 2.** Advantages of anti-angiogenic Cancer Gene Therapy

<sup>1.</sup> Effective for almost all cancers

<sup>2.</sup> Inhibition of tumor growth and prevention of metastases

<sup>3.</sup> Targeting gene transfer is unnecessary

<sup>4.</sup> No resistance

#### **2.3. Why muscle directed?**

of cancer, targeted biological therapies that selectively interfere with tumor angiogenesis have the potential to improve survival among patients, and a number of angiogenesis inhibitors are currently being tested in clinical trials (Shojaei 2012). In addition, preclinical studies using purified anti-angiogenic factors have indicated the ability of anti-angiogenic compounds to minimize the size of established tumors. However, although they are capable of significantly inhibiting tumor cell growth in animal models, the clinical efficacy of administering purified anti-angiogenic factors would likely be limited by the peptides' short half-life. On the other hand, synthesis and secretion of anti-angiogenic factors following gene transfer may overcome that limitation. If so, gene therapy could play an important role in this field, as anti-angiogenic

**Strategy Target cells**

cells Antigen presenting

cells,

factors need to be delivered for long periods to control the progression of tumors.

1) Suppression of oncogenes RNAi, antisense etc. Cancer cells 2) Activation of tumor suppressor genes p53 etc. Cancer cells 3) Suicide gene therapy HSV-TK Cancer cells

2) Anti-angiogenic gene therapy Endostatin etc. All cells

1) Immuno gene therapy Cytokine genes etc. Lymphocytes, Cancer

Anti-angiogenic gene therapy has several advantageous features (Table 2). As mentioned above, this strategy can potentially suppress both the main tumor and small metastatic tumors. Moreover, since angiogenesis is essential for the development of all tumors, this strategy could be applied to a wide variety of cancers. In contrast to genetic therapies targeting tumor cells directly, anti-angiogenic gene therapy does not require the targeting and transduction of can‐ cer cells. Instead, systemic levels of anti-angiogenic factors may be achieved by targeting nontumor cells, which provide a stable platform for transgene expression and subsequent secretion of the translated proteins. Finally, cancers do not develop resistance to anti-angiogenic therapy, and the patients experience no side effects or side effects that are much milder than those

1. Local gene therapy

120 Novel Gene Therapy Approaches

2. Systemic gene therapy

**Table 1.** Strategy of cancer gene therapy

associated with conventional anti-cancer therapies.

2. Inhibition of tumor growth and prevention of metastases

**Table 2.** Advantages of anti-angiogenic Cancer Gene Therapy

**2.2. Why anti-angiogenic?**

1. Effective for almost all cancers

4. No resistance 5. No or mild side-effect

3. Targeting gene transfer is unnecessary

To express therapeutic proteins, skeletal muscles are considered an attractive target for gene delivery because they are large, have a good capacity for protein synthesis and are easily ac‐ cessible for intramuscular injection. In addition, muscle fibers are capable of expressing and secreting biologically active gene products that they do not normally synthesize (Arruda et al. 2001). Thus, direct injection of viral vectors into muscles has been widely used for both the treatment of muscular disorders and for expression of therapeutic proteins used for the treat‐ ment of metabolic disease, genetic bleeding disorders and malignant diseases (Liu et al. 2004; Noro et al. 2004; Yan et al. 2005).

#### **2.4. Why an AAV vector?**

The potential of anti-angiogenic gene therapy in cancer is currently being evaluated using viral and non-viral vectors. The development of an effective gene delivery system is absolutely critical to the effectiveness and safety of gene therapy. Among the several gene transfer strat‐ egies being considered at present, the AAV vector appears the most promising, in view of its lack of pathogenicity, wide tropisms and long-term transgene expression *in vivo*. Gene therapy studies using different serotypes of recombinant AAVs as delivery vehicles have demonstrated that AAVs are an effective modality for cancer gene therapy that meet the requirements nec‐ essary for anti-angiogenic therapy.

#### **2.5. A concept of muscle-directed systemic cancer gene therapy**

Figure 1 illustrates the concept of muscle-directed systemic cancer gene therapy. First, an AAV vector encoding anti-angiogenic agents is injected into a muscle. After a single injection, se‐ creted anti-angiogenic agents are circulating throughout the entire body. These circulating factors suppress both the primary tumor and undetectable metastatic lesions through inhibi‐ tion of tumor angiogenesis. If the gene therapy alone is not sufficient to suppress all the tumors, other therapies such as radiation, chemotherapy or immunotherapy can be added to enhance the effect.

**Figure 1.** A concept of muscle-directed systemic cancer gene therapy

## **3. Applications of systemic cancer gene therapy**

#### **3.1. Systemic anti-angiogenic cancer gene therapy**

To assess the feasibility of anti-angiogenic gene therapy using an AAV vector, we constructed an AAV vector encoding murine endostatin (AAV/mEnd) (Figure 2A), which is a tumor-de‐ rived angiogenesis inhibitor and is the first endogenous inhibitor of angiogenesis to be iden‐ tified in a matrix protein (O'Reilly et al. 1997). We then attempted to use the vector to treat pancreatic cancer in an orthotopic model. When PGHAM-1 derived from chemically induced hamster pancreatic cancer cells is injected into the pancreas of hamsters, a ductal adenocarci‐ noma develops that closely resembles the human disease and, like its human counterpart, it frequently metastasizes to the liver (Matsushita et al. 2001; Yanagi et al. 2000). After AAV/ mEnd (5x1010 vector genomes) was intramuscularly injected into the left quadriceps, we as‐ sessed the ability of AAV-mediated systemic delivery of endostatin to suppress metastatic pancreatic cancer. We found that intramuscular injection of the AAV/mEnd vector increased serum endostatin levels, as compared to a control AAV vector encoding GFP (Figure 2B). In addition, the size of the primary pancreatic tumors and the sizes and number of liver meta‐ stases were all reduced in the treated animals (Figure 2C). This suggests that AAV-mediated systemic delivery of endostatin represents a potentially effective treatment for pancreatic can‐ cer and liver metastases.

To achieve the anti-angiogenic state in the model animals used in this experiment, we used classical AAV serotype 2 vectors (Noro et al. 2004). One problem with that vector is that it takes several weeks to reach the maximal level of transgene expression. Therefore, to evaluate the efficacy of cancer gene therapy in a transplantation model animal, the AAV vector must be injected before tumor inoculation. Recently, several different AAV serotypes have been char‐ acterized, and we investigated which AAV serotype would maximize the efficiency of the gene

Development of Muscle-Directed Systemic Cancer Gene Therapy

http://dx.doi.org/10.5772/54779

123

A number of novel AAV serotypes have been isolated from nonhuman primates (Gao et al. 2002), and we endeavored to determine which AAV vector most efficiently mediates muscle expression of anti-angiogenic proteins useful for treating cancer. Included among these were AAV serotypes 1, 7 and 8, which, when developed as vectors, mediate gene transfer into var‐ ious tissues much more efficiently than those based on previously described serotypes. There‐ fore, to determine the AAV serotype that most efficiently mediates muscle expression of antiangiogenic proteins, we injected 4 different AAV vector serotypes (AAV1, AAV2, AAV7 and AAV8) encoding mEnd together with GFP into a quadriceps muscle in C57BL/6 mice. The highest GFP expression and plasma mEnd levels were observed one week after injection in mice administered AAV8 (8>7>1>2) (Figure 3A). Moreover, expression of mEnd was sustained for at least 6 months. We then confirmed the transduction efficiency into the muscle using an AAV vector harboring the luciferase gene (AAV/Luc). *In vivo* imaging showed that the greatest expression occurred in mice administered AAV8 (Figure 3B). Taken together, these results clearly demonstrate that AAV8 is able to efficiently mediate gene transfer into muscle tissue, leading to prolonged expression and secretion of the gene product (Isotani et al. 2011).

**3.3. Melanoma differentiation-associated gene-7/interleukin-24 (***mda-7***/***IL24***)**

Another candidate gene for systemic cancer gene therapy is melanoma differentiation-associ‐ ated gene-7/interleukin-24 (*mda-7*/*IL24*), which has anti-angiogenic properties (MDA-7/IL24 bioactivity was 20- to 50-fold greater than endostatin or angiostatin) as well as several other features useful for cancer gene therapy. *Mda-7*/*IL24* selectively induces apoptosis in cancer cells without harming normal cells, and it exerts both immunomodulatory effects and potent antitumor bystander effects. These multifunctional tumor-specific cytotoxic effects of MDA-7/ IL24 make this molecule a promising gene-based therapeutic agent for the treatment of cancer. To assess the *in vivo* effects of AAV-mediated systemic delivery of MDA-7/IL24, we constructed an AAV vector encoding MDA-7/IL24 (AAV/IL24). A single intravenous injection of AAV/IL24 (2.0 x 1011 vector genomes) into a subcutaneous tumor induced by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice significantly inhibited tumor growth and increased survival among the AAV/IL24-treated mice (Figure 4). In addition, TUNEL and immunohis‐ tochemical analyses showed significant induction of tumor cell-specific apoptosis and a re‐ duction in microvessel formation within the tumors (Tahara et al. 2007). These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treat‐

transfer into muscle.

ment for cancer.

**3.2. The best AAV serotype**

**Figure 2.** (A) Construction of recombinant AAV vector plasmids expressing murine endostatin (AAV/mEnd) and con‐ trol GFP expressing vector (AAV/GFP). ITR: inverted terminal repeats, CAGp: CAG promoter, B19p: B19 promoter, TKp: thymidine kinase promoter, neoR: neomycin resistance gene (B) Serum levels of endostatin after intramuscular injec‐ tion of AAV/End (42 days after vector injection). Intramuscular injection of AAV/End increased the serum endostatin level. (C) Effects of AAV-mediated endostatin expression on pancreatic tumor growth and liver metastasis.

To achieve the anti-angiogenic state in the model animals used in this experiment, we used classical AAV serotype 2 vectors (Noro et al. 2004). One problem with that vector is that it takes several weeks to reach the maximal level of transgene expression. Therefore, to evaluate the efficacy of cancer gene therapy in a transplantation model animal, the AAV vector must be injected before tumor inoculation. Recently, several different AAV serotypes have been char‐ acterized, and we investigated which AAV serotype would maximize the efficiency of the gene transfer into muscle.

### **3.2. The best AAV serotype**

**3. Applications of systemic cancer gene therapy**

To assess the feasibility of anti-angiogenic gene therapy using an AAV vector, we constructed an AAV vector encoding murine endostatin (AAV/mEnd) (Figure 2A), which is a tumor-de‐ rived angiogenesis inhibitor and is the first endogenous inhibitor of angiogenesis to be iden‐ tified in a matrix protein (O'Reilly et al. 1997). We then attempted to use the vector to treat pancreatic cancer in an orthotopic model. When PGHAM-1 derived from chemically induced hamster pancreatic cancer cells is injected into the pancreas of hamsters, a ductal adenocarci‐ noma develops that closely resembles the human disease and, like its human counterpart, it frequently metastasizes to the liver (Matsushita et al. 2001; Yanagi et al. 2000). After AAV/ mEnd (5x1010 vector genomes) was intramuscularly injected into the left quadriceps, we as‐ sessed the ability of AAV-mediated systemic delivery of endostatin to suppress metastatic pancreatic cancer. We found that intramuscular injection of the AAV/mEnd vector increased serum endostatin levels, as compared to a control AAV vector encoding GFP (Figure 2B). In addition, the size of the primary pancreatic tumors and the sizes and number of liver meta‐ stases were all reduced in the treated animals (Figure 2C). This suggests that AAV-mediated systemic delivery of endostatin represents a potentially effective treatment for pancreatic can‐

**Figure 2.** (A) Construction of recombinant AAV vector plasmids expressing murine endostatin (AAV/mEnd) and con‐ trol GFP expressing vector (AAV/GFP). ITR: inverted terminal repeats, CAGp: CAG promoter, B19p: B19 promoter, TKp: thymidine kinase promoter, neoR: neomycin resistance gene (B) Serum levels of endostatin after intramuscular injec‐ tion of AAV/End (42 days after vector injection). Intramuscular injection of AAV/End increased the serum endostatin

level. (C) Effects of AAV-mediated endostatin expression on pancreatic tumor growth and liver metastasis.

**3.1. Systemic anti-angiogenic cancer gene therapy**

cer and liver metastases.

122 Novel Gene Therapy Approaches

A number of novel AAV serotypes have been isolated from nonhuman primates (Gao et al. 2002), and we endeavored to determine which AAV vector most efficiently mediates muscle expression of anti-angiogenic proteins useful for treating cancer. Included among these were AAV serotypes 1, 7 and 8, which, when developed as vectors, mediate gene transfer into var‐ ious tissues much more efficiently than those based on previously described serotypes. There‐ fore, to determine the AAV serotype that most efficiently mediates muscle expression of antiangiogenic proteins, we injected 4 different AAV vector serotypes (AAV1, AAV2, AAV7 and AAV8) encoding mEnd together with GFP into a quadriceps muscle in C57BL/6 mice. The highest GFP expression and plasma mEnd levels were observed one week after injection in mice administered AAV8 (8>7>1>2) (Figure 3A). Moreover, expression of mEnd was sustained for at least 6 months. We then confirmed the transduction efficiency into the muscle using an AAV vector harboring the luciferase gene (AAV/Luc). *In vivo* imaging showed that the greatest expression occurred in mice administered AAV8 (Figure 3B). Taken together, these results clearly demonstrate that AAV8 is able to efficiently mediate gene transfer into muscle tissue, leading to prolonged expression and secretion of the gene product (Isotani et al. 2011).

#### **3.3. Melanoma differentiation-associated gene-7/interleukin-24 (***mda-7***/***IL24***)**

Another candidate gene for systemic cancer gene therapy is melanoma differentiation-associ‐ ated gene-7/interleukin-24 (*mda-7*/*IL24*), which has anti-angiogenic properties (MDA-7/IL24 bioactivity was 20- to 50-fold greater than endostatin or angiostatin) as well as several other features useful for cancer gene therapy. *Mda-7*/*IL24* selectively induces apoptosis in cancer cells without harming normal cells, and it exerts both immunomodulatory effects and potent antitumor bystander effects. These multifunctional tumor-specific cytotoxic effects of MDA-7/ IL24 make this molecule a promising gene-based therapeutic agent for the treatment of cancer. To assess the *in vivo* effects of AAV-mediated systemic delivery of MDA-7/IL24, we constructed an AAV vector encoding MDA-7/IL24 (AAV/IL24). A single intravenous injection of AAV/IL24 (2.0 x 1011 vector genomes) into a subcutaneous tumor induced by injecting Ehrlich ascites tumor cells into the dorsum of DDY mice significantly inhibited tumor growth and increased survival among the AAV/IL24-treated mice (Figure 4). In addition, TUNEL and immunohis‐ tochemical analyses showed significant induction of tumor cell-specific apoptosis and a re‐ duction in microvessel formation within the tumors (Tahara et al. 2007). These results clearly demonstrate that continuous systemic delivery of MDA-7/IL24 can serve as an effective treat‐ ment for cancer.

mice (Tamai et al. 2011). These mice developed pro-B cell (CD45R/B220+

a potentially important new approach to anticancer therapy.

**4. Summary and future developments**

cer treatment.

**Acknowledgements**

**Author details**

Koichi Miyake and Takashi Shimada

phoma as well as leukemia with high-level HOXA9 expression by 12 months of age, at which time lymphoma cells had infiltrated the liver, lung and spleen. In addition to multiple sites of tumor infiltration, a non-solid hematological malignancy, leukemia, was also present, making local gene therapy entirely impractical. This model is therefore well suited to analyze the utility of AAV-mediated systemic gene therapies. So far, we have observed that after a muscle-di‐ rected single AAV/IL24 injection, infiltration of tumor cells into all organs was suppressed (Tamai et al. 2012). Thus, AAV vector-mediated systemic delivery of MDA-7/IL24 represents

Here we present evidence of the utility of AAV-mediated muscle-directed systemic cancer gene therapy using anti-angiogenic agents together with MDA-7/IL24. This new approach is safe and non-invasive, and could be used to treat primary tumors as well as undetectable metastatic tumors and hematological non-solid tumors without serious side effects. The po‐ tential utility of anti-angiogenic gene therapy in cancer is currently being evaluated in com‐ bination with radiation, chemotherapy or immunotherapy, which appear to provide a synergistic effect. Moreover, systemic cancer gene therapy may enable reduction of the dose of radiation, chemotherapy or immunotherapy needed to be effective, thereby reducing such side effects as bone marrow suppression. Thus, the combination of gene therapy with con‐ ventional anti-cancer therapy may overcome serious problems currently associated with can‐

We thank Dr. James Wilson at the University of Pennsylvania for providing AAV packaging plasmids (p5E18RXC1, p5E18-VD2/7 and p5E18-VD2/8). We also thank Takuya Noro, Ichiro Tahara, Noriko Miyake, Mayu Isotani and Hayato Tamai for helpful discussion and technical assistance. This work was supported in part by grants from the Ministry of Health and Welfare

Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research

of Japan and the Ministry of Education, Science and Culture of Japan.

Center for Advanced Medical Technology, Nippon Medical School, Japan

CD19+

http://dx.doi.org/10.5772/54779

Development of Muscle-Directed Systemic Cancer Gene Therapy

CD43+

) lym‐

125

**Figure 3.** Expression of transgenes following intramuscular administration of AAVs. Following injection of the respec‐ tive AAV serotypes of the AAV/mEnd vectors, which express murine endostatin together with GFP, into quadriceps muscles, expression of GFP and plasma concentrations of mEnd were analyzed (A). Four AAV serotypes of AAV/Luc, which express lucigerase, were injected into the quadriceps muscles of DDY mice. Four weeks after injection, these mice were analyzed by *in vivo* imaging system.

**Figure 4.** Inhibition of tumor growth after administration of AAV/IL24. One week after subcutaneous injection of Ehr‐ lich ascites tumor cells, mice were treated with AAV/IL24 or control AAV/GFP. The mean tumor volumes 56 days after injection were significantly smaller in animals that received AAV/IL24 than in those that received AAV/GFP (*P* < 0.001).

To then assess the feasibility of AAV8-mediated muscle-directed cancer gene therapy using MDA-7/IL24, we established mixed-lineage leukemia (MLL)/AF4 transgenic (MLL/AF4 Tg) mice (Tamai et al. 2011). These mice developed pro-B cell (CD45R/B220+ CD19+ CD43+ ) lym‐ phoma as well as leukemia with high-level HOXA9 expression by 12 months of age, at which time lymphoma cells had infiltrated the liver, lung and spleen. In addition to multiple sites of tumor infiltration, a non-solid hematological malignancy, leukemia, was also present, making local gene therapy entirely impractical. This model is therefore well suited to analyze the utility of AAV-mediated systemic gene therapies. So far, we have observed that after a muscle-di‐ rected single AAV/IL24 injection, infiltration of tumor cells into all organs was suppressed (Tamai et al. 2012). Thus, AAV vector-mediated systemic delivery of MDA-7/IL24 represents a potentially important new approach to anticancer therapy.

## **4. Summary and future developments**

Here we present evidence of the utility of AAV-mediated muscle-directed systemic cancer gene therapy using anti-angiogenic agents together with MDA-7/IL24. This new approach is safe and non-invasive, and could be used to treat primary tumors as well as undetectable metastatic tumors and hematological non-solid tumors without serious side effects. The po‐ tential utility of anti-angiogenic gene therapy in cancer is currently being evaluated in com‐ bination with radiation, chemotherapy or immunotherapy, which appear to provide a synergistic effect. Moreover, systemic cancer gene therapy may enable reduction of the dose of radiation, chemotherapy or immunotherapy needed to be effective, thereby reducing such side effects as bone marrow suppression. Thus, the combination of gene therapy with con‐ ventional anti-cancer therapy may overcome serious problems currently associated with can‐ cer treatment.

## **Acknowledgements**

**Figure 3.** Expression of transgenes following intramuscular administration of AAVs. Following injection of the respec‐ tive AAV serotypes of the AAV/mEnd vectors, which express murine endostatin together with GFP, into quadriceps muscles, expression of GFP and plasma concentrations of mEnd were analyzed (A). Four AAV serotypes of AAV/Luc, which express lucigerase, were injected into the quadriceps muscles of DDY mice. Four weeks after injection, these

**Figure 4.** Inhibition of tumor growth after administration of AAV/IL24. One week after subcutaneous injection of Ehr‐ lich ascites tumor cells, mice were treated with AAV/IL24 or control AAV/GFP. The mean tumor volumes 56 days after injection were significantly smaller in animals that received AAV/IL24 than in those that received AAV/GFP (*P* < 0.001).

To then assess the feasibility of AAV8-mediated muscle-directed cancer gene therapy using MDA-7/IL24, we established mixed-lineage leukemia (MLL)/AF4 transgenic (MLL/AF4 Tg)

mice were analyzed by *in vivo* imaging system.

124 Novel Gene Therapy Approaches

We thank Dr. James Wilson at the University of Pennsylvania for providing AAV packaging plasmids (p5E18RXC1, p5E18-VD2/7 and p5E18-VD2/8). We also thank Takuya Noro, Ichiro Tahara, Noriko Miyake, Mayu Isotani and Hayato Tamai for helpful discussion and technical assistance. This work was supported in part by grants from the Ministry of Health and Welfare of Japan and the Ministry of Education, Science and Culture of Japan.

### **Author details**

Koichi Miyake and Takashi Shimada

Department of Biochemistry and Molecular Biology, Division of Gene Therapy Research Center for Advanced Medical Technology, Nippon Medical School, Japan

## **References**

[1] Arruda V. R., Hagstrom J. N., Deitch J., Heiman-Patterson T., Camire R. M., Chu K., Fields P. A., Herzog R. W., Couto L. B., Larson P. J. & High K. A. (2001). Posttranslational modifications of recombinant myotube-synthesized human factor IX. Blood 97,130-138. [13] Tamai H., Miyake K., Takatori M., Miyake N., Yamaguchi H., Dan K., Shimada T. & Inokuchi K. (2011). Activated K-Ras protein accelerates human MLL/AF4-induced leu‐ kemo-lymphomogenicity in a transgenic mouse model. Leukemia : official journal of

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[13] Tamai H., Miyake K., Takatori M., Miyake N., Yamaguchi H., Dan K., Shimada T. & Inokuchi K. (2011). Activated K-Ras protein accelerates human MLL/AF4-induced leu‐ kemo-lymphomogenicity in a transgenic mouse model. Leukemia : official journal of the Leukemia Society of America, Leukemia Research Fund, U.K 25,888-891.

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[1] Arruda V. R., Hagstrom J. N., Deitch J., Heiman-Patterson T., Camire R. M., Chu K., Fields P. A., Herzog R. W., Couto L. B., Larson P. J. & High K. A. (2001). Posttranslational modifications of recombinant myotube-synthesized human factor IX. Blood 97,130-138.

[2] Folkman J. (1971). Tumor angiogenesis: therapeutic implications. The New England

[3] Gao G. P., Alvira M. R., Wang L., Calcedo R., Johnston J. & Wilson J. M. (2002). Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy.

[4] Isotani M., Miyake K., Miyake N., Hirai Y. & Shimada T. (2011). Direct comparison of four adeno-associated virus serotypes in mediating the production of antiangiogenic

[5] Kerbel R. & Folkman J. (2002). Clinical translation of angiogenesis inhibitors. Nature

[6] Liu Y. L., Mingozzi F., Rodriguez-Colon S. M., Joseph S., Dobrzynski E., Suzuki T., High K. A. & Herzog R. W. (2004). Therapeutic levels of factor IX expression using a musclespecific promoter and adeno-associated virus serotype 1 vector. Hum Gene Ther

[7] Matsushita A., Onda M., Uchida E., Maekawa R. & Yoshioka T. (2001). Antitumor effect of a new selective matrix metalloproteinase inhibitor, MMI-166, on experimental pan‐ creatic cancer. International journal of cancer. Journal international du cancer

[8] McCormick F. (2001). Cancer gene therapy: fringe or cutting edge? Nature reviews.

[9] Noro T., Miyake K., Suzuki-Miyake N., Igarashi T., Uchida E., Misawa T., Yamazaki Y. & Shimada T. (2004). Adeno-associated viral vector-mediated expression of endostatin inhibits tumor growth and metastasis in an orthotropic pancreatic cancer model in

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**Chapter 7**

**Replicating Retroviral Vectors for**

**Gene Therapy of Solid Tumors**

Additional information is available at the end of the chapter

Despite recent progress in the treatment of solid tumours by conventional therapeutic options including surgery, chemotherapy, and radiotherapy, development of more efficient strategies is urgently needed due to delimited efficacy and occurrence of severe side effects in current treatment regimens. Cancer gene therapy can be defined as the introduction of genetic material into the patient´s body for the purpose of reducing tumour burden, increasing life expectancy, and improving the quality of life of the treated individual. It is most commonly intended to either initiate tumour self-destruction, down-regulate angiogenesis and/or metastasis, enhance anti-tumour activity of the immune system, suppress function of an activated

Viral vectors are the most widely used tools for the delivery of therapeutic genetic material into host cells in a clinical setting. More than 65 % of gene therapy clinical trials worldwide are making use of viral vectors (http://www.wiley.com). With almost 370 trials (~20 % of all gene therapy clinical trials), gamma-retroviruses and in particular the murine leukaemia virus (MLV)-based vectors are the second most used gene transfer system employed in recent years. These vectors are able to transduce most cell types, as long as they are actively dividing. However, most of these retroviral vectors are designed to be replication-deficient, resulting in poor transduction efficiencies in vivo. This might be one, if not the reason for the poor therapeutic success observed so far in clinical trials for cancer [5–7]. Thus, nowadays, replica‐ tion-deficient retroviral vectors are mainly used in ex vivo gene transfer for the treatment of

However, to increase in vivo transduction efficiency and the poor therapeutic outcome observed using replication-deficient retroviral (RDR) vectors, replication-competent retroviral (RCR) vectors were created which allow vector production in the infected tumour cell and

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

© 2013 Renner and Hlavaty; 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

oncogene, or restore expression and/or function of tumour suppressor genes [1-4].

inherited monogenic disorders [8–10], rather than for in vivo tumour therapy.

Matthias Renner and Juraj Hlavaty

http://dx.doi.org/10.5772/54861

**1. Introduction**

**Chapter 7**

## **Replicating Retroviral Vectors for Gene Therapy of Solid Tumors**

Matthias Renner and Juraj Hlavaty

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/54861

## **1. Introduction**

Despite recent progress in the treatment of solid tumours by conventional therapeutic options including surgery, chemotherapy, and radiotherapy, development of more efficient strategies is urgently needed due to delimited efficacy and occurrence of severe side effects in current treatment regimens. Cancer gene therapy can be defined as the introduction of genetic material into the patient´s body for the purpose of reducing tumour burden, increasing life expectancy, and improving the quality of life of the treated individual. It is most commonly intended to either initiate tumour self-destruction, down-regulate angiogenesis and/or metastasis, enhance anti-tumour activity of the immune system, suppress function of an activated oncogene, or restore expression and/or function of tumour suppressor genes [1-4].

Viral vectors are the most widely used tools for the delivery of therapeutic genetic material into host cells in a clinical setting. More than 65 % of gene therapy clinical trials worldwide are making use of viral vectors (http://www.wiley.com). With almost 370 trials (~20 % of all gene therapy clinical trials), gamma-retroviruses and in particular the murine leukaemia virus (MLV)-based vectors are the second most used gene transfer system employed in recent years. These vectors are able to transduce most cell types, as long as they are actively dividing. However, most of these retroviral vectors are designed to be replication-deficient, resulting in poor transduction efficiencies in vivo. This might be one, if not the reason for the poor therapeutic success observed so far in clinical trials for cancer [5–7]. Thus, nowadays, replica‐ tion-deficient retroviral vectors are mainly used in ex vivo gene transfer for the treatment of inherited monogenic disorders [8–10], rather than for in vivo tumour therapy.

However, to increase in vivo transduction efficiency and the poor therapeutic outcome observed using replication-deficient retroviral (RDR) vectors, replication-competent retroviral (RCR) vectors were created which allow vector production in the infected tumour cell and

© 2013 Renner and Hlavaty; 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

thus, as a consequence, efficient delivery of the therapeutic gene eventually to almost all target cells (for review see [11-13]). Several research groups were involved in the design and construction of such MLV-based RCR vectors and were able to show that these vectors are well suited for efficient transduction of tumour cells and thus represent an efficacious treatment option for tumour therapy.

with the genomic RNA which is recognized by Gag via a packaging signal present immediately downstream of the 5´-LTR. Newly synthesized virus particles exit the cell via budding through

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Thus, due to its non-lytic nature retroviruses per se cannot be used as so-called oncolytic viruses, which are able to kill tumour cells by their productive infection only, but require additional gene sequences to exert a tumour destroying effect. Such therapeutic replicationcompetent MLV vectors can only be generated by adding therapeutic sequences in addition to the viral genes, which are all essential for virus replication, making the design of such vectors

Early attempts to produce replication-competent retroviral vectors have been already made in the late 80´s of the last century, when various groups inserted a transgene expression cassette into the 3´-LTR of replicating MLV to generate a research tool for analyses in whole-animal models [16-18]. During infection and reverse transcription of the proviral mRNA message, the transgene expression cassette was duplicated and, now present in the 5´- and 3´-LTR, inde‐ pendently expressed from the respective heterologous promoter. An RCR containing a mutant form of the dihydrofolate reductase (DHFR) gene was shown to stably transmit methotrexate resistance to infected fibroblasts upon multiple rounds of virus replication in vitro in the

Later, the group of Finn Skou Pedersen adopted this concept and inserted the transgene within the U3 region of the 3´-LTR of the Akv strain of MLV, mediating expression of the eGFPtransgene via an internal ribosomal entry site (IRES) of the encephalomyocarditis virus (EMCV) (Figure 1, (B)) [19]. This design again resulted in doubling of the IRES-transgene cassette in the infected cell, albeit, only the eGFP gene located in the 3´-LTR, but not the transgene present in the 5´-LTR, was expressed from the regulatory elements in the MLV 5´- LTR. Intraperitoneal injection of this vector at a concentration of 10e4 colony forming units into 3-4 days old mice led to more than 50 % eGFP-expressing spleen cells 4 days after injection. The level of eGFP-positive cells remained constant till day 7, but dramatically dropped from day 12 onwards, most likely to genetic instability of the vector and reversion to wild-type (wt)

Due to the highly compact nature of the MLV genome, however, the positions into which heterologous sequences can be inserted without impacting on viral replication are limited. Thus, up to now only few vector designs in which the transgene is located at different positions and/or its expression is facilitated by different mechanism have been created and are currently

Kasahara and colleagues favoured insertion of the transgene right downstream of the en‐ velope reading frame, as well linked via an ECMV IRES element (Figure 1, (C)) [21,22].

absence of drug selection and was produced at high titres by fibroblast cells [16].

Env protein-rich regions of the host cell membrane without lysis of the cell.

challenging and their genomic stability critical due to genomic overlength.

**3.1. Vector designs, spread kinetics and genome stability**

**3. Replicating MLV vectors**

virus lacking the marker gene [20].

under in-depth evaluation.

In the following sections we will provide an overview on MLV-derived RCR vectors and their therapeutic principle. Emphasis will be put on the different vector designs available and their influence on vector spread kinetics, vector genome stability, and transgene expression levels. Furthermore, strategies to target the vector by either selective infection of distinct cell types or selective expression and replication of the vector genome and expression of the delivered transgene in distinct cell types will be presented. Data from in vivo studies employing a set of different therapeutic genes and targeting different tumour types in various animal models will be reviewed and the therapeutic efficacy in these indications discussed. Finally issues regard‐ ing the safety of these vectors such as data from biodistribution and toxicological studies as well as potential risks associated with such a therapy are addressed in the following.

### **2. Biology of the murine leukaemia virus**

The murine leukaemia virus belongs to the genus of gamma-retroviruses which are small, enveloped viruses carrying two copies of a single-stranded RNA genome within an icosaedric core. The unique feature of retroviruses is their replication cycle, as their RNA genome is reverse transcribed into DNA, which then integrates into the host DNA before being transcri‐ bed to give rise to new virus genomes and viral proteins. MLV is a so-called simple retrovirus carrying only 3 genes in its genome, encoding the viral Gag, Pol and Env polyproteins. The group-specific antigen Gag is processed by the viral protease (PR) to the matrix (MA), capsid (CA) and nucleocapsid (NC) proteins which all form the viral core. The surface (SU) and transmembrane (TM) proteins are processed from the Env protein and are embedded in the host-cell derived lipid-bilayer. The pol gene encodes the viral PR, the reverse transcriptase (RT) and the integrase (IN), which are delivered in the virus particle to the cell to be transduced. After release of the virus core in the cytoplasm of an infected cell, reverse transcription of the single-stranded RNA into double-stranded DNA takes place and the pre-integration complex (PIC) consisting of virus DNA and viral and cellular proteins assembles [14]. As the MLV PIC, in contrast to lentiviruses such as HIV, is not able to cross the nuclear membrane, productive infection only occurs when the nuclear membrane is disrupted, as in dividing cells. Integration of the viral DNA into the host genome occurs randomly, however an integration preference of MLV-based vectors into the 5´-proximity of transcriptionally active genes was observed [15].

During reverse transcription identical long terminal repeats (LTRs) consisting of the so-called U3, R, and U5 region and flanking the viral genes are created which carry the viral promoter in the U3 region and the poly(A) site downstream of the R region. Expression from this promoter leads to two RNA species, a genomic one also encoding the viral proteins Gag and Pol, and the subgenomic env coding message. The Gag and Pol proteins assemble together with the genomic RNA which is recognized by Gag via a packaging signal present immediately downstream of the 5´-LTR. Newly synthesized virus particles exit the cell via budding through Env protein-rich regions of the host cell membrane without lysis of the cell.

Thus, due to its non-lytic nature retroviruses per se cannot be used as so-called oncolytic viruses, which are able to kill tumour cells by their productive infection only, but require additional gene sequences to exert a tumour destroying effect. Such therapeutic replicationcompetent MLV vectors can only be generated by adding therapeutic sequences in addition to the viral genes, which are all essential for virus replication, making the design of such vectors challenging and their genomic stability critical due to genomic overlength.

## **3. Replicating MLV vectors**

thus, as a consequence, efficient delivery of the therapeutic gene eventually to almost all target cells (for review see [11-13]). Several research groups were involved in the design and construction of such MLV-based RCR vectors and were able to show that these vectors are well suited for efficient transduction of tumour cells and thus represent an efficacious

In the following sections we will provide an overview on MLV-derived RCR vectors and their therapeutic principle. Emphasis will be put on the different vector designs available and their influence on vector spread kinetics, vector genome stability, and transgene expression levels. Furthermore, strategies to target the vector by either selective infection of distinct cell types or selective expression and replication of the vector genome and expression of the delivered transgene in distinct cell types will be presented. Data from in vivo studies employing a set of different therapeutic genes and targeting different tumour types in various animal models will be reviewed and the therapeutic efficacy in these indications discussed. Finally issues regard‐ ing the safety of these vectors such as data from biodistribution and toxicological studies as

well as potential risks associated with such a therapy are addressed in the following.

The murine leukaemia virus belongs to the genus of gamma-retroviruses which are small, enveloped viruses carrying two copies of a single-stranded RNA genome within an icosaedric core. The unique feature of retroviruses is their replication cycle, as their RNA genome is reverse transcribed into DNA, which then integrates into the host DNA before being transcri‐ bed to give rise to new virus genomes and viral proteins. MLV is a so-called simple retrovirus carrying only 3 genes in its genome, encoding the viral Gag, Pol and Env polyproteins. The group-specific antigen Gag is processed by the viral protease (PR) to the matrix (MA), capsid (CA) and nucleocapsid (NC) proteins which all form the viral core. The surface (SU) and transmembrane (TM) proteins are processed from the Env protein and are embedded in the host-cell derived lipid-bilayer. The pol gene encodes the viral PR, the reverse transcriptase (RT) and the integrase (IN), which are delivered in the virus particle to the cell to be transduced. After release of the virus core in the cytoplasm of an infected cell, reverse transcription of the single-stranded RNA into double-stranded DNA takes place and the pre-integration complex (PIC) consisting of virus DNA and viral and cellular proteins assembles [14]. As the MLV PIC, in contrast to lentiviruses such as HIV, is not able to cross the nuclear membrane, productive infection only occurs when the nuclear membrane is disrupted, as in dividing cells. Integration of the viral DNA into the host genome occurs randomly, however an integration preference of MLV-based vectors into the 5´-proximity of transcriptionally active genes was observed [15].

During reverse transcription identical long terminal repeats (LTRs) consisting of the so-called U3, R, and U5 region and flanking the viral genes are created which carry the viral promoter in the U3 region and the poly(A) site downstream of the R region. Expression from this promoter leads to two RNA species, a genomic one also encoding the viral proteins Gag and Pol, and the subgenomic env coding message. The Gag and Pol proteins assemble together

treatment option for tumour therapy.

130 Novel Gene Therapy Approaches

**2. Biology of the murine leukaemia virus**

### **3.1. Vector designs, spread kinetics and genome stability**

Early attempts to produce replication-competent retroviral vectors have been already made in the late 80´s of the last century, when various groups inserted a transgene expression cassette into the 3´-LTR of replicating MLV to generate a research tool for analyses in whole-animal models [16-18]. During infection and reverse transcription of the proviral mRNA message, the transgene expression cassette was duplicated and, now present in the 5´- and 3´-LTR, inde‐ pendently expressed from the respective heterologous promoter. An RCR containing a mutant form of the dihydrofolate reductase (DHFR) gene was shown to stably transmit methotrexate resistance to infected fibroblasts upon multiple rounds of virus replication in vitro in the absence of drug selection and was produced at high titres by fibroblast cells [16].

Later, the group of Finn Skou Pedersen adopted this concept and inserted the transgene within the U3 region of the 3´-LTR of the Akv strain of MLV, mediating expression of the eGFPtransgene via an internal ribosomal entry site (IRES) of the encephalomyocarditis virus (EMCV) (Figure 1, (B)) [19]. This design again resulted in doubling of the IRES-transgene cassette in the infected cell, albeit, only the eGFP gene located in the 3´-LTR, but not the transgene present in the 5´-LTR, was expressed from the regulatory elements in the MLV 5´- LTR. Intraperitoneal injection of this vector at a concentration of 10e4 colony forming units into 3-4 days old mice led to more than 50 % eGFP-expressing spleen cells 4 days after injection. The level of eGFP-positive cells remained constant till day 7, but dramatically dropped from day 12 onwards, most likely to genetic instability of the vector and reversion to wild-type (wt) virus lacking the marker gene [20].

Due to the highly compact nature of the MLV genome, however, the positions into which heterologous sequences can be inserted without impacting on viral replication are limited. Thus, up to now only few vector designs in which the transgene is located at different positions and/or its expression is facilitated by different mechanism have been created and are currently under in-depth evaluation.

Kasahara and colleagues favoured insertion of the transgene right downstream of the en‐ velope reading frame, as well linked via an ECMV IRES element (Figure 1, (C)) [21,22]. These RCR vectors are based on the Moloney strain of MLV and are equipped with the amphotropic MLV envelope gene, both of which are features allowing infection of hu‐ man and other mammalian cells. The effect of insert size and sequence on the genetic stability and spread efficacy of the vector revealed a strong negative correlation between insert size and deletion of the introduced sequence. Insertion of 1.6 kb in length led to greatly attenuated replication kinetics relative to wild-type virus and loss of the insert within a single infection cycle, whereas inserts up to 1.3 kb were well tolerated with slightly attenuated replication kinetics. In addition, the genomic integrity was maintained over multiple serial infection cycles [21,22].

round of infection to finally end up with more than 20 rounds of infection, and virus propa‐ gation for up to 100 days. The obtained data revealed a clear advantage of the Moloney-MLV strain over the Akv-MLV strain in respect to spread kinetics, transgene expression and vector stability and demonstrated that location of the transgene immediately downstream of the env gene is preferred in respect to genomic stability of the vector. These observations have been confirmed in spread and stability analyses after virus injection in tumour xenografts of mice [23]. Unexpectedly, our results also indicated that the host cell can influence the ability of MLVbased RCR vectors to stably propagate the expression of heterologous genes, since all vectors, regardless of design, lost the ability to express eGFP in NIH-3T3 cells much more rapidly than in HEK-293 cells [23]. Differences in vector genome stability between infected cell lines seem to be dependent neither on species nor on different replication kinetics of the vector in the respective cell lines. It rather might be due to differences in other virus and/or host-cell features including fidelity of the virus reverse transcriptase linked with the p53 status of the infected cells, expression of the anti-viral mechanisms such as APOBEC and TRIM family members, availability and balance of intracellular dNTP pools, and in general, due to an overall genetic

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Employing this design in which the heterologous sequences are located in the 3´-untranslated region immediately downstream of the env reading frame, expression of the transgene could also be facilitated by introducing, instead of the IRES, a splice acceptor site upstream of the transgene, which would result in a transgene specific mRNA message (Figure 1 (D)) [25]. Propagation of such vectors in cell culture however, revealed a much slower vector spread as compared to the IRES-carrying vector, which led to almost 100% infected cells after 3 days

RCR vectors based on Mo-MLV carrying a therapeutic gene in the 3´-untranslated region resemble currently the most advanced RCR vector design for tumour therapy and are already

A different approach in the design of RCR vectors has been pursued by the group of Christian Buchholz from the Paul-Ehrlich-Institut, as they inserted heterologous sequences including a 3´-terminally located furin cleavage site in frame into the envelope gene of the virus between the signal peptide and surface protein domain coding region (Figure 1, (E)) [26,27]. During production of the Env protein in virus vector transduced cells, the heterologous amino acid sequence will be cleaved off while the Env protein is processed through the secretory pathway and eventually will be secreted from the infected cell. Proof-of-concept for this vector design was shown with the immune stimulatory cytokine GM-CSF and the laminin-specific or T-cell specific single-chain antibody variable region fragment (scFv) [27]. The resulting viruses infected a variety of human cell lines and infectious virus particles were detected in superna‐ tants of infected cells. Moreover, these cells were able to efficiently process the encoded Envfusion proteins and to release reasonable amounts of protein molecules of GM-CSF, lamininspecific or T-cell specific scFvs into the cell culture media. Furthermore, the replicating viruses were genetically stable for at least 12 serial cycles of propagation. Thus, these vectors are ideally suited for production of therapeutic factors which need to be secreted, but less suitable in case

instability of certain cell types and cell lines [24].

employed in the first clinical trial for the treatment of cancer.

the protein produced is intended to be active in the infected cell.

after infection [25].

**Figure 1. Schematic depiction of the different RCR vector designs ((B) – (F)).** The inserted transgene is located at different positions in the viral vector and expression is facilitated by different means. (A) represents the genomic or‐ ganisation of the wt-MLV provirus. LTR = long terminal repeat; gag = viral group specific antigen gene; pol = viral poly‐ merase gene; env = viral envelope gene; IRES = internal ribosomal entry site; TG = transgene; F = protein cleavage site; SD = splice donor; SA = splice enhancer; ψ = psi packaging site; Pol III Prom = Polymerase III dependent promoter.

To further unravel the effects of viral strain and transgene position in the vector, as well as the impact the target cell type might have on spread kinetics and on the genetic stability of the virus vector in particular, we have independently compared the different parameters in serial rounds of infection in different cultured cell types as well as in vivo in tumour bearing animals [23]. To this end, various cell lines have been inoculated with RCR vectors based on the Akv and Moloney strain of MLV and carrying an IRES-EGFP transgene cassette either in the U3 region of the 3´-LTR or immediately downstream of the env gene, and passaged and monitored over time. Supernatant of the infected cells was also used to infected fresh cells for a further round of infection to allow exponential spread of virus vector until a maximum of EGFPexpressing cells was reached. Supernatant of the freshly infected cells was then used for a next round of infection to finally end up with more than 20 rounds of infection, and virus propa‐ gation for up to 100 days. The obtained data revealed a clear advantage of the Moloney-MLV strain over the Akv-MLV strain in respect to spread kinetics, transgene expression and vector stability and demonstrated that location of the transgene immediately downstream of the env gene is preferred in respect to genomic stability of the vector. These observations have been confirmed in spread and stability analyses after virus injection in tumour xenografts of mice [23]. Unexpectedly, our results also indicated that the host cell can influence the ability of MLVbased RCR vectors to stably propagate the expression of heterologous genes, since all vectors, regardless of design, lost the ability to express eGFP in NIH-3T3 cells much more rapidly than in HEK-293 cells [23]. Differences in vector genome stability between infected cell lines seem to be dependent neither on species nor on different replication kinetics of the vector in the respective cell lines. It rather might be due to differences in other virus and/or host-cell features including fidelity of the virus reverse transcriptase linked with the p53 status of the infected cells, expression of the anti-viral mechanisms such as APOBEC and TRIM family members, availability and balance of intracellular dNTP pools, and in general, due to an overall genetic instability of certain cell types and cell lines [24].

These RCR vectors are based on the Moloney strain of MLV and are equipped with the amphotropic MLV envelope gene, both of which are features allowing infection of hu‐ man and other mammalian cells. The effect of insert size and sequence on the genetic stability and spread efficacy of the vector revealed a strong negative correlation between insert size and deletion of the introduced sequence. Insertion of 1.6 kb in length led to greatly attenuated replication kinetics relative to wild-type virus and loss of the insert within a single infection cycle, whereas inserts up to 1.3 kb were well tolerated with slightly attenuated replication kinetics. In addition, the genomic integrity was maintained

**Figure 1. Schematic depiction of the different RCR vector designs ((B) – (F)).** The inserted transgene is located at different positions in the viral vector and expression is facilitated by different means. (A) represents the genomic or‐ ganisation of the wt-MLV provirus. LTR = long terminal repeat; gag = viral group specific antigen gene; pol = viral poly‐ merase gene; env = viral envelope gene; IRES = internal ribosomal entry site; TG = transgene; F = protein cleavage site; SD = splice donor; SA = splice enhancer; ψ = psi packaging site; Pol III Prom = Polymerase III dependent promoter.

To further unravel the effects of viral strain and transgene position in the vector, as well as the impact the target cell type might have on spread kinetics and on the genetic stability of the virus vector in particular, we have independently compared the different parameters in serial rounds of infection in different cultured cell types as well as in vivo in tumour bearing animals [23]. To this end, various cell lines have been inoculated with RCR vectors based on the Akv and Moloney strain of MLV and carrying an IRES-EGFP transgene cassette either in the U3 region of the 3´-LTR or immediately downstream of the env gene, and passaged and monitored over time. Supernatant of the infected cells was also used to infected fresh cells for a further round of infection to allow exponential spread of virus vector until a maximum of EGFPexpressing cells was reached. Supernatant of the freshly infected cells was then used for a next

over multiple serial infection cycles [21,22].

132 Novel Gene Therapy Approaches

Employing this design in which the heterologous sequences are located in the 3´-untranslated region immediately downstream of the env reading frame, expression of the transgene could also be facilitated by introducing, instead of the IRES, a splice acceptor site upstream of the transgene, which would result in a transgene specific mRNA message (Figure 1 (D)) [25]. Propagation of such vectors in cell culture however, revealed a much slower vector spread as compared to the IRES-carrying vector, which led to almost 100% infected cells after 3 days after infection [25].

RCR vectors based on Mo-MLV carrying a therapeutic gene in the 3´-untranslated region resemble currently the most advanced RCR vector design for tumour therapy and are already employed in the first clinical trial for the treatment of cancer.

A different approach in the design of RCR vectors has been pursued by the group of Christian Buchholz from the Paul-Ehrlich-Institut, as they inserted heterologous sequences including a 3´-terminally located furin cleavage site in frame into the envelope gene of the virus between the signal peptide and surface protein domain coding region (Figure 1, (E)) [26,27]. During production of the Env protein in virus vector transduced cells, the heterologous amino acid sequence will be cleaved off while the Env protein is processed through the secretory pathway and eventually will be secreted from the infected cell. Proof-of-concept for this vector design was shown with the immune stimulatory cytokine GM-CSF and the laminin-specific or T-cell specific single-chain antibody variable region fragment (scFv) [27]. The resulting viruses infected a variety of human cell lines and infectious virus particles were detected in superna‐ tants of infected cells. Moreover, these cells were able to efficiently process the encoded Envfusion proteins and to release reasonable amounts of protein molecules of GM-CSF, lamininspecific or T-cell specific scFvs into the cell culture media. Furthermore, the replicating viruses were genetically stable for at least 12 serial cycles of propagation. Thus, these vectors are ideally suited for production of therapeutic factors which need to be secreted, but less suitable in case the protein produced is intended to be active in the infected cell.

An additional site for integration of foreign sequences into the vector genome without impairing virus life cycle is the proline-rich region tract within the Env protein (Figure 1, (F)). Insertion of the eGFP marker gene into this site in a Mo-MLV-based RCR vector resulted in spread through almost 100% of cultured NIH-3T3 cells within one week after initial transfec‐ tion with virus sequences [28,29]. PCR analysis of integrated virus vector DNA from chroni‐ cally infected cells indicated no genetic recombination in the modified env gene region. An additional insertion of a Pol III promoter-shRNA expression cassette in antisense orientation into the 3´-untranslated region of the virus vector resulted in only slightly reduced spread kinetics as compared to the parental vector and in delivery and functional expression of the shRNA in most of the cells [30]. Again PCR analysis did not reveal any recombination events over 4 infection cycles.

**Figure 2. Principle of transcriptional targeting of RCR vectors.** (A) schematically depicts the RCR proviral genome as present in a virus producer cell. The tumour- or tissue-specific regulatory sequence (specific promoter) is located in the 3´-LTR of the viral vector. After infection and reverse transcription of the virus genome, the 3´-LTR U3 region is duplicated to the 5´-LTR and is thus able to drive expression of the viral genes and of the inserted transgene (B). For

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In early studies, the murine liver-specific transthyretin promoter/enhancer was inserted into the LTR U3 region, lacking the endogenous enhancer, of a replication-competent MLV [34]. When compared to wt-MLV however, the recombinant virus did not reveal an improved rate

Transcriptional targeting of MLV-based RCR vectors harbouring modifications in the U3 region by insertion of a heterologous promoter was demonstrated initially by Kasahara and colleagues [35]. In these vectors, hybrid LTRs were constructed by replacement of the MLV 3´-LTR U3 region from the very 5' end to either the CAAT box, the TATA box, or the transcriptional start site (TSS) by a heterologous promoter complementing the deleted boxes, respectively. Using a highly prostate-specific rat probasin (PB) proximal promoter and a synthetic variant of this promoter containing several copies of the androgen respon‐ sive region (ARR2PB), respectively, virus gene expression and virus production was shown to be restricted to prostate cancer cells in vitro [35]. Replication of vectors in which the heterologous promoter was fused directly to the MLV TSS was greatly impaired rela‐ tive to that of vectors in which the viral CAAT and TATA box, or the viral TATA box on‐ ly, was retained. The configuration in which the MLV TATA box was preserved, but all upstream elements had been replaced by heterologous regulatory sequences was found to be ideal in respect to transgene expression, vector spread and specificity [35]. The use of the stronger ARR2PB promoter resulted in a greatly improved efficacy of vector replica‐ tion [35]. Moreover, results from biodistribution studies in immunocompetent and immu‐ nodeficient mice indicated that this targeting strategy prevents the productive spread of

of infectivity of hepatocytes in vitro or a restricted tissue tropism in vivo [34].

RCR vectors to spleen and bone marrow of systemically infected mice [12].

Using a different set of promoter/enhancer elements, Metzl et al. were able to demonstrate that MLV-based RCR vectors can also be targeted to liver cancer cells and to tumour cells har‐ bouring a deregulated ß-catenin signalling pathway [36]. Vectors equipped with a chimeric promoter consisting of the hepatitis B virus enhancer II fused to the human α1-antitrypsin promoter (EIIPa1AT promoter) revealed a substantial spread in the liver cancer cell lines HepG2, AKH12, AKH13, but replicated only scarcely in the colon carcinoma cell lines SW480 and DLD-1, the cervical cancer cell line HeLa and the human embryonic kidney cell line

abbreviations please see legend to Figure 1.

#### **3.2. Targeting of infection and expression**

MLV-based RCR vectors can be accounted for being intrinsically tumour-selective due to the specific nature of MLV to replicate in dividing cells only. Nevertheless, it would be desirable to further improve the vector safety profile. This can be achieved by introducing transcriptional control elements that restrict RCR gene expression and subsequent virus vector replication to tumour cells - so-called transcriptional targeting; or by modulating the interaction of the RCR vector with host cells at the very early step of the infection process, known as physical targeting, via adaptation of the virus envelope glycoprotein to selectively bind to surface molecules exclusively or predominantly present on cancer cells. Alternatively, initial targeting could also be enabled by the use of delivery vehicles to facilitate transport or homing of the RCR vectors to the tumour site.

#### *3.2.1. Transcriptional targeting*

To allow transcriptional targeting of MLV-based RCR vectors, the most reasonable approach is the exchange of the ubiquitously active viral promoter located in the U3 region of the viral LTR by a tissue- or tumour-specific promoter delimiting its activity and thus virus vector replication to a specific cell type. Due to the particularities of retroviral reverse transcription, modifications of this promoter must be introduced into the U3 region of the 3'-LTR. This allows, after initial vector production and infection, duplication of the regulatory elements into the 5´-LTR (Figure 2). This strategy has been successfully employed previously in conventional replication-defective retroviral vectors to direct transgene expression to a particular cell type [31–33]. In RCR vectors however, not only expression of the transgene sequences is mediated by these regulatory elements, but also expression of viral genes which are needed to ensure efficient RCR vector replication in infected target cells and which have to be produced in an ample but well balanced manner. Moreover, as the LTR contains regulatory elements important for reverse transcription, RNA processing, and virus genome integration, modifications in this area may interfere with or may disrupt these elements and may thus negatively affect virus replication kinetics. This altogether renders the transcriptional targeting approach for RCR vectors rather complex.

An additional site for integration of foreign sequences into the vector genome without impairing virus life cycle is the proline-rich region tract within the Env protein (Figure 1, (F)). Insertion of the eGFP marker gene into this site in a Mo-MLV-based RCR vector resulted in spread through almost 100% of cultured NIH-3T3 cells within one week after initial transfec‐ tion with virus sequences [28,29]. PCR analysis of integrated virus vector DNA from chroni‐ cally infected cells indicated no genetic recombination in the modified env gene region. An additional insertion of a Pol III promoter-shRNA expression cassette in antisense orientation into the 3´-untranslated region of the virus vector resulted in only slightly reduced spread kinetics as compared to the parental vector and in delivery and functional expression of the shRNA in most of the cells [30]. Again PCR analysis did not reveal any recombination events

MLV-based RCR vectors can be accounted for being intrinsically tumour-selective due to the specific nature of MLV to replicate in dividing cells only. Nevertheless, it would be desirable to further improve the vector safety profile. This can be achieved by introducing transcriptional control elements that restrict RCR gene expression and subsequent virus vector replication to tumour cells - so-called transcriptional targeting; or by modulating the interaction of the RCR vector with host cells at the very early step of the infection process, known as physical targeting, via adaptation of the virus envelope glycoprotein to selectively bind to surface molecules exclusively or predominantly present on cancer cells. Alternatively, initial targeting could also be enabled by the use of delivery vehicles to facilitate transport or homing of the RCR vectors

To allow transcriptional targeting of MLV-based RCR vectors, the most reasonable approach is the exchange of the ubiquitously active viral promoter located in the U3 region of the viral LTR by a tissue- or tumour-specific promoter delimiting its activity and thus virus vector replication to a specific cell type. Due to the particularities of retroviral reverse transcription, modifications of this promoter must be introduced into the U3 region of the 3'-LTR. This allows, after initial vector production and infection, duplication of the regulatory elements into the 5´-LTR (Figure 2). This strategy has been successfully employed previously in conventional replication-defective retroviral vectors to direct transgene expression to a particular cell type [31–33]. In RCR vectors however, not only expression of the transgene sequences is mediated by these regulatory elements, but also expression of viral genes which are needed to ensure efficient RCR vector replication in infected target cells and which have to be produced in an ample but well balanced manner. Moreover, as the LTR contains regulatory elements important for reverse transcription, RNA processing, and virus genome integration, modifications in this area may interfere with or may disrupt these elements and may thus negatively affect virus replication kinetics. This altogether renders the transcriptional

over 4 infection cycles.

134 Novel Gene Therapy Approaches

to the tumour site.

*3.2.1. Transcriptional targeting*

targeting approach for RCR vectors rather complex.

**3.2. Targeting of infection and expression**

**Figure 2. Principle of transcriptional targeting of RCR vectors.** (A) schematically depicts the RCR proviral genome as present in a virus producer cell. The tumour- or tissue-specific regulatory sequence (specific promoter) is located in the 3´-LTR of the viral vector. After infection and reverse transcription of the virus genome, the 3´-LTR U3 region is duplicated to the 5´-LTR and is thus able to drive expression of the viral genes and of the inserted transgene (B). For abbreviations please see legend to Figure 1.

In early studies, the murine liver-specific transthyretin promoter/enhancer was inserted into the LTR U3 region, lacking the endogenous enhancer, of a replication-competent MLV [34]. When compared to wt-MLV however, the recombinant virus did not reveal an improved rate of infectivity of hepatocytes in vitro or a restricted tissue tropism in vivo [34].

Transcriptional targeting of MLV-based RCR vectors harbouring modifications in the U3 region by insertion of a heterologous promoter was demonstrated initially by Kasahara and colleagues [35]. In these vectors, hybrid LTRs were constructed by replacement of the MLV 3´-LTR U3 region from the very 5' end to either the CAAT box, the TATA box, or the transcriptional start site (TSS) by a heterologous promoter complementing the deleted boxes, respectively. Using a highly prostate-specific rat probasin (PB) proximal promoter and a synthetic variant of this promoter containing several copies of the androgen respon‐ sive region (ARR2PB), respectively, virus gene expression and virus production was shown to be restricted to prostate cancer cells in vitro [35]. Replication of vectors in which the heterologous promoter was fused directly to the MLV TSS was greatly impaired rela‐ tive to that of vectors in which the viral CAAT and TATA box, or the viral TATA box on‐ ly, was retained. The configuration in which the MLV TATA box was preserved, but all upstream elements had been replaced by heterologous regulatory sequences was found to be ideal in respect to transgene expression, vector spread and specificity [35]. The use of the stronger ARR2PB promoter resulted in a greatly improved efficacy of vector replica‐ tion [35]. Moreover, results from biodistribution studies in immunocompetent and immu‐ nodeficient mice indicated that this targeting strategy prevents the productive spread of RCR vectors to spleen and bone marrow of systemically infected mice [12].

Using a different set of promoter/enhancer elements, Metzl et al. were able to demonstrate that MLV-based RCR vectors can also be targeted to liver cancer cells and to tumour cells har‐ bouring a deregulated ß-catenin signalling pathway [36]. Vectors equipped with a chimeric promoter consisting of the hepatitis B virus enhancer II fused to the human α1-antitrypsin promoter (EIIPa1AT promoter) revealed a substantial spread in the liver cancer cell lines HepG2, AKH12, AKH13, but replicated only scarcely in the colon carcinoma cell lines SW480 and DLD-1, the cervical cancer cell line HeLa and the human embryonic kidney cell line HEK-293 [36]. Similarly, vectors equipped with the synthetic beta-catenin/T-cell factor dependent CTP4 promoter replicated in the ß-catenin deregulated cancer cell lines HepG2, SW480, and DLD-1, but not in the cell lines AKH12, AKH13, HeLa, and HEK-293, which revealed a normal ß-catenin signalling pathway. When the heterologous promoters were used to replace almost the entire U3 region, including the MLV TATA box and TSS (TATAreplacement (TR) design), vector replication was inefficient as virus particle production from infected cells was clearly reduced by factor 100 as compared to a vector harbouring the wt-MLV 3´-LTR. On the contrary, fusion of the heterologous promoter lacking the TATA box to the MLV TATA box (TATA-fusion (TF) design) generated vectors which replicated with almost wt-MLV kinetics throughout permissive cells despite the fact that virion production from infected cells was reduced by 10-fold as compared to the prototype vector ACE-GFP. As expected, these TF-vectors exhibited low or negligible spread in non-permissive cells. The genomic stability of the TF-vectors, however, was shown to be comparable to those containing wt-MLV LTRs [36].

in splicing of the viral RNA. However, employing an in vitro evolution strategy, extended passaging of cells exposed to the chimeric virus resulted in selection of virus mutants with rapid replication kinetics. Different variants arose from different sets of infection experiments. None of the revertants exhibited mutations in the GALV env gene itself, but rather in other areas of the virus to retain the ratio of spliced to unspliced viral messages which had been

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An alternative strategy for changing the tropism of MLV-based RCR vectors is via direct engineering of the specific targeting ligand sequence within the env gene. Again, several strategies have been demonstrated for RDR vectors, including the incorporation of ligands or single chain antibodies into the env gene to allow targeting to an alternative receptor [43-45]. However, these approaches were of limited success in respect to transciptional targeting, as, although retargeting of the vector was achieved, infection efficiency was greatly reduced, since the conformational change in the envelope protein necessary for proper virus-cell fusion failed to happen subsequently to the binding of the modified envelope to the alternate receptor [46].

In another approach to allow the physical targeting of RCR vectors, two tandem repeats of the immunoglobulin G-binding Z-domains of Staphylococcus protein A were inserted into the proline-rich region of amphotropic or ecotropic MLV envelope proteins present in MLV-based RCR vector particles, respectively [47]. This approach should allow virus particles to be conjugated to an antibody of choice, which can then be used for the selective binding of virus to cells (over)-expressing the chosen antibody target. Modified envelopes were efficiently expressed and incorporated into virions, while infectivity was markedly reduced by this

For RCR vectors the most efficient physical targeting system up to now is based on proteaseactivatable envelope proteins [48]. Rather than attempting to redirect infection to target cells by incorporation of specific binding domains into the envelope protein, here, the virus remains non-infectious until the Env proteins become activated by cleavage by a secreted or membranebound cellular protease recognizing the protease target site present in the engineered Env molecule. A directed evolution-based approach was employed for the selection of retroviruses activatable by matrix metalloproteinases (MMPs) which are specifically expressed by tumour cells [26,49,50]. RCR vectors generated to express either the epidermal growth factor (EGF) or the CD40 ligand, linked via MMP cleavage sites as fusion to the N-terminal of the MLV 4070A envelope protein, were sequestered by the EGF receptor or the CD40 receptor, which are ubiquitously-expressed on potential host cells. By that the envelope protein was prevented from interacting with its natural Pit-2 receptor resulting in poor infection efficiencies and thus de-targeting of the RCR vectors from non-tumour cells [49,50]. Infection efficiency however, is restored in cells which express high levels of MMPs, such as many tumour cell types, due to Env-ligand cleavage and interaction of the Env protein with its natural receptor [50]. In a comparative study Duerner and colleagues analysed the spread of targeted and non-targeted MLV RCR variants in *s.c.* tumours derived from HT1080 and U-87MG cells, respectively, and in extratumoral organs after systemic tail vein injection of the vector into SCID mice [48]. Both virus types were able to efficiently infect tumour cells however, the non-targeted virus efficiently infected also extratumoral organs such as bone marrow, spleen, and liver. Quanti‐

pertubated by the substitution of the env gene [42].

pseudotyping [47].

Both studies indicated that the precise manner in which the heterologous promoters are inserted into the U3 region of the 3´-LTR is of paramount importance. Only vectors retaining the MLV TATA box in its natural position exhibited both regulated gene expression and rapid replication kinetics [35,36].

#### *3.2.2. Physical targeting*

For decades it was generally accepted, that with MLV, and as a consequence, with MLV-based vectors, infection can only take place in dividing cells. Therefore, such vectors can be *a priori* accounted as intrinsically tumour-selective in the appropriate environment. However, very recently Liu et al. reported infection of neurons and growth arrested neuroendocrine cells with MLV-based RCR and RDR vectors harbouring an amphotropic envelope at efficiency similar to that of lentiviral vectors, which are known to infect also non-dividing cells [37]. This new and unexpected observation, if confirmed, will further raise the need for retrovirus vectors with a specific and/or targeted infection and/or replication range.

Retroviruses are unique among viral vectors in their capacity to incorporate a wide range of envelope proteins from other retroviruses and even from completely unrelated virus types. Insertion of heterologous envelope proteins into the outer shell of viral particles, so called pseudotyping, to exert an infection targeting approach has already been demonstrated in RDR vectors for a variety of envelope molecules [38–40]. The MLV-based RCR vectors used in cancer gene therapy applications to date are mostly based on the Moloney strain of MLV, which naturally expresses the ecotropic MLV envelope and thus are able to only infect rodent cells. To allow infection of human cells, the vectors are pseudotyped with the amphotropic envelope gene from the 4070A strain of MLV, which can infect most mammalian cell types via the ubiquitously-expressed Pit-2 receptor [41]. Logg and colleagues also replaced the native env sequence with that of the gibbon ape leukaemia virus (GALV) retaining a short portion of the signal peptide coding sequence of the MLV Env to avoid alteration of the MLV polymerase reading frame which is overlapping with the 5´-end of the envelope reading frame [42]. This env gene replacement greatly attenuated viral replication, most probably by a large clearance in splicing of the viral RNA. However, employing an in vitro evolution strategy, extended passaging of cells exposed to the chimeric virus resulted in selection of virus mutants with rapid replication kinetics. Different variants arose from different sets of infection experiments. None of the revertants exhibited mutations in the GALV env gene itself, but rather in other areas of the virus to retain the ratio of spliced to unspliced viral messages which had been pertubated by the substitution of the env gene [42].

HEK-293 [36]. Similarly, vectors equipped with the synthetic beta-catenin/T-cell factor dependent CTP4 promoter replicated in the ß-catenin deregulated cancer cell lines HepG2, SW480, and DLD-1, but not in the cell lines AKH12, AKH13, HeLa, and HEK-293, which revealed a normal ß-catenin signalling pathway. When the heterologous promoters were used to replace almost the entire U3 region, including the MLV TATA box and TSS (TATAreplacement (TR) design), vector replication was inefficient as virus particle production from infected cells was clearly reduced by factor 100 as compared to a vector harbouring the wt-MLV 3´-LTR. On the contrary, fusion of the heterologous promoter lacking the TATA box to the MLV TATA box (TATA-fusion (TF) design) generated vectors which replicated with almost wt-MLV kinetics throughout permissive cells despite the fact that virion production from infected cells was reduced by 10-fold as compared to the prototype vector ACE-GFP. As expected, these TF-vectors exhibited low or negligible spread in non-permissive cells. The genomic stability of the TF-vectors, however, was shown to be comparable to those containing

Both studies indicated that the precise manner in which the heterologous promoters are inserted into the U3 region of the 3´-LTR is of paramount importance. Only vectors retaining the MLV TATA box in its natural position exhibited both regulated gene expression and rapid

For decades it was generally accepted, that with MLV, and as a consequence, with MLV-based vectors, infection can only take place in dividing cells. Therefore, such vectors can be *a priori* accounted as intrinsically tumour-selective in the appropriate environment. However, very recently Liu et al. reported infection of neurons and growth arrested neuroendocrine cells with MLV-based RCR and RDR vectors harbouring an amphotropic envelope at efficiency similar to that of lentiviral vectors, which are known to infect also non-dividing cells [37]. This new and unexpected observation, if confirmed, will further raise the need for retrovirus vectors

Retroviruses are unique among viral vectors in their capacity to incorporate a wide range of envelope proteins from other retroviruses and even from completely unrelated virus types. Insertion of heterologous envelope proteins into the outer shell of viral particles, so called pseudotyping, to exert an infection targeting approach has already been demonstrated in RDR vectors for a variety of envelope molecules [38–40]. The MLV-based RCR vectors used in cancer gene therapy applications to date are mostly based on the Moloney strain of MLV, which naturally expresses the ecotropic MLV envelope and thus are able to only infect rodent cells. To allow infection of human cells, the vectors are pseudotyped with the amphotropic envelope gene from the 4070A strain of MLV, which can infect most mammalian cell types via the ubiquitously-expressed Pit-2 receptor [41]. Logg and colleagues also replaced the native env sequence with that of the gibbon ape leukaemia virus (GALV) retaining a short portion of the signal peptide coding sequence of the MLV Env to avoid alteration of the MLV polymerase reading frame which is overlapping with the 5´-end of the envelope reading frame [42]. This env gene replacement greatly attenuated viral replication, most probably by a large clearance

with a specific and/or targeted infection and/or replication range.

wt-MLV LTRs [36].

136 Novel Gene Therapy Approaches

replication kinetics [35,36].

*3.2.2. Physical targeting*

An alternative strategy for changing the tropism of MLV-based RCR vectors is via direct engineering of the specific targeting ligand sequence within the env gene. Again, several strategies have been demonstrated for RDR vectors, including the incorporation of ligands or single chain antibodies into the env gene to allow targeting to an alternative receptor [43-45]. However, these approaches were of limited success in respect to transciptional targeting, as, although retargeting of the vector was achieved, infection efficiency was greatly reduced, since the conformational change in the envelope protein necessary for proper virus-cell fusion failed to happen subsequently to the binding of the modified envelope to the alternate receptor [46].

In another approach to allow the physical targeting of RCR vectors, two tandem repeats of the immunoglobulin G-binding Z-domains of Staphylococcus protein A were inserted into the proline-rich region of amphotropic or ecotropic MLV envelope proteins present in MLV-based RCR vector particles, respectively [47]. This approach should allow virus particles to be conjugated to an antibody of choice, which can then be used for the selective binding of virus to cells (over)-expressing the chosen antibody target. Modified envelopes were efficiently expressed and incorporated into virions, while infectivity was markedly reduced by this pseudotyping [47].

For RCR vectors the most efficient physical targeting system up to now is based on proteaseactivatable envelope proteins [48]. Rather than attempting to redirect infection to target cells by incorporation of specific binding domains into the envelope protein, here, the virus remains non-infectious until the Env proteins become activated by cleavage by a secreted or membranebound cellular protease recognizing the protease target site present in the engineered Env molecule. A directed evolution-based approach was employed for the selection of retroviruses activatable by matrix metalloproteinases (MMPs) which are specifically expressed by tumour cells [26,49,50]. RCR vectors generated to express either the epidermal growth factor (EGF) or the CD40 ligand, linked via MMP cleavage sites as fusion to the N-terminal of the MLV 4070A envelope protein, were sequestered by the EGF receptor or the CD40 receptor, which are ubiquitously-expressed on potential host cells. By that the envelope protein was prevented from interacting with its natural Pit-2 receptor resulting in poor infection efficiencies and thus de-targeting of the RCR vectors from non-tumour cells [49,50]. Infection efficiency however, is restored in cells which express high levels of MMPs, such as many tumour cell types, due to Env-ligand cleavage and interaction of the Env protein with its natural receptor [50]. In a comparative study Duerner and colleagues analysed the spread of targeted and non-targeted MLV RCR variants in *s.c.* tumours derived from HT1080 and U-87MG cells, respectively, and in extratumoral organs after systemic tail vein injection of the vector into SCID mice [48]. Both virus types were able to efficiently infect tumour cells however, the non-targeted virus efficiently infected also extratumoral organs such as bone marrow, spleen, and liver. Quanti‐ tative analyses revealed an up to 500-fold higher selective infection of tumour tissue with the targeted viruses than with the non-targeted counterpart [50].

tion of metabolites in the tumour cells results in a more contained and specific effect as

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Initial data demonstrating therapeutic efficacy of an RCR vector mediated suicide therapy approach have been shown using the yeast cytosine deaminase (yCD) gene in a mouse model of human glioma [56]. In cells infected with the yCD expressing RCR vector ACE-CD, the nontoxic prodrug 5-fluorocytosine (5-FC) was converted into the toxic component 5-fluorouracil (5-FU), leading to cell death not only in infected cells but also in surrounding non-infected dividing cells due to intercellular diffusion of 5-FU. Stereotactic intratumoral injection of only 1x10e4 infectious ACE-CD particles into pre-established intracranial U-87MG human glioma xenografts in nude mice followed by daily intraperitoneal (*i.p.*) administration of 500 mg 5-FC per kg body weight eight days later for 15 days (ACE-CD + 5-FC) led to survival of all treated animals for a follow up period of 60 days, whereas mice of both control groups, vector only (ACE-CD + PBS) and prodrug only (PBS + 5-FC), died within 40 days [56]. Subsequent studies, however, revealed that this treatment regimen is insufficient to get complete eradication of U-87MG tumours in the treated animals, resulting in death of the animals after 70 and more days [57]. Retrospective immunohistochemical analyses showed that in most of the treated animals still small areas of tumour tissue were present indicating an ectopic spread of glioma cells in the brain. Despite the fact that all of the surviving tumour cells stained positive for viral envelope protein, these tumour cells had not been killed by the administration of 5-FC. This suggests that these cells either have been infected with a suicide gene deletion variant of the vector or the therapeutic gene, although present, is not expressed in these cells or that the cells are refractory to chemotherapy [57]. Tai et al. also examined whether the efficacy of this treatment could be increased by administering multiple cycles of 5-FC [57]. To this end, after injection of 1x10e4 transducing units of vector ACE-CD into preformed intracranial tumours, mice received multiple cycles of 5-FC for 8 consecutive days with 3-week intervals between the treatment periods [57]. Again, all control animals receiving virus vector only and prodrug only, respectively, died within 40 days, whereas all mice treated with vector and prodrug survived for more than 100 days, demonstrating that the multi-cycle strategy provides a significant therapeutic benefit compared to a single cycle of prodrug administration [57].

To investigate the effect of the immune system on the effectiveness of the RCR vector-mediated glioma therapy, the rat glioma cell line RG2 was used to establish syngeneic intracranial tumours in Fischer 344 rats [58]. Three days after tumour implantation, 1x10e6 infectious ACE-CD virus particles were stereotactically injected into the growing tumours. Ten days after virus injection, 5-FC at 500 mg/kg body weight was administered *i.p*. for 7 consecutive days and after a 10 days interval the treatment cycle was repeated. Animals treated with vector ACE-CD and 5-FC survived for up to 35 days, whereas control animals (ACE-CD + PBS) died within 21 days. Despite the higher initial virus vector load injected into the tumour and the shorter intervals between the treatment cycles a significantly shorter survival time of treated Fischer 344 rats was observed as compared to data of vector/prodrug treated U-87MG tumour bearing nude mice [58]. This observation might be, to a certain extent, due to reduced spread kinetics in the RG2 tumour as compared to the U-87MG tumours, as only 65 % of RG2 tumour cells got transduced after 21 days after initial virus infection. On the other hand, animals with tumours

compared to the, in most cases, systemically given chemotherapy.

#### *3.2.3. Alternative ways for RCR vector targeting*

Recently, a fully functional chimeric vector system that uses a helper-dependent adenovirus 5 (Ad) vector as a first stage carrier to express and deliver a fully functional RCR vector has been described [51]. The RCR vectors are produced *in situ* from initially adenovirus-transduced cells, thereby combining benefits of both vector systems – high titers reached with adenovi‐ ruses and stable integration of retroviruses. At equivalent initial transduction levels, more secondary RCR progeny were produced from Ad-RCR-transduced cells as compared to RCRtransduced cells, resulting in further acceleration of RCR replication kinetics [51]. In preestablished *s.c.* human breast cancer xenografts in mice, suicide gene therapy with high titre chimeric Ad-RCR vectors achieved, in a dose dependent manner, an enhanced efficacy compared to delivery of respective RCR-only vectors [52]. As the target cell binding tropism of adenoviral vectors can be altered by modifications to the fibre knob [53], the strategy of employing a chimeric Ad-RCR vector system might represent a promising step forward towards a targeted and efficacious cancer gene therapy.

As a future option, delivery of RCR vectors to tumours could also be facilitated by the use of tumour-homing cells as vector carriers. Mesenchymal stromal cells, for example, which have been shown to be able to home to malignant areas, have been loaded with replicating adeno‐ viral vectors to deliver them to the tumour tissue to execute their oncolytic potential [54], an approach which might be also applicable to RCR vectors.

#### **3.3. Therapeutic application of MLV-based RCR vectors**

Despite the fact that the first experiments employing MLV-based RCR vectors have been commenced more than 20 years ago, the utilization of these vectors for cancer gene therapy is still at an early stage. The vectors are not oncolytic *per se* and the choice of therapeutic genes which can be used is limited, as large heterologous sequences cannot be inserted into the MLV genome without impairing vector stability and replication efficiency. Nonetheless a number of therapeutic sequences such as those encoding suicide genes, cytokines and interfering RNAs have been demonstrated to be stably propagated over several infection cycles by MLV-based RCR vectors.

#### *3.3.1. Suicide genes*

Suicide genes, also called prodrug-converting genes, encode proteins which are not toxic *per se*, but which are able to catalyse the formation of toxic metabolite(s) from a non-toxic or lowtoxic prodrug. By the delivery of suicide genes to and their expression in tumour cells, conversion of a systemically administered prodrug by the respective suicide gene product results in a highly specific and effective anti-tumor therapy [55]. Although the toxic metabolites generated are quite often identical to those of classical chemotherapy, here, the local produc‐ tion of metabolites in the tumour cells results in a more contained and specific effect as compared to the, in most cases, systemically given chemotherapy.

tative analyses revealed an up to 500-fold higher selective infection of tumour tissue with the

Recently, a fully functional chimeric vector system that uses a helper-dependent adenovirus 5 (Ad) vector as a first stage carrier to express and deliver a fully functional RCR vector has been described [51]. The RCR vectors are produced *in situ* from initially adenovirus-transduced cells, thereby combining benefits of both vector systems – high titers reached with adenovi‐ ruses and stable integration of retroviruses. At equivalent initial transduction levels, more secondary RCR progeny were produced from Ad-RCR-transduced cells as compared to RCRtransduced cells, resulting in further acceleration of RCR replication kinetics [51]. In preestablished *s.c.* human breast cancer xenografts in mice, suicide gene therapy with high titre chimeric Ad-RCR vectors achieved, in a dose dependent manner, an enhanced efficacy compared to delivery of respective RCR-only vectors [52]. As the target cell binding tropism of adenoviral vectors can be altered by modifications to the fibre knob [53], the strategy of employing a chimeric Ad-RCR vector system might represent a promising step forward

As a future option, delivery of RCR vectors to tumours could also be facilitated by the use of tumour-homing cells as vector carriers. Mesenchymal stromal cells, for example, which have been shown to be able to home to malignant areas, have been loaded with replicating adeno‐ viral vectors to deliver them to the tumour tissue to execute their oncolytic potential [54], an

Despite the fact that the first experiments employing MLV-based RCR vectors have been commenced more than 20 years ago, the utilization of these vectors for cancer gene therapy is still at an early stage. The vectors are not oncolytic *per se* and the choice of therapeutic genes which can be used is limited, as large heterologous sequences cannot be inserted into the MLV genome without impairing vector stability and replication efficiency. Nonetheless a number of therapeutic sequences such as those encoding suicide genes, cytokines and interfering RNAs have been demonstrated to be stably propagated over several infection cycles by MLV-based

Suicide genes, also called prodrug-converting genes, encode proteins which are not toxic *per se*, but which are able to catalyse the formation of toxic metabolite(s) from a non-toxic or lowtoxic prodrug. By the delivery of suicide genes to and their expression in tumour cells, conversion of a systemically administered prodrug by the respective suicide gene product results in a highly specific and effective anti-tumor therapy [55]. Although the toxic metabolites generated are quite often identical to those of classical chemotherapy, here, the local produc‐

targeted viruses than with the non-targeted counterpart [50].

towards a targeted and efficacious cancer gene therapy.

approach which might be also applicable to RCR vectors.

**3.3. Therapeutic application of MLV-based RCR vectors**

RCR vectors.

*3.3.1. Suicide genes*

*3.2.3. Alternative ways for RCR vector targeting*

138 Novel Gene Therapy Approaches

Initial data demonstrating therapeutic efficacy of an RCR vector mediated suicide therapy approach have been shown using the yeast cytosine deaminase (yCD) gene in a mouse model of human glioma [56]. In cells infected with the yCD expressing RCR vector ACE-CD, the nontoxic prodrug 5-fluorocytosine (5-FC) was converted into the toxic component 5-fluorouracil (5-FU), leading to cell death not only in infected cells but also in surrounding non-infected dividing cells due to intercellular diffusion of 5-FU. Stereotactic intratumoral injection of only 1x10e4 infectious ACE-CD particles into pre-established intracranial U-87MG human glioma xenografts in nude mice followed by daily intraperitoneal (*i.p.*) administration of 500 mg 5-FC per kg body weight eight days later for 15 days (ACE-CD + 5-FC) led to survival of all treated animals for a follow up period of 60 days, whereas mice of both control groups, vector only (ACE-CD + PBS) and prodrug only (PBS + 5-FC), died within 40 days [56]. Subsequent studies, however, revealed that this treatment regimen is insufficient to get complete eradication of U-87MG tumours in the treated animals, resulting in death of the animals after 70 and more days [57]. Retrospective immunohistochemical analyses showed that in most of the treated animals still small areas of tumour tissue were present indicating an ectopic spread of glioma cells in the brain. Despite the fact that all of the surviving tumour cells stained positive for viral envelope protein, these tumour cells had not been killed by the administration of 5-FC. This suggests that these cells either have been infected with a suicide gene deletion variant of the vector or the therapeutic gene, although present, is not expressed in these cells or that the cells are refractory to chemotherapy [57]. Tai et al. also examined whether the efficacy of this treatment could be increased by administering multiple cycles of 5-FC [57]. To this end, after injection of 1x10e4 transducing units of vector ACE-CD into preformed intracranial tumours, mice received multiple cycles of 5-FC for 8 consecutive days with 3-week intervals between the treatment periods [57]. Again, all control animals receiving virus vector only and prodrug only, respectively, died within 40 days, whereas all mice treated with vector and prodrug survived for more than 100 days, demonstrating that the multi-cycle strategy provides a significant therapeutic benefit compared to a single cycle of prodrug administration [57].

To investigate the effect of the immune system on the effectiveness of the RCR vector-mediated glioma therapy, the rat glioma cell line RG2 was used to establish syngeneic intracranial tumours in Fischer 344 rats [58]. Three days after tumour implantation, 1x10e6 infectious ACE-CD virus particles were stereotactically injected into the growing tumours. Ten days after virus injection, 5-FC at 500 mg/kg body weight was administered *i.p*. for 7 consecutive days and after a 10 days interval the treatment cycle was repeated. Animals treated with vector ACE-CD and 5-FC survived for up to 35 days, whereas control animals (ACE-CD + PBS) died within 21 days. Despite the higher initial virus vector load injected into the tumour and the shorter intervals between the treatment cycles a significantly shorter survival time of treated Fischer 344 rats was observed as compared to data of vector/prodrug treated U-87MG tumour bearing nude mice [58]. This observation might be, to a certain extent, due to reduced spread kinetics in the RG2 tumour as compared to the U-87MG tumours, as only 65 % of RG2 tumour cells got transduced after 21 days after initial virus infection. On the other hand, animals with tumours derived from pre-infected and 100 % CD-expressing cells treated with 5-FC died due to tumour burden at a significantly later time point, however still within 50 days [58]. This indicates that other factors might contribute to the poorer treatment efficacy, such as insufficient levels of 5- FC/5-FU in the tumour, differences in resistance to 5-FU, or effects triggered by the immune system. Regarding this latter point, however, no immune response against the RCR vector in the brain has been detected [58].

and 5-FC showed stable or decreased levels of bioluminescence, indicating that the de‐ velopment of metastases was inhibited [61]. The locoregional delivery of the CD suicide gene by RCR vectors infused into the portal circulation thus resulted in progressive transduction of multiple tumour foci in the liver without evidence of spread to adjacent normal parenchyma or extrahepatic tissues as shown by qPCR analyses of MLV-specific sequences in the DNA of liver tumour tissue, normal liver, and bone marrow cells [61].

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For proof-of-concept in a further indication, Kikuchi and colleagues evaluated the transduction and therapeutic efficacy of intravesically administered RCR vectors in orthotopic bladder tumours in mice [62]. Tumours were established by implantation of either MBT-2 murine bladder cancer cells into immunocompetent syngeneic C3H/HeJ mice or of KU-19-19 human bladder cancer cells into nude mice. RCR vector particles carrying the eGFP transgene were delivered intravesically *via* a catheter inserted into the bladder 5 days after tumour formation. Upon injection of 3.2x10e5 infectious virus particles, vector spread in the human xenograft model reached almost all of the tumour cells within 35 days post infection [62]. In the orthotopic syngeneic model, vector spread resulted in, on average, 30 % of infected tumour cells 27 days after vector instillation, as revealed by immunohistochemical analyses of transgene expression [62]. The effects of the respective therapeutic vector ACE-CD were demonstrated in the MBT-2 bladder cancer model, whereby 3.2x10e5 infectious virus particles were instilled intravesically into the bladder of mice containing preformed tumours. Twelve days following infection, animals received daily 5-FC at 500 mg/kg given *i.p.* for 15 days [62]. A single course of 5-FC treatment led to significantly reduced tumour growth and 50 % of the animals survived for more than 70 days. In contrast, all of the animals in vector only or vehicle control groups died within 40 days. Again, the locoregional delivery of intravesically administered RCR vectors was shown to achieve significant tumour growth inhibition by efficient delivery of the therapeutic gene into the orthotopic bladder tumour cells without any evidence of spread to

Recently, Kawasaki et al. investigated RCR vector mediated gene therapy for the treatment of human malignant mesothelioma [63]. Cells of this tumour type were found to be highly permissive for RCR infection [63]. After a single intratumoral injection of 1x10e4 ACE-CD virus vector particles into pre-established subcutaneous MSTO-211H human mesothelioma tumour xenografts in nude mice, followed by daily 5-FC administration at 500 mg/kg body weight given *i.p*. from day 12 to day 32 post-infection, significant inhibition of tumour growth was observed in ACE-CD/5-FC treated mice compared to control groups as indicated by a tumour

was also investigated in a peritoneally disseminated human mesothelioma xenograft model [63]. Here, MSTO-211H cells stably expressing the mCherry fluorescence protein and trans‐ duced with vector ACE-CD were mixed at a ratio of 1:100 with non-transduced mCherry expressing MSTO-211H cells and the mixture was injected i.p. into nude mice. After confir‐ mation of tumour formation by in vivo imaging, daily 5-FC administration was initiated (500 mg/kg *i.p*.) for 15 consecutive days. In 4 out of 9 mice of the vector/5-FC receiving group no visible tumours were observed and mice survived until day 100 after cell injection. On the contrary, all non-treated mice had died at day 61 at the latest. In summary, the 5-FC treated

, respectively, at day 32 after infection. Efficacy of the treatment

adjacent distant organs [62].

volume of 200 mm3 *vs.* 800 mm3

Recently, we have critically analysed a panel of 15 different human and rodent glioma and glioblastoma cell lines in respect to spread of RCR vectors derived from vector ACE-CD to their sensitivity towards the 5-FC/5-FU suicide system, and to their orthotopic growth characteristics in mice to identify suitable preclinical animal models as test bed for the development and evaluation of RCR vector mediated treatment of glioblastoma [59]. Rapid virus spread was observed in eight out of nine human cell lines tested in vi‐ tro. As expected, only CD-expressing cells became sensitive to 5-FC. All LD50 values were within the range of concentrations obtained in human body fluids after convention‐ al 5-FC administration. In addition, a significant bystander effect was observed in all hu‐ man glioma cell lines tested, supporting the potential of this suicide gene therapy for the treatment of brain tumours [59].

This therapeutic concept has also been experimentally employed to subcutaneous and or‐ thotopic liver metastasis of colorectal cancer in an immunocompetent rodent model [60,61]. To this end, murine CT26 cells pre-transduced with vector ACE-CD were mixed with non-transduced CT26 cells at a ratio of 1:200 prior to implantation into BALB/c mice. Twelve days later, daily 5-FC treatment at 500 mg/kg, given *i.p.* twice a day, was started and continued until the end of the experiment. 5-FC treated animals showed sig‐ nificant inhibition of tumour growth resulting in an average tumour size of approx. 100 mm3 at day 24, whereas in animals whose drinking water was supplemented with 0.4 mg/ml of azidothymidine (AZT) to prevent MLV vector replication, tumour growth was inhibited only moderately with an average tumour size of approx. 500 mm3 at day 24 [61]. Tumour size in untreated animals reached more than 800 mm3 at day 24 [61]. The effect on tumour size observed in the AZT-treated group most probably was due to the presence of a bystander effect, in which toxic metabolites produced by the CD-positive cells can freely diffuse into neighbouring, CD-negative cells to cause cell death and thereby eventually lead to a delay in tumour growth. These observations clearly indicate that the efficacy of the ACE-CD/5-FC treatment is dependent on a sustained vector spread. Proof-of-concept of this therapy was also shown in a multifocal hepatic metasta‐ sis model [61]. Here, CT26 cells stably expressing the firefly luciferase marker gene were infused into the portal system via intrasplenic injection, became trapped within the hep‐ atic microcirculation, and seeded metastases. Three days later, 2x10e4 ACE-CD virus vec‐ tor particles were instilled into tumour-bearing mice, again via intrasplenic injection, followed by daily 5-FC administration at 500 mg/kg given *i.p.* twice a day, initiated 14 days after tumour cells inoculation [61]. In contrast to untreated animals or animals which received vector and PBS only, and which revealed increasing bioluminescence throughout the 28 day experiment, 5 out of 7 animals treated with both vector ACE-CD and 5-FC showed stable or decreased levels of bioluminescence, indicating that the de‐ velopment of metastases was inhibited [61]. The locoregional delivery of the CD suicide gene by RCR vectors infused into the portal circulation thus resulted in progressive transduction of multiple tumour foci in the liver without evidence of spread to adjacent normal parenchyma or extrahepatic tissues as shown by qPCR analyses of MLV-specific sequences in the DNA of liver tumour tissue, normal liver, and bone marrow cells [61].

derived from pre-infected and 100 % CD-expressing cells treated with 5-FC died due to tumour burden at a significantly later time point, however still within 50 days [58]. This indicates that other factors might contribute to the poorer treatment efficacy, such as insufficient levels of 5- FC/5-FU in the tumour, differences in resistance to 5-FU, or effects triggered by the immune system. Regarding this latter point, however, no immune response against the RCR vector in

Recently, we have critically analysed a panel of 15 different human and rodent glioma and glioblastoma cell lines in respect to spread of RCR vectors derived from vector ACE-CD to their sensitivity towards the 5-FC/5-FU suicide system, and to their orthotopic growth characteristics in mice to identify suitable preclinical animal models as test bed for the development and evaluation of RCR vector mediated treatment of glioblastoma [59]. Rapid virus spread was observed in eight out of nine human cell lines tested in vi‐ tro. As expected, only CD-expressing cells became sensitive to 5-FC. All LD50 values were within the range of concentrations obtained in human body fluids after convention‐ al 5-FC administration. In addition, a significant bystander effect was observed in all hu‐ man glioma cell lines tested, supporting the potential of this suicide gene therapy for the

This therapeutic concept has also been experimentally employed to subcutaneous and or‐ thotopic liver metastasis of colorectal cancer in an immunocompetent rodent model [60,61]. To this end, murine CT26 cells pre-transduced with vector ACE-CD were mixed with non-transduced CT26 cells at a ratio of 1:200 prior to implantation into BALB/c mice. Twelve days later, daily 5-FC treatment at 500 mg/kg, given *i.p.* twice a day, was started and continued until the end of the experiment. 5-FC treated animals showed sig‐ nificant inhibition of tumour growth resulting in an average tumour size of approx. 100 mm3 at day 24, whereas in animals whose drinking water was supplemented with 0.4 mg/ml of azidothymidine (AZT) to prevent MLV vector replication, tumour growth was inhibited only moderately with an average tumour size of approx. 500 mm3 at day 24 [61]. Tumour size in untreated animals reached more than 800 mm3 at day 24 [61]. The effect on tumour size observed in the AZT-treated group most probably was due to the presence of a bystander effect, in which toxic metabolites produced by the CD-positive cells can freely diffuse into neighbouring, CD-negative cells to cause cell death and thereby eventually lead to a delay in tumour growth. These observations clearly indicate that the efficacy of the ACE-CD/5-FC treatment is dependent on a sustained vector spread. Proof-of-concept of this therapy was also shown in a multifocal hepatic metasta‐ sis model [61]. Here, CT26 cells stably expressing the firefly luciferase marker gene were infused into the portal system via intrasplenic injection, became trapped within the hep‐ atic microcirculation, and seeded metastases. Three days later, 2x10e4 ACE-CD virus vec‐ tor particles were instilled into tumour-bearing mice, again via intrasplenic injection, followed by daily 5-FC administration at 500 mg/kg given *i.p.* twice a day, initiated 14 days after tumour cells inoculation [61]. In contrast to untreated animals or animals which received vector and PBS only, and which revealed increasing bioluminescence throughout the 28 day experiment, 5 out of 7 animals treated with both vector ACE-CD

the brain has been detected [58].

140 Novel Gene Therapy Approaches

treatment of brain tumours [59].

For proof-of-concept in a further indication, Kikuchi and colleagues evaluated the transduction and therapeutic efficacy of intravesically administered RCR vectors in orthotopic bladder tumours in mice [62]. Tumours were established by implantation of either MBT-2 murine bladder cancer cells into immunocompetent syngeneic C3H/HeJ mice or of KU-19-19 human bladder cancer cells into nude mice. RCR vector particles carrying the eGFP transgene were delivered intravesically *via* a catheter inserted into the bladder 5 days after tumour formation. Upon injection of 3.2x10e5 infectious virus particles, vector spread in the human xenograft model reached almost all of the tumour cells within 35 days post infection [62]. In the orthotopic syngeneic model, vector spread resulted in, on average, 30 % of infected tumour cells 27 days after vector instillation, as revealed by immunohistochemical analyses of transgene expression [62]. The effects of the respective therapeutic vector ACE-CD were demonstrated in the MBT-2 bladder cancer model, whereby 3.2x10e5 infectious virus particles were instilled intravesically into the bladder of mice containing preformed tumours. Twelve days following infection, animals received daily 5-FC at 500 mg/kg given *i.p.* for 15 days [62]. A single course of 5-FC treatment led to significantly reduced tumour growth and 50 % of the animals survived for more than 70 days. In contrast, all of the animals in vector only or vehicle control groups died within 40 days. Again, the locoregional delivery of intravesically administered RCR vectors was shown to achieve significant tumour growth inhibition by efficient delivery of the therapeutic gene into the orthotopic bladder tumour cells without any evidence of spread to adjacent distant organs [62].

Recently, Kawasaki et al. investigated RCR vector mediated gene therapy for the treatment of human malignant mesothelioma [63]. Cells of this tumour type were found to be highly permissive for RCR infection [63]. After a single intratumoral injection of 1x10e4 ACE-CD virus vector particles into pre-established subcutaneous MSTO-211H human mesothelioma tumour xenografts in nude mice, followed by daily 5-FC administration at 500 mg/kg body weight given *i.p*. from day 12 to day 32 post-infection, significant inhibition of tumour growth was observed in ACE-CD/5-FC treated mice compared to control groups as indicated by a tumour volume of 200 mm3 *vs.* 800 mm3 , respectively, at day 32 after infection. Efficacy of the treatment was also investigated in a peritoneally disseminated human mesothelioma xenograft model [63]. Here, MSTO-211H cells stably expressing the mCherry fluorescence protein and trans‐ duced with vector ACE-CD were mixed at a ratio of 1:100 with non-transduced mCherry expressing MSTO-211H cells and the mixture was injected i.p. into nude mice. After confir‐ mation of tumour formation by in vivo imaging, daily 5-FC administration was initiated (500 mg/kg *i.p*.) for 15 consecutive days. In 4 out of 9 mice of the vector/5-FC receiving group no visible tumours were observed and mice survived until day 100 after cell injection. On the contrary, all non-treated mice had died at day 61 at the latest. In summary, the 5-FC treated group showed a significantly prolonged median survival time as compared to the control group (81 days *vs.* 34 days) [63].

CD, a lead clinical candidate (vocimagene amiretrorepvec, Tocagen Inc., San Diego, CA) has been constructed carrying a human codon-optimised thermostable yeast CD gene [68]. Comparison with the prototype vector ACE-CD harbouring the wildtype yeast CD gene revealed a three-fold increased CD-specific conversion of 5-FC to 5-FU in infected cells and a markedly higher genetic stability of the clinical vector candidate Toca 511 [68]. To further support the production of toxic metabolites in infected cells, the modified CD gene was further linked as fusion to the gene encoding the yeast uracil phosphoribosyl transferase (UPRT) or, alternatively, to the human orotate phosphoribosyltransferase (OPRT) gene. It has been reported that expression of the UPRT gene in the CD-expressing cells leads to increased sensitivity to 5-FC, as the UPRT converts 5-FU directly to 5-FUMP, from which the active metabolites 5-FdUMP and 5-FUTP are formed [69,70]. The human OPRT protein, as part of a multifunctional UMP synthase, is a human analogue of the UPRT, converting 5-FU directly to 5-FUMP with direct impact on the cellular sensitivity towards 5-FU, since downregulation of this endogenous enzyme was found in tumour cells resistant to 5-FU [71,72]. Moreover, exogenous expression of the OPRT gene led to increased 5-FU sensitivity in cancer cell lines in vitro [73]. Despite better individual in vitro cell killing with vectors carrying these fusion genes, it remains unclear, whether this would be beneficial for therapy, as the highly efficient 5-FC salvage to phosphorylated nucleotides may diminish 5-FU diffusion and thereby the effects exerted by the bystander mechanism. Furthermore, initial killing of most of the infected cells might hinder vector spread during the 5-FC-rest period, leading to a reduced antitumor activity in vivo [68,74]. In addition, serial passaging of these infectious viruses on human U-87MG glioma cells revealed a decreased genomic stability of vectors containing fusion constructs as compared to Toca 511, probably due to the size and nature of the inserted transgene(s) sequences (~500 bp in Toca 511 *vs.* ~1250 bp in the fusion constructs) [68].

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The therapeutic potential of Toca 511 was evaluated in two different intracranial brain tumour models in immune competent mice (CT26-BALB/c and Tu-2449-B6C3F1) [75]. Treatment of CT26 brain metastases with three different virus doses (1x10e6, 1x10e5, 1x10e4 transforming units (TU)/g brain weight) plus 5-FC administration initiated nine days after intratumoral virus injection at 500 mg/kg given *i.p.* twice a day for always 7 days, followed by 10 days off until termination of the study, revealed a statistically significant prolongation in median survival of mice treated with the mid (1x10e5) and high (1x10e6) vector dose as compared to their PBStreated counterparts [75]. In a similar experiment mouse Tu-2449 gliomas were treated using different virus doses (1x10e6, 1x10e5, 1x10e4 and 1x10e3 TU/g brain weight) and 5-FC doses (500 mg/kg and 50 mg/kg body weight) [75]. 5-FC treatment or PBS treatment as control was initiated 9 days after vector injection and was given twice a day for 4 days, followed by a 10 day recovery phase. Cycles were repeated until termination of the study [75]. All vector doses in combination with 5-FC treatment at 500 mg/kg resulted in prolonged survival, as compared to PBS controls. Mice treated with vector doses of 1x10e4 and 1x10e5 and high dose 5-FC revealed a significantly prolonged survival when compared to the PBS controls [75]. However, even the 1x10e5 vector dose level with a rather low prodrug dose of 50 mg/kg 5-FC resulted in a survival advantage when compared to control. Histological analyses of Tu-2449 tumours taken before the first, second, and fourth treatment cycle with 500 mg/kg 5-FC revealed tumour

Beside the CD/5-FC suicide gene/prodrug combination, which is the most prominent and ex‐ tensively studied system in context of RCR vectors, other therapeutic genes have been intro‐ duced in this vector system and have been analysed. The bacterial purine nucleoside phosphorylase (PNP) gene, for example, converts the prodrugs fludarabine phosphate (F-ara‐ AMP) or 6-methylpurine 2´-deoxyriboside (MeP-dR) into its toxic metabolites and represents a reasonable alternative to the CD/5-FC suicide system. Kikuchi et al. demonstrated the thera‐ peutic efficacy of vector ACE-PNP, an MLV-based RCR vector expressing the *E.*coli PNP gene, in a subcutaneous model of KU-19-19 human bladder cancer cells in nude mice [64]. Five days following tumour cell implantation, 3.2x10e5 infectious virus particles were injected di‐ rectly into the tumour and 10 days later F-araAMP at 75 mg/m2 body surface was adminis‐ tered *i.p*. every other day for a total of seven injections. Significant tumour growth inhibition could be demonstrated with a tumour mass of 600 mm3 *vs.* ≥ 2,200 mm<sup>3</sup> at day 26 post virus in‐ jection in treated mice *vs.* vector and vehicle control groups [64]. These results indicate that the PNP-RCR system is a reasonable alternative to the use of CD-expressing RCR vectors, in par‐ ticular as enzymatic products generated by PNP seem to be more cytotoxic than 5-FU [65,66].

In in vitro experiments using human U-87MG glioma cells, Tai and colleagues were able to show that transduction of even only 1 % of cells with the ACE-PNP vector and subsequent systemic prodrug administration is sufficient to achieve significant cell killing over time [67]. Thereby, the rapidity of cell killing is highly dependent on the initial level of transduction. Treatment of pre-established *s.c.* U-87MG tumour xenografts with 1x10e5 ACE-PNP vector particles followed by F-araAMP prodrug administration 14 days after virus injection at a concentration of 80 mg/kg given *i.p.* once every other day for five times resulted in significantly inhibited tumour growth [67]. A second cycle of prodrug administration reduced tumour growth even further. The potential of an ACE-PNP RCR vector-based therapy to improve survival of nude mice bearing intracranial U-87MG tumours was evaluated by inoculating pre-established xenografts with 1x10e4 vector particles 7 days after tumour onset. Eight days later, the mice received F-araAMP (40 mg/kg given i.p. once every other day) for a total of eight treatments. The median survival time was 59 days *vs.* 30 and 28 days in treated *vs.* control groups [67]. In a second experiment, the same experimental setting was employed, but a second round of F-araAMP treatment was done after a 14 days recovery period. The median survival time of treated mice was further improved to 73 days *vs.* 33 days in the control groups [67]. This data again demonstrated the potential for additional survival benefit from multiple cycles of prodrug administration.

In summary, the published data of non-preclinical studies using suicide gene-expressing MLVbased RCR vectors have demonstrated that such vectors are therapeutically efficacious in solid tumours and/or metastases of a range of different tumour types (glioma, colorectal and bladder cancer, mesothelioma) in both immunodeficient and immunocompetent mouse and rat models, and using different therapeutic genes (yCD and PNP). On grounds of these nonclinical data the therapeutic concept of replicating retroviral vectors was moved towards clinical application. To this end, based on the design of the extensively evaluated vector ACE- CD, a lead clinical candidate (vocimagene amiretrorepvec, Tocagen Inc., San Diego, CA) has been constructed carrying a human codon-optimised thermostable yeast CD gene [68]. Comparison with the prototype vector ACE-CD harbouring the wildtype yeast CD gene revealed a three-fold increased CD-specific conversion of 5-FC to 5-FU in infected cells and a markedly higher genetic stability of the clinical vector candidate Toca 511 [68]. To further support the production of toxic metabolites in infected cells, the modified CD gene was further linked as fusion to the gene encoding the yeast uracil phosphoribosyl transferase (UPRT) or, alternatively, to the human orotate phosphoribosyltransferase (OPRT) gene. It has been reported that expression of the UPRT gene in the CD-expressing cells leads to increased sensitivity to 5-FC, as the UPRT converts 5-FU directly to 5-FUMP, from which the active metabolites 5-FdUMP and 5-FUTP are formed [69,70]. The human OPRT protein, as part of a multifunctional UMP synthase, is a human analogue of the UPRT, converting 5-FU directly to 5-FUMP with direct impact on the cellular sensitivity towards 5-FU, since downregulation of this endogenous enzyme was found in tumour cells resistant to 5-FU [71,72]. Moreover, exogenous expression of the OPRT gene led to increased 5-FU sensitivity in cancer cell lines in vitro [73]. Despite better individual in vitro cell killing with vectors carrying these fusion genes, it remains unclear, whether this would be beneficial for therapy, as the highly efficient 5-FC salvage to phosphorylated nucleotides may diminish 5-FU diffusion and thereby the effects exerted by the bystander mechanism. Furthermore, initial killing of most of the infected cells might hinder vector spread during the 5-FC-rest period, leading to a reduced antitumor activity in vivo [68,74]. In addition, serial passaging of these infectious viruses on human U-87MG glioma cells revealed a decreased genomic stability of vectors containing fusion constructs as compared to Toca 511, probably due to the size and nature of the inserted transgene(s) sequences (~500 bp in Toca 511 *vs.* ~1250 bp in the fusion constructs) [68].

group showed a significantly prolonged median survival time as compared to the control

Beside the CD/5-FC suicide gene/prodrug combination, which is the most prominent and ex‐ tensively studied system in context of RCR vectors, other therapeutic genes have been intro‐ duced in this vector system and have been analysed. The bacterial purine nucleoside phosphorylase (PNP) gene, for example, converts the prodrugs fludarabine phosphate (F-ara‐ AMP) or 6-methylpurine 2´-deoxyriboside (MeP-dR) into its toxic metabolites and represents a reasonable alternative to the CD/5-FC suicide system. Kikuchi et al. demonstrated the thera‐ peutic efficacy of vector ACE-PNP, an MLV-based RCR vector expressing the *E.*coli PNP gene, in a subcutaneous model of KU-19-19 human bladder cancer cells in nude mice [64]. Five days following tumour cell implantation, 3.2x10e5 infectious virus particles were injected di‐

tered *i.p*. every other day for a total of seven injections. Significant tumour growth inhibition

jection in treated mice *vs.* vector and vehicle control groups [64]. These results indicate that the PNP-RCR system is a reasonable alternative to the use of CD-expressing RCR vectors, in par‐ ticular as enzymatic products generated by PNP seem to be more cytotoxic than 5-FU [65,66].

In in vitro experiments using human U-87MG glioma cells, Tai and colleagues were able to show that transduction of even only 1 % of cells with the ACE-PNP vector and subsequent systemic prodrug administration is sufficient to achieve significant cell killing over time [67]. Thereby, the rapidity of cell killing is highly dependent on the initial level of transduction. Treatment of pre-established *s.c.* U-87MG tumour xenografts with 1x10e5 ACE-PNP vector particles followed by F-araAMP prodrug administration 14 days after virus injection at a concentration of 80 mg/kg given *i.p.* once every other day for five times resulted in significantly inhibited tumour growth [67]. A second cycle of prodrug administration reduced tumour growth even further. The potential of an ACE-PNP RCR vector-based therapy to improve survival of nude mice bearing intracranial U-87MG tumours was evaluated by inoculating pre-established xenografts with 1x10e4 vector particles 7 days after tumour onset. Eight days later, the mice received F-araAMP (40 mg/kg given i.p. once every other day) for a total of eight treatments. The median survival time was 59 days *vs.* 30 and 28 days in treated *vs.* control groups [67]. In a second experiment, the same experimental setting was employed, but a second round of F-araAMP treatment was done after a 14 days recovery period. The median survival time of treated mice was further improved to 73 days *vs.* 33 days in the control groups [67]. This data again demonstrated the potential for additional survival benefit from multiple cycles

In summary, the published data of non-preclinical studies using suicide gene-expressing MLVbased RCR vectors have demonstrated that such vectors are therapeutically efficacious in solid tumours and/or metastases of a range of different tumour types (glioma, colorectal and bladder cancer, mesothelioma) in both immunodeficient and immunocompetent mouse and rat models, and using different therapeutic genes (yCD and PNP). On grounds of these nonclinical data the therapeutic concept of replicating retroviral vectors was moved towards clinical application. To this end, based on the design of the extensively evaluated vector ACE-

body surface was adminis‐

at day 26 post virus in‐

rectly into the tumour and 10 days later F-araAMP at 75 mg/m2

could be demonstrated with a tumour mass of 600 mm3 *vs.* ≥ 2,200 mm<sup>3</sup>

group (81 days *vs.* 34 days) [63].

142 Novel Gene Therapy Approaches

of prodrug administration.

The therapeutic potential of Toca 511 was evaluated in two different intracranial brain tumour models in immune competent mice (CT26-BALB/c and Tu-2449-B6C3F1) [75]. Treatment of CT26 brain metastases with three different virus doses (1x10e6, 1x10e5, 1x10e4 transforming units (TU)/g brain weight) plus 5-FC administration initiated nine days after intratumoral virus injection at 500 mg/kg given *i.p.* twice a day for always 7 days, followed by 10 days off until termination of the study, revealed a statistically significant prolongation in median survival of mice treated with the mid (1x10e5) and high (1x10e6) vector dose as compared to their PBStreated counterparts [75]. In a similar experiment mouse Tu-2449 gliomas were treated using different virus doses (1x10e6, 1x10e5, 1x10e4 and 1x10e3 TU/g brain weight) and 5-FC doses (500 mg/kg and 50 mg/kg body weight) [75]. 5-FC treatment or PBS treatment as control was initiated 9 days after vector injection and was given twice a day for 4 days, followed by a 10 day recovery phase. Cycles were repeated until termination of the study [75]. All vector doses in combination with 5-FC treatment at 500 mg/kg resulted in prolonged survival, as compared to PBS controls. Mice treated with vector doses of 1x10e4 and 1x10e5 and high dose 5-FC revealed a significantly prolonged survival when compared to the PBS controls [75]. However, even the 1x10e5 vector dose level with a rather low prodrug dose of 50 mg/kg 5-FC resulted in a survival advantage when compared to control. Histological analyses of Tu-2449 tumours taken before the first, second, and fourth treatment cycle with 500 mg/kg 5-FC revealed tumour growth between the first and the second dosing. Most of the tumour tissue however, was no longer visible and gliosis was evident by the start of the fourth treatment [75].

into the 3' UTR of the vector backbone to drive expression of an shRNA sequence targeted against the epidermal growth factor receptor (EGFR) gene or the STAT3 gene [30]. To allow monitoring of infection efficiency, these vectors also contain the eGFP gene fused into the virus envelope gene. Insertion of both expression cassettes did not interfere significantly with virus fitness, and the receptor specificity of the Env protein was not impaired by the introduced eGFP sequences [28]. The modified vectors replicated rapidly and were genetically stable over several infection cycles. In addition, silencing of EGFR and STAT3 target gene expression in

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145

An improved, second generation MLV-based RNAi transfer vector suitable for in vivo application was recently described [79]. This RCR vector encodes miRNA modified shRNA sequences specifically targeting the eGFP and luciferase reporter genes under control of the small nuclear U6 promoter inserted in antisense orientation into the 3' UTR of the vector. In HT1080 cells stably expressing eGFP or luciferase, marker gene expression was suppressed by more than 80 %, even when only 0.1 % of the cells were initially infected with the RCR vectors [79]. In vivo systemic tail vein administration of 2.9x10e7 of shLuc expressing vector particles in animals with pre-established subcutaneous HT1080-Luc tumours led to more than 80 % reduction in luciferase activity compared to uninfected tumours at day 25 post infection [79]. To investigate the effects of downregulation of tumour-promoting proteins, PLK1- and MMP14-specific shRNA expression cassettes were inserted into the vector. Upon infection of target cells, PLK1 and MMP14 mRNA and protein levels were reduced [79]. MLV-shPLKinfected cells were trapped in the G2-phase of the cell cycle at day 3 post infection, followed by induction of apoptosis at day 5 post infection. MLV-shMMP14 infected cells showed reduced MMP2 activity consistent with a reduced invasion capacity by ~75 % as compared to non-infected cells [79]. Tumour growth of MLV-shMMP14 infected HT1080-Rec-1 cells in immunodeficient mice was significantly and substantially reduced in comparison to controls. Similarly, direct intratumoral application of 1x10e6 of shPLK1 expressing vector particles in animals with pre-established subcutaneous HT1080 xenografts led to a significantly reduced

Up to now, a variety of different therapeutic approaches have been utilized in context of RCR vectors. High levels of vector spread, infection efficiency, and therapeutic gene expression have been detected leading to an efficacious therapeutic option for the treatment of cancer. From data on the eradication of tumour mass in animal models existing so far, a clear benefit of the RCR mediated tumour therapy is indicated. However, the use of replicating retroviral

In particular, 3 major concerns directly related to the use of RCR vectors have to be taken into consideration: (i) the risk of insertional mutagenesis due to integration of the vector genome into the host cell DNA, a step that can trigger the transformation of normal cells into tumour cells, (ii) the spread of viral vectors throughout the body of the patient causing viraemia, and (iii) the infection of dividing non-tumour cells and their loss due to therapeutic intervention

vectors also bears a number of risks which have to be identified and analysed.

cells infected to levels of 80 – 95 % was shown to be highly efficient [30].

tumour growth in comparison to the controls [79].

**3.4. Safety of MLV-based RCR vectors**

leading to severe side effects.

Currently, Toca 511 is being investigated in clinical trials in the United States in subjects with recurrent high-grade glioma either as a direct intratumoral vector injection (Phase I/II Study; NCT01156584; http://www.clinicaltrials.gov) or vector injection at the time of tumour removal (Phase I Study; NCT01470794 http://www.clinicaltrials.gov).

### *3.3.2. Secreted therapeutic molecules*

Beside direct killing of tumour cells mediated by suicide gene products, different other therapeutic approaches, e.g. based on the secretion of therapeutic molecules such as cytokines or single chain antibodies (scFv) directed against specific tumour antigens could be facilitated by RCR vectors.

To allow secretion of therapeutic molecules from infected cells, a replicating retroviral vector was constructed by inserting specific transgene sequences to the first codon of the MLV env gene via a furin cleavage site sequence [26]. The respective fusion protein will be cleaved by furin proteases in the Golgi and the scFv will be secreted upon release of the new virus particles from infected cells (for details see above). The resulting vectors were capable of efficiently transducing susceptible cells, were genetically stable for more than 12 passages and were able to efficiently mediate intracellular production and secretion of the GM-CSF cytokine and the functional laminin-specific or T-cell-specific scFv antibody, respectively [27].

Sun and colleagues demonstrated the potential of the human chemokine interferon-gammainducible protein 10 (IP10) gene, delivered and expressed from a MLV-based RCR vectors, to inhibit tumour growth *in vivo* [76]. IP10 is known to be a potent inhibitor of angiogenesis, tumour growth, and metastasis [77,78]. Using human fibrosarcoma HT1080 cells transduced with the IP10 RCR vector designed as described above in an *s.c*. xenograft tumour model in nude mice, significant tumour growth inhibition and a marked reduction in microvessel density was observed as compared to non-infected HT1080 control mice (tumour volume 190 mm3 *vs.* 510 mm3 ) [76]. In addition, both, growth of *s.c.* tumour xenografts established from pre-infected murine Lewis lung carcinoma (LLC) cells as well as the formation of lung metastases from pre-infected murine melanoma B16F10 cells in immunocompetent C57BL/6 mice was significantly reduced [76].

#### *3.3.3. RNA interference*

In cancer gene therapy applications, RNAi-expressing RCR vectors can be used to inhibit tumour growth, invasion and metastasis. The length of the RNA duplex required for efficient RNAi is not longer than 21-23 bp. Therefore, the insertion of RNAi expression cassettes into RCR vectors should be well tolerated in respect to genetic stability and spread kinetics of the vector. The expression of duplex RNA is usually achieved using an expression cassette consisting of the RNA Pol III promoter transcribing a sequence designed to form a short hairpin RNA structure (shRNA). MLV-based RCR vectors were constructed encompassing a tran‐ scription cassette consisting of an H1-RNA Pol III promoter inserted in antisense orientation into the 3' UTR of the vector backbone to drive expression of an shRNA sequence targeted against the epidermal growth factor receptor (EGFR) gene or the STAT3 gene [30]. To allow monitoring of infection efficiency, these vectors also contain the eGFP gene fused into the virus envelope gene. Insertion of both expression cassettes did not interfere significantly with virus fitness, and the receptor specificity of the Env protein was not impaired by the introduced eGFP sequences [28]. The modified vectors replicated rapidly and were genetically stable over several infection cycles. In addition, silencing of EGFR and STAT3 target gene expression in cells infected to levels of 80 – 95 % was shown to be highly efficient [30].

An improved, second generation MLV-based RNAi transfer vector suitable for in vivo application was recently described [79]. This RCR vector encodes miRNA modified shRNA sequences specifically targeting the eGFP and luciferase reporter genes under control of the small nuclear U6 promoter inserted in antisense orientation into the 3' UTR of the vector. In HT1080 cells stably expressing eGFP or luciferase, marker gene expression was suppressed by more than 80 %, even when only 0.1 % of the cells were initially infected with the RCR vectors [79]. In vivo systemic tail vein administration of 2.9x10e7 of shLuc expressing vector particles in animals with pre-established subcutaneous HT1080-Luc tumours led to more than 80 % reduction in luciferase activity compared to uninfected tumours at day 25 post infection [79]. To investigate the effects of downregulation of tumour-promoting proteins, PLK1- and MMP14-specific shRNA expression cassettes were inserted into the vector. Upon infection of target cells, PLK1 and MMP14 mRNA and protein levels were reduced [79]. MLV-shPLKinfected cells were trapped in the G2-phase of the cell cycle at day 3 post infection, followed by induction of apoptosis at day 5 post infection. MLV-shMMP14 infected cells showed reduced MMP2 activity consistent with a reduced invasion capacity by ~75 % as compared to non-infected cells [79]. Tumour growth of MLV-shMMP14 infected HT1080-Rec-1 cells in immunodeficient mice was significantly and substantially reduced in comparison to controls. Similarly, direct intratumoral application of 1x10e6 of shPLK1 expressing vector particles in animals with pre-established subcutaneous HT1080 xenografts led to a significantly reduced tumour growth in comparison to the controls [79].

#### **3.4. Safety of MLV-based RCR vectors**

growth between the first and the second dosing. Most of the tumour tissue however, was no

Currently, Toca 511 is being investigated in clinical trials in the United States in subjects with recurrent high-grade glioma either as a direct intratumoral vector injection (Phase I/II Study; NCT01156584; http://www.clinicaltrials.gov) or vector injection at the time of tumour removal

Beside direct killing of tumour cells mediated by suicide gene products, different other therapeutic approaches, e.g. based on the secretion of therapeutic molecules such as cytokines or single chain antibodies (scFv) directed against specific tumour antigens could be facilitated

To allow secretion of therapeutic molecules from infected cells, a replicating retroviral vector was constructed by inserting specific transgene sequences to the first codon of the MLV env gene via a furin cleavage site sequence [26]. The respective fusion protein will be cleaved by furin proteases in the Golgi and the scFv will be secreted upon release of the new virus particles from infected cells (for details see above). The resulting vectors were capable of efficiently transducing susceptible cells, were genetically stable for more than 12 passages and were able to efficiently mediate intracellular production and secretion of the GM-CSF cytokine and the

Sun and colleagues demonstrated the potential of the human chemokine interferon-gammainducible protein 10 (IP10) gene, delivered and expressed from a MLV-based RCR vectors, to inhibit tumour growth *in vivo* [76]. IP10 is known to be a potent inhibitor of angiogenesis, tumour growth, and metastasis [77,78]. Using human fibrosarcoma HT1080 cells transduced with the IP10 RCR vector designed as described above in an *s.c*. xenograft tumour model in nude mice, significant tumour growth inhibition and a marked reduction in microvessel density was observed as compared to non-infected HT1080 control mice (tumour volume 190

pre-infected murine Lewis lung carcinoma (LLC) cells as well as the formation of lung metastases from pre-infected murine melanoma B16F10 cells in immunocompetent C57BL/6

In cancer gene therapy applications, RNAi-expressing RCR vectors can be used to inhibit tumour growth, invasion and metastasis. The length of the RNA duplex required for efficient RNAi is not longer than 21-23 bp. Therefore, the insertion of RNAi expression cassettes into RCR vectors should be well tolerated in respect to genetic stability and spread kinetics of the vector. The expression of duplex RNA is usually achieved using an expression cassette consisting of the RNA Pol III promoter transcribing a sequence designed to form a short hairpin RNA structure (shRNA). MLV-based RCR vectors were constructed encompassing a tran‐ scription cassette consisting of an H1-RNA Pol III promoter inserted in antisense orientation

) [76]. In addition, both, growth of *s.c.* tumour xenografts established from

functional laminin-specific or T-cell-specific scFv antibody, respectively [27].

longer visible and gliosis was evident by the start of the fourth treatment [75].

(Phase I Study; NCT01470794 http://www.clinicaltrials.gov).

*3.3.2. Secreted therapeutic molecules*

by RCR vectors.

144 Novel Gene Therapy Approaches

mm3 *vs.* 510 mm3

*3.3.3. RNA interference*

mice was significantly reduced [76].

Up to now, a variety of different therapeutic approaches have been utilized in context of RCR vectors. High levels of vector spread, infection efficiency, and therapeutic gene expression have been detected leading to an efficacious therapeutic option for the treatment of cancer. From data on the eradication of tumour mass in animal models existing so far, a clear benefit of the RCR mediated tumour therapy is indicated. However, the use of replicating retroviral vectors also bears a number of risks which have to be identified and analysed.

In particular, 3 major concerns directly related to the use of RCR vectors have to be taken into consideration: (i) the risk of insertional mutagenesis due to integration of the vector genome into the host cell DNA, a step that can trigger the transformation of normal cells into tumour cells, (ii) the spread of viral vectors throughout the body of the patient causing viraemia, and (iii) the infection of dividing non-tumour cells and their loss due to therapeutic intervention leading to severe side effects.

The risk of insertional oncogenesis is an issue associated with the use of retroviral vectors in general, irrespective if they are replication-deficient or replication-competent. With RCR vectors this concern might be more substantial as due to the replicating nature more cells might be affected and multiple infections of the single cell might occur. On the other hand, in tumour therapy it is intended to kill the infected cells. Hence, due to this, insertional oncogenesis should not be an issue unless infected cells are resistant to treatment or got infected with an RCR vector which is reverted to wild-type due to genetic instability and thus is not able to exert its therapeutic potential.

By detection of CD gene-specific sequences by PCR at a detection limit of 400 copies per 100,000 cell genomes (600 ng of gDNA, transduction level 0.4 %), Tai and co-workers were able to determine proviral sequences in the transduced glioma tissue, but observed no extratumoral spread to and in any of the tissue examined (lung, liver, oesophagus and stomach, intestine, spleen, kidney, skin, bone marrow, contralateral normal brain) in an orthotopic glioma model in nude mice intratumorally injected with 1.2x10e4 virus particles [57]. However, all of these studies had been based on the detection of the non-viral transgene (e.g. GFP, CD, PNP, etc.). Hence, putative spread of vectors which have lost the transgene or parts of it will not be

Replicating Retroviral Vectors for Gene Therapy of Solid Tumors

http://dx.doi.org/10.5772/54861

147

In an immunocompetent intracranial RG2-glioma model performed in Fischer 344 rats, tumours were injected with 1x10e6 of CD expressing RCR vector particles followed by 5-FC or PBS treatment initiated 10 days later [58]. Organs from moribund animals were collected and quantitative real-time PCR targeting the MLV env gene was performed [58]. There was no evidence of presence of the env gene from the RCR vector in systemic tissues carrying highly mitotic cells such as lung, liver, kidney, spleen, bone marrow, skin, oesophagus, intestine, and testis [58]. Using this highly sensitive technique which enables detection of 20-35 copies in 50,000 cellular genomes, Hiraoka et al. analysed RCR vector biodistribution in a mouse hepatic metastasis model using BALB/c mice and the syngeneic colon carcinoma cell line CT26 [60,61]. After locoregional delivery of 2x10e4 of RCR vector particles either expressing the eGFP or CD transgene, *via* intrasplenic injection followed by splenectomy, no detectable virus-related signals were observed in genomic DNA extracted from peritumoral normal liver tissue, bone marrow, lung, kidney, small intestine, and colon. As expected, proviral RCR signals were highly abundant in genomic DNA from RCR-transduced tumour tissues with 3,000 – 16,600 proviral copies per 50,000 cells [60,61]. The copy number was increased in DNA obtained from ACE-CD/PBS-treated control animals (11,000 – 16,600 copies/50,000 cells) compared to ACE-CD/5-FC-treated animals (8,000 – 11,000 copies/50,000 cells) [61]. Optical imaging analyses, flow cytometric analyses as well as immunohistochemistry using an anti-eGFP antibody consistently revealed strongly positive eGFP signals in the tumour masses but not in the other

Biodistribution of RCR vectors after intravenous injection into immune deficient as well as immune competent mice was analysed by Solly and colleagues [25]. Two weeks after RCR vector injection, a 4070Aenv-based quantitative real-time PCR revealed presence of proviral genomes in bone marrow and spleen of nude mice. On the contrary, no proviral genomes could be detected in any tissue from immunocompetent animals, which empha‐ sizes the potency of anti-MLV specific immune responses [25]. In vivo biodistribution of wt-MLV and MMP-activatable RCR vectors and their ability to reach tumour tissue after systemic administration was analysed in CB17-SCID mice using an optimized PCR meth‐ od detecting up to 50 copies of proviral DNA in 300 ng of genomic DNA [48]. After in‐ travenous injection of RCR vectors corresponding to 60 U of RT activity into tumour-free mice, tissues were analysed at different time points after virus injection. A strong signal in spleen and weak signals in liver and bone marrow were obtained after administration of wt-MLV vector one day after infection. After two weeks, wt-MLV sequences were

considered in these analyses.

organs mentioned above [60].

The use of replication-competent retroviral vectors might also bear the risk of uncontrolled spread of vectors throughout the human body, resulting in infection of dividing cells other than the tumour cells itself. Non-dividing cells should not be infected as MLV-based vectors are thought to transduce dividing cells only, and, as most cells in the adult human body are non-dividing, virus spread should be limited. Moreover, some cell types, such as dividing human primary T-lymphocytes have only a low capacity to produce MLV-based RCR and, in addition, the produced virions are largely non-infectious [80]. Nevertheless, in case of unintended virus spread and high risk of viraemia, an early systemic intervention with antiretroviral drug(s) could be implemented to limit viral load [81]. Recent findings, on the other hand, suggest that the host range of MLV-based RCR vectors might include also postmitotic and other growth-arrested cells in mammals [37]. Therefore, the issue of RCR-vector dissemination outside of the tumor mass is of particular concern in clinical studies employing RCR vectors and thus should be addressed in respective biodistribution studies.

For RCR biodistribution studies, sensitivity of the analysis is of utmost importance. Techniques based on conventional PCR, real-time PCR, flow-cytometry and immunohistochemical detection were employed so far to analyse the infection range of RCR vectors in animal models. In an early report, Logg and colleagues analysed the presence of the eGFP transgene in the DNA extracted from tumour tissue as well as from a variety of extratumoral tissues including spleen, lung, kidney, liver, and heart obtained from *nu/nu* BALB/c mice harbouring *s.c.* xenografts 7 weeks after intratumoral application of 6x10e3 RCR vector particles [22]. A PCR assay detecting 140 copies of GFP in a background of 100,000 equivalents of untransduced genomic DNA (transduction level 0.14 %) was employed [22]. Transgene sequences have been detected in tumour samples only. These data were further supported by flow cytometric analysis of the same tissues [22]. By improving this PCR assay, Wang et al. were able to detect as few as 35 copies of proviral DNA in 0.5 μg of genomic DNA. In an orthotopic glioma model in nude mice, in which animals were intratumorally inoculated with 1.2x10e4 virus particles, proviral sequences have been detected in tumour tissue, but not in contralateral brain paren‐ chyma, bone marrow, GI tract, liver, kidney, spleen, lung, and skin [56]. Using a PCR driven detection method, eGFP transgene sequences could not be observed in normal tissues sur‐ rounding the injected tumours in a *s.c.* bladder tumour xenograft model in *nu/nu* BALB/c mice [62, 64]. No signals have been obtained in distant organs such as brain, liver, spleen, lung, bladder, kidney, heart, ovary, uterus, and stomach in an orthotopic model of bladder cancer, when RCR vectors were administered intravesically [62, 64].

By detection of CD gene-specific sequences by PCR at a detection limit of 400 copies per 100,000 cell genomes (600 ng of gDNA, transduction level 0.4 %), Tai and co-workers were able to determine proviral sequences in the transduced glioma tissue, but observed no extratumoral spread to and in any of the tissue examined (lung, liver, oesophagus and stomach, intestine, spleen, kidney, skin, bone marrow, contralateral normal brain) in an orthotopic glioma model in nude mice intratumorally injected with 1.2x10e4 virus particles [57]. However, all of these studies had been based on the detection of the non-viral transgene (e.g. GFP, CD, PNP, etc.). Hence, putative spread of vectors which have lost the transgene or parts of it will not be considered in these analyses.

The risk of insertional oncogenesis is an issue associated with the use of retroviral vectors in general, irrespective if they are replication-deficient or replication-competent. With RCR vectors this concern might be more substantial as due to the replicating nature more cells might be affected and multiple infections of the single cell might occur. On the other hand, in tumour therapy it is intended to kill the infected cells. Hence, due to this, insertional oncogenesis should not be an issue unless infected cells are resistant to treatment or got infected with an RCR vector which is reverted to wild-type due to genetic instability and thus is not able to

The use of replication-competent retroviral vectors might also bear the risk of uncontrolled spread of vectors throughout the human body, resulting in infection of dividing cells other than the tumour cells itself. Non-dividing cells should not be infected as MLV-based vectors are thought to transduce dividing cells only, and, as most cells in the adult human body are non-dividing, virus spread should be limited. Moreover, some cell types, such as dividing human primary T-lymphocytes have only a low capacity to produce MLV-based RCR and, in addition, the produced virions are largely non-infectious [80]. Nevertheless, in case of unintended virus spread and high risk of viraemia, an early systemic intervention with antiretroviral drug(s) could be implemented to limit viral load [81]. Recent findings, on the other hand, suggest that the host range of MLV-based RCR vectors might include also postmitotic and other growth-arrested cells in mammals [37]. Therefore, the issue of RCR-vector dissemination outside of the tumor mass is of particular concern in clinical studies employing

RCR vectors and thus should be addressed in respective biodistribution studies.

when RCR vectors were administered intravesically [62, 64].

For RCR biodistribution studies, sensitivity of the analysis is of utmost importance. Techniques based on conventional PCR, real-time PCR, flow-cytometry and immunohistochemical detection were employed so far to analyse the infection range of RCR vectors in animal models. In an early report, Logg and colleagues analysed the presence of the eGFP transgene in the DNA extracted from tumour tissue as well as from a variety of extratumoral tissues including spleen, lung, kidney, liver, and heart obtained from *nu/nu* BALB/c mice harbouring *s.c.* xenografts 7 weeks after intratumoral application of 6x10e3 RCR vector particles [22]. A PCR assay detecting 140 copies of GFP in a background of 100,000 equivalents of untransduced genomic DNA (transduction level 0.14 %) was employed [22]. Transgene sequences have been detected in tumour samples only. These data were further supported by flow cytometric analysis of the same tissues [22]. By improving this PCR assay, Wang et al. were able to detect as few as 35 copies of proviral DNA in 0.5 μg of genomic DNA. In an orthotopic glioma model in nude mice, in which animals were intratumorally inoculated with 1.2x10e4 virus particles, proviral sequences have been detected in tumour tissue, but not in contralateral brain paren‐ chyma, bone marrow, GI tract, liver, kidney, spleen, lung, and skin [56]. Using a PCR driven detection method, eGFP transgene sequences could not be observed in normal tissues sur‐ rounding the injected tumours in a *s.c.* bladder tumour xenograft model in *nu/nu* BALB/c mice [62, 64]. No signals have been obtained in distant organs such as brain, liver, spleen, lung, bladder, kidney, heart, ovary, uterus, and stomach in an orthotopic model of bladder cancer,

exert its therapeutic potential.

146 Novel Gene Therapy Approaches

In an immunocompetent intracranial RG2-glioma model performed in Fischer 344 rats, tumours were injected with 1x10e6 of CD expressing RCR vector particles followed by 5-FC or PBS treatment initiated 10 days later [58]. Organs from moribund animals were collected and quantitative real-time PCR targeting the MLV env gene was performed [58]. There was no evidence of presence of the env gene from the RCR vector in systemic tissues carrying highly mitotic cells such as lung, liver, kidney, spleen, bone marrow, skin, oesophagus, intestine, and testis [58]. Using this highly sensitive technique which enables detection of 20-35 copies in 50,000 cellular genomes, Hiraoka et al. analysed RCR vector biodistribution in a mouse hepatic metastasis model using BALB/c mice and the syngeneic colon carcinoma cell line CT26 [60,61]. After locoregional delivery of 2x10e4 of RCR vector particles either expressing the eGFP or CD transgene, *via* intrasplenic injection followed by splenectomy, no detectable virus-related signals were observed in genomic DNA extracted from peritumoral normal liver tissue, bone marrow, lung, kidney, small intestine, and colon. As expected, proviral RCR signals were highly abundant in genomic DNA from RCR-transduced tumour tissues with 3,000 – 16,600 proviral copies per 50,000 cells [60,61]. The copy number was increased in DNA obtained from ACE-CD/PBS-treated control animals (11,000 – 16,600 copies/50,000 cells) compared to ACE-CD/5-FC-treated animals (8,000 – 11,000 copies/50,000 cells) [61]. Optical imaging analyses, flow cytometric analyses as well as immunohistochemistry using an anti-eGFP antibody consistently revealed strongly positive eGFP signals in the tumour masses but not in the other organs mentioned above [60].

Biodistribution of RCR vectors after intravenous injection into immune deficient as well as immune competent mice was analysed by Solly and colleagues [25]. Two weeks after RCR vector injection, a 4070Aenv-based quantitative real-time PCR revealed presence of proviral genomes in bone marrow and spleen of nude mice. On the contrary, no proviral genomes could be detected in any tissue from immunocompetent animals, which empha‐ sizes the potency of anti-MLV specific immune responses [25]. In vivo biodistribution of wt-MLV and MMP-activatable RCR vectors and their ability to reach tumour tissue after systemic administration was analysed in CB17-SCID mice using an optimized PCR meth‐ od detecting up to 50 copies of proviral DNA in 300 ng of genomic DNA [48]. After in‐ travenous injection of RCR vectors corresponding to 60 U of RT activity into tumour-free mice, tissues were analysed at different time points after virus injection. A strong signal in spleen and weak signals in liver and bone marrow were obtained after administration of wt-MLV vector one day after infection. After two weeks, wt-MLV sequences were found in lung, spleen, liver, heart, bone marrow, and muscle, but not in brain. The in‐ crease in PCR signal intensity over time suggests continuous virus replication. This was further supported by the presence of infectious virus in the blood of these animals. On the contrary, no positive signals were detected in mice infected with the MMP-activata‐ ble RCR vectors [48]. A similar experiment was performed with U-87MG and HT-1080 *s.c*. tumour-bearing mice to quantify the virus load in the tumour tissue. Again, animals were injected intravenously with similar amounts of wt-MLV and MMP-activatable RCR vectors and genomic DNA was analysed 2 weeks later. For all viruses, tumour tissue re‐ vealed the highest virus load reaching 100 % of cells in case of the non-targeted wt-MLV and up to 30 % of cells for MMP-activatable RCR vectors [48]. In addition, wt-MLV in‐ fected on average 32 % of the cells in the bone marrow, 11 % of the cells in spleen, and 6 % of the cells in liver, whereas the MMP-activatable RCR vector infected only 0.02 % of cells in these organs [48]. Interestingly, the load of wt-MLV in bone marrow and the other extratumoral organs in tumour-bearing animals was generally lower compared to those obtained in tumour-free animals [48].

**4. Conclusion**

tumour therapy in humans.

Medicine, Vienna, Austria

and Juraj Hlavaty2,3

Clinical Medicine & Research 2006;4(3): 218–227.

Pharmacology & Therapeutics 2010;32(8): 953–968.

for cancer. Gene Therapy 2011;18(12): 1121–1126.

1 Department of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany

2 Christian-Doppler Laboratory for Innovative Immunotherapy, University of Veterinary

3 Institute of Virology, Department of Pathobiology, University of Veterinary Medicine,

[1] Cross D, Burmester JK. Gene therapy for cancer treatment: past, present and future.

[2] Touchefeu Y, Harrington KJ, Galmiche JP, Vassaux G. Review article: gene therapy, recent developments and future prospects in gastrointestinal oncology. Alimentary

[3] Bader AG, Brown D, Stoudemire J, Lammers P. Developing therapeutic microRNAs

[4] Duarte S, Carle G, Faneca H, de Lima MCP, Pierrefite-Carle V. Suicide gene therapy in

cancer: Where do we stand now. Cancer Letters 2012;324(2): 160–170.

**Author details**

Matthias Renner1

Vienna, Austria

**References**

Very recently, RCR vectors have been employed as a novel gene transfer vehicle for the treatment of cancer. Due to their dense genome organisation and the need for presence of all virus genes to allow vector replication, an only limited capacity for the introduction of foreign sequences is available, rendering the design of such vectors rather challenging. Nevertheless, different vector designs in respect to transgene location and mode of transgene expression have been elaborated. Their analysis in in vitro and in vivo studies revealed that the vectors are genetically stable over several replication cycles and result in an efficient delivery of the therapeutic gene into solid tumours in various animal models. On the other hand, different risks are associated with the use of RCR vectors, such as the risk of insertional tumorigenesis of non-target cells or the risk of inadvertent vector spread resulting in severe side effects. Such risks need to be carefully examined in appropriate non-clinical studies. In case they can be adequately addressed and dispelled, RCR vectors will be a promising option for efficient

Replicating Retroviral Vectors for Gene Therapy of Solid Tumors

http://dx.doi.org/10.5772/54861

149

Recently, Ostertag et al. reported biodistribution of the clinical vector Toca 511 after intra‐ tumoral administration in an immunocompetent mouse model of brain cancer using aqua‐ ntitative real-time PCR technique to detect integrated provirus sequences with high sensitivity (10-25 copies per μg of gDNA) [75]. Two animal models based on different mouse strains had been Involved; BALB/c mice permissive for virus infection, and poorly permissive C57BL/6 mice. In these animal models 10e5 and 10e6 RCR vector particles have been injected intracranially into the tumor mass. Quantitative DNA analyses were performed on samples from mice which survived for 90 days and 180 days for the CT26- BALB/c model and the Tu-2449-B6C3F1 model, respectively. In the CT26-BALB/c model, vector spread to other tissues, particularly to lymphoid organs (thymus, spleen, lymph node, blood) was detectable [75]. Up to 5x10e5 proviral copies/μg gDNA were detected in thymus, up to 1.5x10e5 proviral copies/μg gDNA in samples from salivary gland, oeso‐ phagus, lung, heart, spleen, lymph node, and blood, and less than 5x10e3 proviral copies/μg gDNA in samples from skin, ovary, intestine, liver, kidney, spinal cord, bone marrow, cerebellum and brain [75]. In the Tu-2449-B6C3F1 model vector spread was ob‐ served at low levels only. Less than 5x10e3 proviral copies/μg gDNA were detected and interestingly the oesophagus was the organ in which the proviral copy number was high‐ est. The difference in viral distribution observed in both model systems could be ex‐ plained by the presence of different APOBEC3 alleles in these mouse strains. BALB/c mice have been shown to carry an allele that does not restrict MLV, whereas B6C3F1 mice car‐ ry an active allele [82]. Both mouse strains produced antibodies against Toca 511 [75].

The issue of biodistribution of the vector to non-target sites, as well as the expression of the therapeutic gene in off-target cells could be addressed best by infection targeting of the vector or expression targeting of the therapeutic gene to cells of the tumour. This approach has already been applied using tumour- and tissue-specific regulatory sequences to drive virus replication and transgene expression, and by modifying the viral envelope protein to allow transduction in a tissue/tumour specific manner.

## **4. Conclusion**

found in lung, spleen, liver, heart, bone marrow, and muscle, but not in brain. The in‐ crease in PCR signal intensity over time suggests continuous virus replication. This was further supported by the presence of infectious virus in the blood of these animals. On the contrary, no positive signals were detected in mice infected with the MMP-activata‐ ble RCR vectors [48]. A similar experiment was performed with U-87MG and HT-1080 *s.c*. tumour-bearing mice to quantify the virus load in the tumour tissue. Again, animals were injected intravenously with similar amounts of wt-MLV and MMP-activatable RCR vectors and genomic DNA was analysed 2 weeks later. For all viruses, tumour tissue re‐ vealed the highest virus load reaching 100 % of cells in case of the non-targeted wt-MLV and up to 30 % of cells for MMP-activatable RCR vectors [48]. In addition, wt-MLV in‐ fected on average 32 % of the cells in the bone marrow, 11 % of the cells in spleen, and 6 % of the cells in liver, whereas the MMP-activatable RCR vector infected only 0.02 % of cells in these organs [48]. Interestingly, the load of wt-MLV in bone marrow and the other extratumoral organs in tumour-bearing animals was generally lower compared to

Recently, Ostertag et al. reported biodistribution of the clinical vector Toca 511 after intra‐ tumoral administration in an immunocompetent mouse model of brain cancer using aqua‐ ntitative real-time PCR technique to detect integrated provirus sequences with high sensitivity (10-25 copies per μg of gDNA) [75]. Two animal models based on different mouse strains had been Involved; BALB/c mice permissive for virus infection, and poorly permissive C57BL/6 mice. In these animal models 10e5 and 10e6 RCR vector particles have been injected intracranially into the tumor mass. Quantitative DNA analyses were performed on samples from mice which survived for 90 days and 180 days for the CT26- BALB/c model and the Tu-2449-B6C3F1 model, respectively. In the CT26-BALB/c model, vector spread to other tissues, particularly to lymphoid organs (thymus, spleen, lymph node, blood) was detectable [75]. Up to 5x10e5 proviral copies/μg gDNA were detected in thymus, up to 1.5x10e5 proviral copies/μg gDNA in samples from salivary gland, oeso‐ phagus, lung, heart, spleen, lymph node, and blood, and less than 5x10e3 proviral copies/μg gDNA in samples from skin, ovary, intestine, liver, kidney, spinal cord, bone marrow, cerebellum and brain [75]. In the Tu-2449-B6C3F1 model vector spread was ob‐ served at low levels only. Less than 5x10e3 proviral copies/μg gDNA were detected and interestingly the oesophagus was the organ in which the proviral copy number was high‐ est. The difference in viral distribution observed in both model systems could be ex‐ plained by the presence of different APOBEC3 alleles in these mouse strains. BALB/c mice have been shown to carry an allele that does not restrict MLV, whereas B6C3F1 mice car‐ ry an active allele [82]. Both mouse strains produced antibodies against Toca 511 [75].

The issue of biodistribution of the vector to non-target sites, as well as the expression of the therapeutic gene in off-target cells could be addressed best by infection targeting of the vector or expression targeting of the therapeutic gene to cells of the tumour. This approach has already been applied using tumour- and tissue-specific regulatory sequences to drive virus replication and transgene expression, and by modifying the viral envelope protein to allow transduction

those obtained in tumour-free animals [48].

148 Novel Gene Therapy Approaches

in a tissue/tumour specific manner.

Very recently, RCR vectors have been employed as a novel gene transfer vehicle for the treatment of cancer. Due to their dense genome organisation and the need for presence of all virus genes to allow vector replication, an only limited capacity for the introduction of foreign sequences is available, rendering the design of such vectors rather challenging. Nevertheless, different vector designs in respect to transgene location and mode of transgene expression have been elaborated. Their analysis in in vitro and in vivo studies revealed that the vectors are genetically stable over several replication cycles and result in an efficient delivery of the therapeutic gene into solid tumours in various animal models. On the other hand, different risks are associated with the use of RCR vectors, such as the risk of insertional tumorigenesis of non-target cells or the risk of inadvertent vector spread resulting in severe side effects. Such risks need to be carefully examined in appropriate non-clinical studies. In case they can be adequately addressed and dispelled, RCR vectors will be a promising option for efficient tumour therapy in humans.

## **Author details**

Matthias Renner1 and Juraj Hlavaty2,3

1 Department of Medical Biotechnology, Paul-Ehrlich-Institut, Langen, Germany

2 Christian-Doppler Laboratory for Innovative Immunotherapy, University of Veterinary Medicine, Vienna, Austria

3 Institute of Virology, Department of Pathobiology, University of Veterinary Medicine, Vienna, Austria

## **References**


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**Chapter 8**

**A Novel Therapy for Melanoma and Prostate Cancer**

**Using a Non-Replicating Sendai Virus Particle (HVJ-E)**

The virotherapy approach to cancer therapy uses virus particles. It is based on the case reports since 1950s, which reported the regression of cancers including leukemia, Hodgkin's disease,

The earliest virotherapies involved injection of wild-type viruses and evaluation of their efficacies [1-3]. *Ex vivo* therapies using autologous irradiated tumors infected with oncolytic viruses were also investigated [12-18]. Deletion mutants of oncolytic viruses [19-24], and recombinant viruses carrying a therapeutic gene [25-32] that induce cancer apoptosis or cancer

Oncolytic viruses derived from adenovirus, poxvirus, reovirus, picornavirus, paramyxovirus, and herpes simplex virus are currently available and have been clinically evaluated [33-35]. In China, two adenovirus-based products (Gendicine and Ocorine) have been commercialized [36], while randomized phase III studies of two oncolytic viruses (reovirus and poxvirus) are ongoing in advanced countries [33-35]. Thus, virotherapy is expected to become available as a new approach for cancer treatment, and specific product approval is anticipated in the US,

The major drawback associated with virotherapy is safety since replicating viruses are used in this therapy. In order to reduce the toxicity to normal cells, oncolytic viruses with strict specificity for cancer cells were constructed [29, 38-42]. However, the use of these viruses is still considered to be high risk because it is theoretically possible that a virulent infection may

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

© 2013 Nakajima 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

and Burkitt's lymphoma after the infection of wild type viruses [1-11].

immunity have been developed and evaluated in the clinical setting.

occur after recombination with wild-type viruses [43].

Toshihiro Nakajima, Toshimitsu Itai, Hiroshi Wada,

Additional information is available at the end of the chapter

Toshie Yamauchi, Eiji Kiyohara and

Yasufumi Kaneda

**1. Introduction**

EU, and Japan [37].

http://dx.doi.org/10.5772/55014


## **A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)**

Toshihiro Nakajima, Toshimitsu Itai, Hiroshi Wada, Toshie Yamauchi, Eiji Kiyohara and Yasufumi Kaneda

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55014

## **1. Introduction**

[77] Wang JM, Deng X, Gong W, Su S. Chemokines and their role in tumor growth and

[78] Frederick MJ, Clayman GL. Chemokines in cancer. Expert Reviews in Molecular

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Medicine 2001;3(19): 1–18.

156 Novel Gene Therapy Approaches

Therapy 2003;10(21): 1800–1806.

vectors. Gene Therapy 2011;18(10): 953–960.

human cells. Journal of Virology 1999;73(10): 8813–8816.

genesis in vivo. Journal of Virology 2008;82(22): 10998–11008.

The virotherapy approach to cancer therapy uses virus particles. It is based on the case reports since 1950s, which reported the regression of cancers including leukemia, Hodgkin's disease, and Burkitt's lymphoma after the infection of wild type viruses [1-11].

The earliest virotherapies involved injection of wild-type viruses and evaluation of their efficacies [1-3]. *Ex vivo* therapies using autologous irradiated tumors infected with oncolytic viruses were also investigated [12-18]. Deletion mutants of oncolytic viruses [19-24], and recombinant viruses carrying a therapeutic gene [25-32] that induce cancer apoptosis or cancer immunity have been developed and evaluated in the clinical setting.

Oncolytic viruses derived from adenovirus, poxvirus, reovirus, picornavirus, paramyxovirus, and herpes simplex virus are currently available and have been clinically evaluated [33-35]. In China, two adenovirus-based products (Gendicine and Ocorine) have been commercialized [36], while randomized phase III studies of two oncolytic viruses (reovirus and poxvirus) are ongoing in advanced countries [33-35]. Thus, virotherapy is expected to become available as a new approach for cancer treatment, and specific product approval is anticipated in the US, EU, and Japan [37].

The major drawback associated with virotherapy is safety since replicating viruses are used in this therapy. In order to reduce the toxicity to normal cells, oncolytic viruses with strict specificity for cancer cells were constructed [29, 38-42]. However, the use of these viruses is still considered to be high risk because it is theoretically possible that a virulent infection may occur after recombination with wild-type viruses [43].

© 2013 Nakajima 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

An alternative option to avoid such a risk is to use a non-replicating oncolytic virus [44]. We found that a non-replicating oncolytic virus (HVJ-E: hemagglutinating virus of Japanenvelope) is able to induce cancer cell-specific apoptosis and immunity [45]. The induction of apoptosis and activation of dendritic cells *in vitro*, and anti-tumor activity *in vivo* are similar to the wild-type hemagglutinating virus of Japan (also known as Sendai virus, HVJ) [45].

The hemagglutinating virus of Japan was discovered in Sendai, Japan, in the 1950s [46]. It is a paramyxovirus with a minus-strand RNA genome. The virus has fusogenic activity [47, 48], and is used to prepare hybridoma cells for the production of monoclonal antibodies, and heterokaryons for chromosome analysis [49-51].

The hemagglutinating virus of Japan-envelope is an inactivated HVJ particle [52]. It is manufactured by a process similar to that used for whole virus particle vaccines. Good manufacturing practice (GMP)-regulated processes have been established in their production for use in preclinical and clinical studies [53].

We conducted dose-setting efficacy studies for HVJ-E in a murine cancer model in which dosedependent anti-cancer activity was observed. We also conducted safety studies following good laboratory practices (GLP), including pharmacological safety studies and toxicokinetic (TK) studies in rats and monkeys, as part of an investigational new drug (IND) application.

These observations suggest that a two-step strategy is necessary for the eradication of cancer

**Figure 1. Problems with conventional therapies and a 2-step strategy for cancer treatment.** Conventional anticancer therapies are problematic and a 2-step therapeutic strategy is proposed for effective cancer treatment. Viro‐ therapy possesses ideal characteristics for such a 2-step therapy. Problems associated with conventional anti-cancer

**Removal of residual cancer cells**

**Activation of cancer immunities**

**Removal of cancer**

drugs and immune therapies, including cancer vaccines, are shown.

**Induction of cancer-cell–specific apoptosis**

**Removal of residual cancer cells**

**Killing of cancer cells**

**2. Anticancer agents**

**3. Immune therapies**

159

http://dx.doi.org/10.5772/55014

**Killing of cancer cells**

**Removal of cancer cells**

**Limited efficacy**

**Activation of immunity**

**No cytotoxic effects**

**Relapse of tumor (growth of drug resistant cells)** 

**Cytotoxic effects**

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)

**Immune suppression**

**Killing of cancer cells**

**1. Virotherapy**

During the first step, the direct killing of cancer cells is necessary for reduction of tumor volume. CSCs are usually resistant to conventional anti-cancer drugs and continue to prolif‐ erate during chemotherapy. Therefore, an agent that targets and kills CSCs is required for

During the second step, the removal of residual cancer cells (and CSCs) from the body by a cancer-specific immune response is necessary to avoid relapse of the condition [57]. However, it is difficult for immune cells to recognize and remove CSCs because they exist as a minority population within the tumor, and possess a lower antigenicity than differentiated cancer cells [57]. Oncolytic viruses have the capability of both directly killing cancer cells and inducing

It has been reported that several oncolytic viruses have the capability to kill CSCs [60-67]. The reovirus-based oncolytic virus [61], telomerase-specific oncolytic adenovirus [62], and herpes simplex virus-based oncolytic viruses (G47Delta and Delta 68H-6) [63, 64] reduced CSCs in murine models of breast cancer, esophageal cancer, and malignant glioma, respectively. Thus, a virotherapy approach in patients is expected to kill cancer cells and eradicate cancer cells

A non-replicating virus particle, HVJ-E, is currently being developed as a potential new agent for the treatment of advanced melanoma and CRPC [44, 55, 56]. It is derived from HVJ, a

7

**3. Non-replicating virus particles as anti-cancer agents**

The use of non-replicating virus particles is a new approach in virotherapy.

cells (Figure 1).

**Success of the treatment** 

**Step 1**

**Step 2**

Amendment 3.

cancer immunity.

effective cancer treatment.

**Removal of residual cancer cells ( by combination therapy, cancer immunity…)**

**Killing of cancer cells ( by cytotoxic effects, induction of apoptosis…)**

**Ideal anticancer drug**

including CSCs (Figure 1).

Osaka University Hospital is currently conducting two investigational clinical studies with HVJ-E for the treatment of advanced melanoma and castration-resistant prostate cancer (CRPC) [54-56]. These clinical trials are the first human studies for HVJ-E and will reveal the safety and efficacy of the non-replicating virus (HVJ-E). Virotherapy with a non-replicating oncolytic virus is a new approach that is anticipated to provide a new strategy for cancer therapy.

## **2. A new strategy for cancer therapy**

Most cancers are still incurable and new approaches are required to improve the efficacy of cancer treatments. However, conventional cancer therapies are problematic.

Chemotherapy with anti-cancer agents is useful in achieving tumor regression. However, the immune system, which is important in the removal of residual cancer cells, is also suppressed by these agents (Figure 1). Therefore, surviving cancer cells and cancer stem cells (CSC) eventually acquire drug resistance, resulting in tumor relapse (Figure 1) [57]. Thus, chemo‐ therapy with cytotoxic drugs does not generally result in the necessary eradication of cancer cells required for long-term survival.

Immune therapies for cancer offer a new approach to cancer treatment, and several products, including sipuleucel-T, are currently approved in advanced countries [58, 59]. The aim of these therapies is the removal of cancers by the immune system. Numerous cancer immune therapies are currently under evaluation in clinical studies. However, these agents are not potent because of lack of cytotoxic effect on cancer cells (Figure 1).

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E) http://dx.doi.org/10.5772/55014 159

Amendment 3.

An alternative option to avoid such a risk is to use a non-replicating oncolytic virus [44]. We found that a non-replicating oncolytic virus (HVJ-E: hemagglutinating virus of Japanenvelope) is able to induce cancer cell-specific apoptosis and immunity [45]. The induction of apoptosis and activation of dendritic cells *in vitro*, and anti-tumor activity *in vivo* are similar to the wild-type hemagglutinating virus of Japan (also known as Sendai virus, HVJ) [45].

The hemagglutinating virus of Japan was discovered in Sendai, Japan, in the 1950s [46]. It is a paramyxovirus with a minus-strand RNA genome. The virus has fusogenic activity [47, 48], and is used to prepare hybridoma cells for the production of monoclonal antibodies, and

The hemagglutinating virus of Japan-envelope is an inactivated HVJ particle [52]. It is manufactured by a process similar to that used for whole virus particle vaccines. Good manufacturing practice (GMP)-regulated processes have been established in their production

We conducted dose-setting efficacy studies for HVJ-E in a murine cancer model in which dosedependent anti-cancer activity was observed. We also conducted safety studies following good laboratory practices (GLP), including pharmacological safety studies and toxicokinetic (TK) studies in rats and monkeys, as part of an investigational new drug (IND) application.

Osaka University Hospital is currently conducting two investigational clinical studies with HVJ-E for the treatment of advanced melanoma and castration-resistant prostate cancer (CRPC) [54-56]. These clinical trials are the first human studies for HVJ-E and will reveal the safety and efficacy of the non-replicating virus (HVJ-E). Virotherapy with a non-replicating oncolytic virus is a new approach that is anticipated to provide a new strategy for cancer

Most cancers are still incurable and new approaches are required to improve the efficacy of

Chemotherapy with anti-cancer agents is useful in achieving tumor regression. However, the immune system, which is important in the removal of residual cancer cells, is also suppressed by these agents (Figure 1). Therefore, surviving cancer cells and cancer stem cells (CSC) eventually acquire drug resistance, resulting in tumor relapse (Figure 1) [57]. Thus, chemo‐ therapy with cytotoxic drugs does not generally result in the necessary eradication of cancer

Immune therapies for cancer offer a new approach to cancer treatment, and several products, including sipuleucel-T, are currently approved in advanced countries [58, 59]. The aim of these therapies is the removal of cancers by the immune system. Numerous cancer immune therapies are currently under evaluation in clinical studies. However, these agents are not potent because

cancer treatments. However, conventional cancer therapies are problematic.

heterokaryons for chromosome analysis [49-51].

158 Novel Gene Therapy Approaches

for use in preclinical and clinical studies [53].

**2. A new strategy for cancer therapy**

cells required for long-term survival.

of lack of cytotoxic effect on cancer cells (Figure 1).

therapy.

**Figure 1. Problems with conventional therapies and a 2-step strategy for cancer treatment.** Conventional anticancer therapies are problematic and a 2-step therapeutic strategy is proposed for effective cancer treatment. Viro‐ therapy possesses ideal characteristics for such a 2-step therapy. Problems associated with conventional anti-cancer drugs and immune therapies, including cancer vaccines, are shown.

These observations suggest that a two-step strategy is necessary for the eradication of cancer cells (Figure 1).

During the first step, the direct killing of cancer cells is necessary for reduction of tumor volume. CSCs are usually resistant to conventional anti-cancer drugs and continue to prolif‐ erate during chemotherapy. Therefore, an agent that targets and kills CSCs is required for effective cancer treatment.

During the second step, the removal of residual cancer cells (and CSCs) from the body by a cancer-specific immune response is necessary to avoid relapse of the condition [57]. However, it is difficult for immune cells to recognize and remove CSCs because they exist as a minority population within the tumor, and possess a lower antigenicity than differentiated cancer cells [57]. Oncolytic viruses have the capability of both directly killing cancer cells and inducing cancer immunity.

It has been reported that several oncolytic viruses have the capability to kill CSCs [60-67]. The reovirus-based oncolytic virus [61], telomerase-specific oncolytic adenovirus [62], and herpes simplex virus-based oncolytic viruses (G47Delta and Delta 68H-6) [63, 64] reduced CSCs in murine models of breast cancer, esophageal cancer, and malignant glioma, respectively. Thus, a virotherapy approach in patients is expected to kill cancer cells and eradicate cancer cells including CSCs (Figure 1).

## **3. Non-replicating virus particles as anti-cancer agents**

The use of non-replicating virus particles is a new approach in virotherapy.

A non-replicating virus particle, HVJ-E, is currently being developed as a potential new agent for the treatment of advanced melanoma and CRPC [44, 55, 56]. It is derived from HVJ, a

7

member of the paramyxovirus family (Figure 2). The HVJ-E particle is prepared by inactivating the wild-type virus (HVJ) by treatment with an alkylating agent and UV irradiation [52, 53]. HVJ-E was originally developed as a drug delivery system (vector) for various biopharma‐ ceuticals such as plasmid DNAs, siRNAs, decoy oligonucleotides, antibody proteins, and anticancer drugs [52, 68-75].

Kurooka and Kaneda discovered that the HVJ-E particle itself displayed anti-cancer effects in a murine model of colon cancer [45]. Similar to the live (replicating) virus, HVJ-E induced maturation and differentiation of human and murine dendritic cells (DCs). It also induced infiltration of immune cells into the tumor tissue followed by activation of cancer cell-specific cytotoxic T cells. Furthermore, HVJ-E suppressed the function of regulatory T cells (Treg), which have been reported to be negative regulators of cancer immunity. Thus, HVJ-E activates cancer immunity, and simultaneously suppresses Treg [45].

**1. Structure** 

Amendment 4.

	- **a. The RNA in the particle acts as an RIG-I agonist and induces an induces cancer cell apoptosis**
	- **b. Activates the RIG-I/MAVS pathway and also induces anticancer immune responses**
	- **a. Process adapted to GMP guidelines, and pilot plant for clinical trial is available**
	- **b. Freeze-dried formulation is stable for over 21 months in refrigerator.**

In addition to the induction of cancer immunities, HVJ-E has the capability to induce cancer cell-specific apoptosis. Kawaguchi and Kaneda *et al*. reported that HVJ-E showed a dosedependent, direct killing effect on human prostate cancer cell lines. In contrast, it showed no

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161

HVJ-E also induced apoptosis of prostate cancer cells *in vivo* because it showed an anti-tumor effect in severe combined immunodeficiency (SCID) mice that lack B lymphocyte- and T lymphocyte-mediated immunities [76, 77]. When NK cells were depleted from SCID mice by injection of the asialo-GM1 antibody, intratumoral injection of HVJ-E still showed an antitumor effect in a murine model of CRPC [77]. This result suggested that HVJ-E showed a direct killing activity *in vivo*. HVJ-E-mediated apoptosis of cancer cells was further confirmed in a murine model of prostate cancer using a NOD/SCID mouse, which lacks both innate (NK cell-

Numerous studies have revealed that the non-replicating HVJ-E particle shows anti-tumor effects in murine models of renal cell carcinoma, glioma, colon, bladder and CRPCs [45, 76-79].

In contrast, previous studies have reported that the non-replicating oncolytic Newcastle disease virus (NDV) failed to show an anti-tumor effect *in vivo* [80, 81]. These results are inconsistent with results obtained with HVJ-E [45, 76, 77, 79], and the putative reason is the difference between the number of particles used in the studies. In studies with NDV, 5 × 109 PFU of oncolytic virus were systemically or locally administrated [81], whereas the number of

Another possibility is the difference in the capability of the virus to deliver its RNA fragments to target cancer cells. The Z strain-derived HVJ-E used in our studies has the highest level of membrane fusion activity [52]. Therefore, it is possible that HVJ-E has the ability to deliver more RNA component to the target cancer cells than the UV-inactivated NDV particle. The difference in process of inactivation may be responsible for the activity of non-replicating oncolytic viruses. The Newcastle disease virus was inactivated by UV irradiation, whereas HVJ-E was inactivated by a combination of treatment with an alkylating agent and UV irradiation [53]. Inactivation conditions affect the efficiency of delivery, and strictly regulated

The major target cells of HVJ-E are cancer cells and dendritic cells (DCs) (Figure 3A) [44, 45, 76].

Treatment of cancer cells with HVJ-E enhanced the expression and activation (cleavage) of caspases 3, 8, and 9 (Figure 4A) [77], and induced dose-dependent apoptosis of melanoma, prostate, and other cancer cell lines *in vitro* (Figure 4B) [77]. Interestingly, no apoptotic effects were observed on normal epithelial cells derived from murine prostate [54, 77]. Thus, the

apoptotic activity of HVJ-E is considered to be specific to cancer cells [54, 77].

suppression of normal human prostate epithelium proliferation [77].

mediated) and adaptive (antibody and CTL-mediated) immunities [54].

HVJ-E particles administered was estimated to be higher.

processes are necessary to obtain suitable performance [53].

**4. Mode of actions**

8

**Figure 2. Characteristics and structure of HVJ-E/GEN0101.** The characteristics of HVJ-E/GEN0101 are shown on the left. A schematic structure of the particle is shown on the right.

Fujiwara and Kaneda *et al*. reported that HVJ-E induced innate immunity [76]. Intratumoral injection of HVJ-E promoted infiltration and activation of natural killer (NK) cells by the induction of C-X-C motif chemokine 10 (CXCL10) and type I interferons. When HVJ-E was injected into the tumor of a murine model of renal cell carcinoma (RCC), NK cells exhibited cytotoxic activity against the RCC cell line *in vivo* [76]. The involvement of NK cells in the antitumor effect was also confirmed by showing the depletion of NK cells using an asialo-GM1 antibody [76]. Activated NK cells produced interferon-γ, which induces cancer-specific cytotoxic T cells [76]. These results indicated that HVJ-E is able to induce both innate and adaptive immunities.

In addition to the induction of cancer immunities, HVJ-E has the capability to induce cancer cell-specific apoptosis. Kawaguchi and Kaneda *et al*. reported that HVJ-E showed a dosedependent, direct killing effect on human prostate cancer cell lines. In contrast, it showed no suppression of normal human prostate epithelium proliferation [77].

HVJ-E also induced apoptosis of prostate cancer cells *in vivo* because it showed an anti-tumor effect in severe combined immunodeficiency (SCID) mice that lack B lymphocyte- and T lymphocyte-mediated immunities [76, 77]. When NK cells were depleted from SCID mice by injection of the asialo-GM1 antibody, intratumoral injection of HVJ-E still showed an antitumor effect in a murine model of CRPC [77]. This result suggested that HVJ-E showed a direct killing activity *in vivo*. HVJ-E-mediated apoptosis of cancer cells was further confirmed in a murine model of prostate cancer using a NOD/SCID mouse, which lacks both innate (NK cellmediated) and adaptive (antibody and CTL-mediated) immunities [54].

Numerous studies have revealed that the non-replicating HVJ-E particle shows anti-tumor effects in murine models of renal cell carcinoma, glioma, colon, bladder and CRPCs [45, 76-79].

In contrast, previous studies have reported that the non-replicating oncolytic Newcastle disease virus (NDV) failed to show an anti-tumor effect *in vivo* [80, 81]. These results are inconsistent with results obtained with HVJ-E [45, 76, 77, 79], and the putative reason is the difference between the number of particles used in the studies. In studies with NDV, 5 × 109 PFU of oncolytic virus were systemically or locally administrated [81], whereas the number of HVJ-E particles administered was estimated to be higher.

Another possibility is the difference in the capability of the virus to deliver its RNA fragments to target cancer cells. The Z strain-derived HVJ-E used in our studies has the highest level of membrane fusion activity [52]. Therefore, it is possible that HVJ-E has the ability to deliver more RNA component to the target cancer cells than the UV-inactivated NDV particle. The difference in process of inactivation may be responsible for the activity of non-replicating oncolytic viruses. The Newcastle disease virus was inactivated by UV irradiation, whereas HVJ-E was inactivated by a combination of treatment with an alkylating agent and UV irradiation [53]. Inactivation conditions affect the efficiency of delivery, and strictly regulated processes are necessary to obtain suitable performance [53].

## **4. Mode of actions**

8

member of the paramyxovirus family (Figure 2). The HVJ-E particle is prepared by inactivating the wild-type virus (HVJ) by treatment with an alkylating agent and UV irradiation [52, 53]. HVJ-E was originally developed as a drug delivery system (vector) for various biopharma‐ ceuticals such as plasmid DNAs, siRNAs, decoy oligonucleotides, antibody proteins, and anti-

Kurooka and Kaneda discovered that the HVJ-E particle itself displayed anti-cancer effects in a murine model of colon cancer [45]. Similar to the live (replicating) virus, HVJ-E induced maturation and differentiation of human and murine dendritic cells (DCs). It also induced infiltration of immune cells into the tumor tissue followed by activation of cancer cell-specific cytotoxic T cells. Furthermore, HVJ-E suppressed the function of regulatory T cells (Treg), which have been reported to be negative regulators of cancer immunity. Thus, HVJ-E activates

**Figure 2. Characteristics and structure of HVJ-E/GEN0101.** The characteristics of HVJ-E/GEN0101 are shown on the

Fujiwara and Kaneda *et al*. reported that HVJ-E induced innate immunity [76]. Intratumoral injection of HVJ-E promoted infiltration and activation of natural killer (NK) cells by the induction of C-X-C motif chemokine 10 (CXCL10) and type I interferons. When HVJ-E was injected into the tumor of a murine model of renal cell carcinoma (RCC), NK cells exhibited cytotoxic activity against the RCC cell line *in vivo* [76]. The involvement of NK cells in the antitumor effect was also confirmed by showing the depletion of NK cells using an asialo-GM1 antibody [76]. Activated NK cells produced interferon-γ, which induces cancer-specific cytotoxic T cells [76]. These results indicated that HVJ-E is able to induce both innate and

**Schematic structure** 

**ssRNA ( Agonist of nucleic acid receptors such as RIG-I )**

> P protein

M protein

NP protein **200–300 nm**

HN protein

L protein

Lipid membrane

**F protein ( Treg repression mediated by IL-6 )**

cancer immunity, and simultaneously suppresses Treg [45].

**a. Spherical particle of diameter 200–300 nm b. Contains single strand RNA (ssRNA) c. Contains proteins+lipid for delivery** 

**induces cancer cell apoptosis**

**immune responses**

**trial is available**

**refrigerator.**

adaptive immunities.

**(functions as a natural DDS for nucleic acid)**

**a. The RNA in the particle acts as an RIG-I agonist and induces an** 

**b. Activates the RIG-I/MAVS pathway and also induces anticancer** 

**a. Process adapted to GMP guidelines, and pilot plant for clinical** 

**b. Freeze-dried formulation is stable for over 21 months in** 

left. A schematic structure of the particle is shown on the right.

cancer drugs [52, 68-75].

160 Novel Gene Therapy Approaches

Amendment 4.

**1. Structure** 

**2. Mode of action**

**3. Manufacture**

The major target cells of HVJ-E are cancer cells and dendritic cells (DCs) (Figure 3A) [44, 45, 76].

Treatment of cancer cells with HVJ-E enhanced the expression and activation (cleavage) of caspases 3, 8, and 9 (Figure 4A) [77], and induced dose-dependent apoptosis of melanoma, prostate, and other cancer cell lines *in vitro* (Figure 4B) [77]. Interestingly, no apoptotic effects were observed on normal epithelial cells derived from murine prostate [54, 77]. Thus, the apoptotic activity of HVJ-E is considered to be specific to cancer cells [54, 77].

(a)

**Fluoescence**

**Caspase activity (A375)**

**Caspase activity (B16/BL6)**

http://dx.doi.org/10.5772/55014

163

no treat 10 30 100 340 **HVJ-E (mU/mL)**

**B16/BL6 WST-8 (72hr)**

0 10 20 40 80 160 320 **HVJ-E (mU/well)**

0.0E+00 1.0E+06 2.0E+06 3.0E+06 4.0E+06 5.0E+06 6.0E+06 7.0E+06

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)

**Figure 4. HVJ-E/GEN0101 induces apoptosis of human and murine melanoma cells. (A)** Induction of caspase ac‐ tivity after treatment of melanoma cells with HVJ-E/GEN0101. Human (A375) and murine (B16/BL6) melanoma cells were treated with various amounts of HVJ-E/GEN0101, and caspase activities were measured 24 hours later. A dosedependent activation was observed. **(B)** Survival of melanoma cells after treatment with various amounts of HVJ-E/ GEN0101. Human (A375) and murine (B16/BL6) melanoma cells were treated with various amounts of HVJ-E/

Matsushima and Kaneda *et al*. conducted an investigation to determine the active component for anti-cancer effects, and identified RNA fragments within the particle [54]. Moreover, Kaneda *et al*. analyzed the signaling pathway involved and revealed that retinoic acid inducible gene-I (RIG-I) is a key factor for signal transduction [44, 54, 77]. Retinoic acid inducible gene-I is a cytosolic nucleic acid receptor and was originally identified as a sensor that recognizes infection by single strand RNA viruses [82, 83]. Thus, RIG-I has been recognized as an inducer

The RNA fragments delivered by HVJ-E bind the helicase domain of RIG-I in the cytoplasm and change the conformation to unmask the caspase activation and recruitment domain (CARD) (Figure 3B). After binding with the RNA fragments, RIG-I interacts with the mito‐ chondrial antiviral signaling (MAVS) protein on the mitochondrial membrane (Figure 3B) [84]. The mitochondrial antiviral signaling protein forms a complex with an adaptor protein

**Relative Survival (%)**

**Fluoecsence**

no treat 10 30 100 340 **HVJ-E (mU/mL)**

**A375 WST-8 (72hr)**

0 10 20 40 80 160 320 **HVJ-E (mU/well)**

GEN0101 and cell survival was measured by WST-8 assay 72 hours later.

of immune response against infected viruses [82-84].

(b)

**Relative Survival (%)**

0.0E+00 1.0E+06 2.0E+06 3.0E+06 4.0E+06 5.0E+06 6.0E+06 7.0E+06

**Figure 3. The mechanism of anti-cancer effects of HVJ-E/GEN0101.** A novel type of virotherapy agent (HVJ-E/ GEN0101) has a multi-mode of action that is ideal for 2-step cancer treatment. **(A)** Target cells and sequential anticancer effects of HVJ-E/GEN0101. **(B)** Signaling pathway induced by stimulation with HVJ-E/GEN0101. The RIG-I/ MAVS pathway is the major pathway involved. RIG-I is a cytosolic receptor for nucleic acids: it usually functions as a sensor to recognize viral infection. The nucleic acids in the HVJ-E/GEN0101 particle act as an agonist for RIG-I and in‐ duce cancer cell-specific gene expression followed by the induction of apoptosis.

**CARD CARD**

Attachment to cell membrane

**Differenciation and maturation of dendritic cells**

**1. Induction of an apoptosis of cancer cells (direct killing)**

RNA fragments are directly delivered to cytoplasm by membrane fusion

Conformational

**Cancer cell specific gene expressions followed by the induction of apoptosis.**

duce cancer cell-specific gene expression followed by the induction of apoptosis.

**CARD CARD**

Association of RIG-I/MAVS

**lymphocyte (Treg**

(CARD domains are unmasked)

**Dendritic cell Regulatory T** 

**Ag presentation**

**2. Induction of an innate immunity NK kills cancer cells**

**Induction of IL-6**

**Type I Interferons**

**CARD Pro**

**MABS**

**Cellular membrane of target cells**

Dimerzation

**Figure 3. The mechanism of anti-cancer effects of HVJ-E/GEN0101.** A novel type of virotherapy agent (HVJ-E/ GEN0101) has a multi-mode of action that is ideal for 2-step cancer treatment. **(A)** Target cells and sequential anticancer effects of HVJ-E/GEN0101. **(B)** Signaling pathway induced by stimulation with HVJ-E/GEN0101. The RIG-I/ MAVS pathway is the major pathway involved. RIG-I is a cytosolic receptor for nucleic acids: it usually functions as a sensor to recognize viral infection. The nucleic acids in the HVJ-E/GEN0101 particle act as an agonist for RIG-I and in‐

**CARD**

**TRAF 3**

**Pro**

**Pro**

**CARD**

**TRAF 5**

**IRF3/7 NFkB**

**Kinases Kinases** Activation Activation

Expression of Type-I interferons Expression of cytokines and chemokines

Cancer cell-specific gene expression by epigenic regulation

Normal cells (e.x. endothelial cells, blood cells) - (no change) - ( no change) Cancer cells +++ (induction) +++ (induction) type TRAIL/TRAIL R Cell Noxa

Enhancer/Promoter IFNs Enhancer/Promoter Cytokines/chemokines

Mitochondria

**Th1 cells Death of**

**4. Maintenance of a cancer-specific immunity Supression of Treg induces sustained immunity**

**(CTL)**

**NKcells**

**Cancer cells Killer T** 

**3. Induction of a cancer-specific immunity CTL kills cancer cells**

> **CARD Pro**

**TRAF 5 TRAF 5**

**CARD Pro**

**Helicase domain** (CARD domains are masked)

**HVJ-E**

(b)

**HVJ-E/GEN0101**

(a)

162 Novel Gene Therapy Approaches

Bind to helicase domain change RNA fragments

**RIG-I**

**Figure 4. HVJ-E/GEN0101 induces apoptosis of human and murine melanoma cells. (A)** Induction of caspase ac‐ tivity after treatment of melanoma cells with HVJ-E/GEN0101. Human (A375) and murine (B16/BL6) melanoma cells were treated with various amounts of HVJ-E/GEN0101, and caspase activities were measured 24 hours later. A dosedependent activation was observed. **(B)** Survival of melanoma cells after treatment with various amounts of HVJ-E/ GEN0101. Human (A375) and murine (B16/BL6) melanoma cells were treated with various amounts of HVJ-E/ GEN0101 and cell survival was measured by WST-8 assay 72 hours later.

Matsushima and Kaneda *et al*. conducted an investigation to determine the active component for anti-cancer effects, and identified RNA fragments within the particle [54]. Moreover, Kaneda *et al*. analyzed the signaling pathway involved and revealed that retinoic acid inducible gene-I (RIG-I) is a key factor for signal transduction [44, 54, 77]. Retinoic acid inducible gene-I is a cytosolic nucleic acid receptor and was originally identified as a sensor that recognizes infection by single strand RNA viruses [82, 83]. Thus, RIG-I has been recognized as an inducer of immune response against infected viruses [82-84].

The RNA fragments delivered by HVJ-E bind the helicase domain of RIG-I in the cytoplasm and change the conformation to unmask the caspase activation and recruitment domain (CARD) (Figure 3B). After binding with the RNA fragments, RIG-I interacts with the mito‐ chondrial antiviral signaling (MAVS) protein on the mitochondrial membrane (Figure 3B) [84]. The mitochondrial antiviral signaling protein forms a complex with an adaptor protein (TRAF3/5), stimulates transcription factors (IRF3 and IRF7), and promotes the expression of interferon-α and β (Figure 3B) [85, 86]. It also stimulates kinases of regulator protein of transcription factor NK-κB, resulting in increased expression of cytokines, chemokines, and other genes (Figure 3B) [45, 54, 76, 77].

ent similar to other non-viral anti-cancer agents (Figure 5B). This feature is important for the development of non-replicating oncolytic virus as a novel therapeutic agent; identifica‐ tion of the optimal dose for non-replicating oncolytic viruses is easier than determining the optimal dose for replicating oncolytic viruses as the latter are subject to change (increase) resulting from replication in target cancer cells. The effects of administration in a xeno‐ graft model of human CRPC were also examined (Figure 5A). Efficacy after subcutane‐ ous administration was revealed (Figure 5C). It is known that the SCID mice lacks B lymphocyte- and T lymphocyte-mediated immunities such as antibody production, and induction of cytotoxic T cells but retains monocytes and NK cells important for innate immunity. Thus, the non-replicating virus (HVJ-E/GEN0101) is able to induce innate immunity, and show anti-cancer activity *in vivo* even in the absence of direct killing of cancer cells. Efficacy by subcutaneous administration is important for the development of a non-replicating virus because subcutaneous administration is more common than intratumoral administration. Intratumoral administration kills cancer cells directly and promotes the release of tumor antigens, which are recognized by immune cells. Subcutane‐ ous administration is expected to enhance and sustain the immune response. Therefore, the combination of intratumoral and subcutaneous administration is suggested as a suitable

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In summary, the results of efficacy studies have indicated that the anti-cancer effect of the non-replicating oncolytic virus HVJ-E/GEN0101 is dose-dependent similar to conventional anti-cancer drugs, and subcutaneous administration may be the preferred administration

Safety of the non-replicating oncolytic virus (HVJ-E/GEN0101) has been confirmed in nonprimate (rat) and primate (Cynomolgus monkey) animals. Lists of the studies that have been

Results from single dose general toxicity studies revealed that no death or severe finding was observed even in the maximum dosage groups. Similar to the single dose studies, no severe

Results from immunological and genetic toxicity studies in rat and monkeys revealed that no abnormal symptoms related to the test agent were observed. The levels of IL-6 and IFN-γ in monkey serum were analyzed after subcutaneous injection of HVJ-E/GEN0101, and the levels of both cytokines were determined to be within the normal range (data not shown). A core battery of safety pharmaceutical studies was performed to determine the effects on major organs (central nervous, respiratory, and cardiovascular systems); no abnormal effect was

finding was observed in repeated dose, general toxicity studies (Table 1A).

observed with the exception of transient and non-severe pyrexia (Table 1B).

regimen in the clinical setting.

**6. Safety studies**

route for the viral and non-replicating viral agents.

conducted are shown in Table 1A and B.

It has been reported that the RIG-I/MAVS pathway is activated after cancer cells are treated with HVJ-E [54]. Transfection of isolated RNA fragments from HVJ-E particle also induced apoptosis of cancer cells *in vitro* [54]. Thus, HVJ-E uses a natural oligonucleotide (RNA fragment from the inactivated virus genome) as an active ingredient, and a natural virus particle (envelope of HVJ) as a delivery system for a nucleic acid medicine (RNA fragments). The apoptosis induced by HVJ-E was cancer cell-specific because normal endothelial cells showed no apoptosis after the treatment with HVJ-E [54, 77].

Involvement of RIG-I in cancer cell-specific apoptosis has also been indicated in ovarian cancers and melanoma. Kübler and Barchet *et al*. reported that the RIG-I agonist (Poly(dAdT)) induced apoptosis and expression of MHC class I molecules in ovarian cancer cells [87, 88]. Van and Bell *et al*. also reported apoptosis of ovarian cancer cells after treatment with dsRNA [89]. Analysis by shRNA-mediated knockdown revealed that RIG-I and other receptors for dsRNA (TLR3 and MDA-5) were involved in the caspase 8/9 mediated apoptosis of cancer cells. Similar to sensitivity of HVJ-E, epithelial cells derived from ovarian surface was resistant to apoptosis mediated by RIG-I signal pathway. When combined with conventional chemotherapy (carboplatin/paclitaxel), treatment with dsRNA showed a synergistic suppression of ovarian cancer cell viability [89]. Besch *et al*. report‐ ed that stimulation of RIG-I and MDA-5 induced apoptosis of human melanoma cells [90]. The authors used pppRNA and poly(I:C) as ligands for RIG-I and MDA-5, and showed the involvement of caspase-9 and Apaf-1 during apoptosis. They also reported the reduction of lung metastasis by treatment with ligands for RIG-I and MDA-5 in the NOD/SCID mouse [90]. Details of the underlying pathways are currently being analyzed using siRNAs of apoptosis-related factors [54]. It has been suggested that differences in the expression of apoptotic genes such as Noxa, TRAIL, and TRAIL receptors in cancer cells and normal cells determine the specificity of apoptosis induced by HVJ-E (Figure 3B) [54].

HVJ-E also induced differentiation and maturation of murine and human DCs [45]. It induced the expression of surface markers on mature DCs, and the production of various cytokines and chemokines from DCs [45, 76, 77]. Activated DCs induce both innate (NK cell-mediated) and adaptive (cytotoxic T cell-mediated) immunities (Figure 3A) [45, 76]. It has been reported that an RIG-I agonist (Poly(dAdT)) induced the production of cytokines (IL-6 and TNF-α) and chemokines (CXCL1-and CCL5/RANTES) [87, 88] in human ovarian cancer cells.

## **5. Efficacy in preclinical studies**

The efficacy of the non-replicating oncolytic virus (HVJ-E/GEN0101) was examined in murine models of melanoma and prostate cancer (Figure 5A). GEN0101 is the identifica‐ tion code for the agent. The data have indicated that the anti-tumor effect is dose-depend‐ ent similar to other non-viral anti-cancer agents (Figure 5B). This feature is important for the development of non-replicating oncolytic virus as a novel therapeutic agent; identifica‐ tion of the optimal dose for non-replicating oncolytic viruses is easier than determining the optimal dose for replicating oncolytic viruses as the latter are subject to change (increase) resulting from replication in target cancer cells. The effects of administration in a xeno‐ graft model of human CRPC were also examined (Figure 5A). Efficacy after subcutane‐ ous administration was revealed (Figure 5C). It is known that the SCID mice lacks B lymphocyte- and T lymphocyte-mediated immunities such as antibody production, and induction of cytotoxic T cells but retains monocytes and NK cells important for innate immunity. Thus, the non-replicating virus (HVJ-E/GEN0101) is able to induce innate immunity, and show anti-cancer activity *in vivo* even in the absence of direct killing of cancer cells. Efficacy by subcutaneous administration is important for the development of a non-replicating virus because subcutaneous administration is more common than intratumoral administration. Intratumoral administration kills cancer cells directly and promotes the release of tumor antigens, which are recognized by immune cells. Subcutane‐ ous administration is expected to enhance and sustain the immune response. Therefore, the combination of intratumoral and subcutaneous administration is suggested as a suitable regimen in the clinical setting.

In summary, the results of efficacy studies have indicated that the anti-cancer effect of the non-replicating oncolytic virus HVJ-E/GEN0101 is dose-dependent similar to conventional anti-cancer drugs, and subcutaneous administration may be the preferred administration route for the viral and non-replicating viral agents.

## **6. Safety studies**

(TRAF3/5), stimulates transcription factors (IRF3 and IRF7), and promotes the expression of interferon-α and β (Figure 3B) [85, 86]. It also stimulates kinases of regulator protein of transcription factor NK-κB, resulting in increased expression of cytokines, chemokines, and

It has been reported that the RIG-I/MAVS pathway is activated after cancer cells are treated with HVJ-E [54]. Transfection of isolated RNA fragments from HVJ-E particle also induced apoptosis of cancer cells *in vitro* [54]. Thus, HVJ-E uses a natural oligonucleotide (RNA fragment from the inactivated virus genome) as an active ingredient, and a natural virus particle (envelope of HVJ) as a delivery system for a nucleic acid medicine (RNA fragments). The apoptosis induced by HVJ-E was cancer cell-specific because normal endothelial cells

Involvement of RIG-I in cancer cell-specific apoptosis has also been indicated in ovarian cancers and melanoma. Kübler and Barchet *et al*. reported that the RIG-I agonist (Poly(dAdT)) induced apoptosis and expression of MHC class I molecules in ovarian cancer cells [87, 88]. Van and Bell *et al*. also reported apoptosis of ovarian cancer cells after treatment with dsRNA [89]. Analysis by shRNA-mediated knockdown revealed that RIG-I and other receptors for dsRNA (TLR3 and MDA-5) were involved in the caspase 8/9 mediated apoptosis of cancer cells. Similar to sensitivity of HVJ-E, epithelial cells derived from ovarian surface was resistant to apoptosis mediated by RIG-I signal pathway. When combined with conventional chemotherapy (carboplatin/paclitaxel), treatment with dsRNA showed a synergistic suppression of ovarian cancer cell viability [89]. Besch *et al*. report‐ ed that stimulation of RIG-I and MDA-5 induced apoptosis of human melanoma cells [90]. The authors used pppRNA and poly(I:C) as ligands for RIG-I and MDA-5, and showed the involvement of caspase-9 and Apaf-1 during apoptosis. They also reported the reduction of lung metastasis by treatment with ligands for RIG-I and MDA-5 in the NOD/SCID mouse [90]. Details of the underlying pathways are currently being analyzed using siRNAs of apoptosis-related factors [54]. It has been suggested that differences in the expression of apoptotic genes such as Noxa, TRAIL, and TRAIL receptors in cancer cells and normal cells

other genes (Figure 3B) [45, 54, 76, 77].

164 Novel Gene Therapy Approaches

showed no apoptosis after the treatment with HVJ-E [54, 77].

determine the specificity of apoptosis induced by HVJ-E (Figure 3B) [54].

chemokines (CXCL1-and CCL5/RANTES) [87, 88] in human ovarian cancer cells.

**5. Efficacy in preclinical studies**

HVJ-E also induced differentiation and maturation of murine and human DCs [45]. It induced the expression of surface markers on mature DCs, and the production of various cytokines and chemokines from DCs [45, 76, 77]. Activated DCs induce both innate (NK cell-mediated) and adaptive (cytotoxic T cell-mediated) immunities (Figure 3A) [45, 76]. It has been reported that an RIG-I agonist (Poly(dAdT)) induced the production of cytokines (IL-6 and TNF-α) and

The efficacy of the non-replicating oncolytic virus (HVJ-E/GEN0101) was examined in murine models of melanoma and prostate cancer (Figure 5A). GEN0101 is the identifica‐ tion code for the agent. The data have indicated that the anti-tumor effect is dose-depend‐ Safety of the non-replicating oncolytic virus (HVJ-E/GEN0101) has been confirmed in nonprimate (rat) and primate (Cynomolgus monkey) animals. Lists of the studies that have been conducted are shown in Table 1A and B.

Results from single dose general toxicity studies revealed that no death or severe finding was observed even in the maximum dosage groups. Similar to the single dose studies, no severe finding was observed in repeated dose, general toxicity studies (Table 1A).

Results from immunological and genetic toxicity studies in rat and monkeys revealed that no abnormal symptoms related to the test agent were observed. The levels of IL-6 and IFN-γ in monkey serum were analyzed after subcutaneous injection of HVJ-E/GEN0101, and the levels of both cytokines were determined to be within the normal range (data not shown). A core battery of safety pharmaceutical studies was performed to determine the effects on major organs (central nervous, respiratory, and cardiovascular systems); no abnormal effect was observed with the exception of transient and non-severe pyrexia (Table 1B).

days after transplant). A time course study of change in tumor volume was performed and differences in tumor vol‐ ume between the two groups were statistically analyzed at the end of study. Significant differences were determined by t-test and significant differences to the medium were observed in both routes (p < 0.01 and p < 0.05). **(B)** Efficacy study in a murine model of melanoma. Dose-dependency of the anti-tumor effect of HVJ-E/GEN0101 was revealed. **(C)** Efficacy study in a murine model of prostate cancer. A time course study of change in tumor volume was per‐ formed and differences in tumor volume between the two groups were statistically analyzed at the end of the study. Significant differences were determined by t-test, and significant differences to the medium were observed for both

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)

Dosing Regimen Species Route Dose

Study Method Species Route Dose

TK Q-PCR Rat sc 6 times 2 weeks Genetic toxicity Micronucleus Rat sc single Antibody production ELISA Rat sc 6 times 2 weeks

Two clinical studies using the non-replicating oncolytic virus (HVJ-E/GEN0101) are currently being conducted in Osaka University Hospital. The target diseases are advanced melanoma (stage IIIC and stage IV) and CRPC [55, 56]. These proof-of-concept studies in melanoma and CRPC using the non-replicating oncolytic virus were initiated in July 2009 and July 2011, respectively [55, 56]. The respective summaries of both studies are shown in Table 2A and B. The primary endpoints of these studies were safety and tolerability based on the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, whereas the secondary endpoints were efficacy and confirmation of the mode of action. The major difference between the regimens for the melanoma and CRPC studies was the route of administration and number of administrations. A combination of intratumoral and subcutaneous routes of administration (one intratumoral and three subcutaneous injections) was adopted in the CRPC study. In addition, a new injection system developed by Okayama University was used in the CRPC

Rat iv single Rat sc single Cynomolgus monkey iv single Cynomolgus monkey sc single

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167

Rat iv 7 days Rat sc 6 times in 2 weeks Cynomolgus monkey sc 6 times in 2 weeks

Rat FOB Rat sc single Respiratory Rat sc single Cardiovascular Cynomolgus monkey sc single

administration routes (p < 0.01 and p < 0.05).

Single Dose

Repeated Dose

Safety pharmacology

**Table 1.** Summary of toxicological studies

**7. Clinical studies**

**a) Toxicological study (1): General toxicity study**

**b) Toxicological study (2): Safety pharmacology and other studies**

**Figure 5. Efficacy studies of HVJ-E/GEN0101 in a murine model of melanoma and prostate cancer.(A)** Protocols for the efficacy study using murine models of melanoma and prostate cancer. Left: C57/BL6 mice were transplanted with B16/BL6 cells and 3 intratumoral injections of HVJ-E/GEN0101 were administered after tumor formation (4 days after transplant). A time course study of change in tumor volume was performed, and the difference in tumor volume among the three groups was statistically analyzed at the end of the study. Significant differences were determined by Dunnett's multigroup test, and significant differences to the medium (control) group were observed (p < 0.01). The survival rate after the injection of HVJ-E/GEN0101 was also monitored in the melanoma model. A survival study was performed and the difference among the three groups was statistically analyzed at the end of the study. Significant differences were determined by log-rank analysis. Significant differences to the medium group were observed (p < 0.001). Right: Protocol for the efficacy study in a murine model of prostate cancer. Human CRPC (PC-3)-bearing mice were used for the study. A summary of the study protocol is shown. The anti-tumor effect of HVJ-E/GEN0101 for each administration route was examined. Severe combined immunodeficiency mice were transplanted with PC-3 cells. Two intratumoral injections or 6 subcutaneous injections of HVJ-E/GEN0101 were performed after tumor formation (4

#### A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E) http://dx.doi.org/10.5772/55014 167

days after transplant). A time course study of change in tumor volume was performed and differences in tumor vol‐ ume between the two groups were statistically analyzed at the end of study. Significant differences were determined by t-test and significant differences to the medium were observed in both routes (p < 0.01 and p < 0.05). **(B)** Efficacy study in a murine model of melanoma. Dose-dependency of the anti-tumor effect of HVJ-E/GEN0101 was revealed. **(C)** Efficacy study in a murine model of prostate cancer. A time course study of change in tumor volume was per‐ formed and differences in tumor volume between the two groups were statistically analyzed at the end of the study. Significant differences were determined by t-test, and significant differences to the medium were observed for both administration routes (p < 0.01 and p < 0.05).


**Table 1.** Summary of toxicological studies

## **7. Clinical studies**

(a)

(b)

**Tumor volume (mm**

**3)** Intradermal inoculation (right dorsal)

C57/BL6 mice (n = 10)

166 Novel Gene Therapy Approaches

melanoma cells

**Day 0 Day 4 Day 4, 6, 8 (3 times)**

Randomization HVJ-E/GEN0101 administration (intratumoral 6 times 100 uL/mouse)

> :0 mNAU (medium) :100 mNAU :1000 mNAU

B16/BL6 **survival rate**

**Calculation of tumor vol. (mm3) = length x (width)2 x 0.5**

**Days after transplant**

**Intratumoral administration**

**: HVJ-E/GEN0101 : 5% trehalose**

Intratumoral injections

4 8 12 16 20 24 28 32 36 40 44 48 **Days after inoculation**

4 6 8 10 12 14 16 18 20

**\*\*: p < 0.01 (Dunnett multigroup test)**

Group

Intratumoral injections

**Day 4, 6, 8, 11, 13, 15 (6 times)**

**Day 4 - 20 Measurement of tumor size, body weight and** 

> **\*\* \*\***

**Tumor volume Survival**

**Survival rate (%)**

\*\*

**Figure 5. Efficacy studies of HVJ-E/GEN0101 in a murine model of melanoma and prostate cancer.(A)** Protocols for the efficacy study using murine models of melanoma and prostate cancer. Left: C57/BL6 mice were transplanted with B16/BL6 cells and 3 intratumoral injections of HVJ-E/GEN0101 were administered after tumor formation (4 days after transplant). A time course study of change in tumor volume was performed, and the difference in tumor volume among the three groups was statistically analyzed at the end of the study. Significant differences were determined by Dunnett's multigroup test, and significant differences to the medium (control) group were observed (p < 0.01). The survival rate after the injection of HVJ-E/GEN0101 was also monitored in the melanoma model. A survival study was performed and the difference among the three groups was statistically analyzed at the end of the study. Significant differences were determined by log-rank analysis. Significant differences to the medium group were observed (p < 0.001). Right: Protocol for the efficacy study in a murine model of prostate cancer. Human CRPC (PC-3)-bearing mice were used for the study. A summary of the study protocol is shown. The anti-tumor effect of HVJ-E/GEN0101 for each administration route was examined. Severe combined immunodeficiency mice were transplanted with PC-3 cells. Two intratumoral injections or 6 subcutaneous injections of HVJ-E/GEN0101 were performed after tumor formation (4

**(n = 10)**

**\*\* : p<0.01(t-test)**

**(n = 5)**

Day 0 Day 4 or 18 Day 4, 8, 11, 12

HVJ-E/GEN0101 administration (intra-tumor or sc, 2 or 3 times 100 uL/mouse) \* Calculation of tumor vol. (mm3) = length x (width)2 x 0.5

**\*\*\*: p < 0.001, (Log-rank test)**

**Subcutaneous administration**

**Days after inoculation**

4 11 18 25 32 39 46

**(n = 8)**

\*

**\* : p<0.05(t-test)**

**Days after transplant**

**: HVJ-E/GEN0101 : 5% trehalose**

24 26 28 30 32 34 36 38

**(n = 10)**

body weight PC-3

Day 4 - 48 Measurement of tumor size,

> **\*\*\* \*\*\***

Group Randomization

:0mNAU (medium) :100mNAU :1000mNAU

**Tumor volume (mm^3)**

Intradermal inoculation (right dorsal)

SCID mice

Prostae cancer cells

(c)

**Tumor volume (mm^3)**

Two clinical studies using the non-replicating oncolytic virus (HVJ-E/GEN0101) are currently being conducted in Osaka University Hospital. The target diseases are advanced melanoma (stage IIIC and stage IV) and CRPC [55, 56]. These proof-of-concept studies in melanoma and CRPC using the non-replicating oncolytic virus were initiated in July 2009 and July 2011, respectively [55, 56]. The respective summaries of both studies are shown in Table 2A and B. The primary endpoints of these studies were safety and tolerability based on the Common Terminology Criteria for Adverse Events (CTCAE) version 4.0, whereas the secondary endpoints were efficacy and confirmation of the mode of action. The major difference between the regimens for the melanoma and CRPC studies was the route of administration and number of administrations. A combination of intratumoral and subcutaneous routes of administration (one intratumoral and three subcutaneous injections) was adopted in the CRPC study. In addition, a new injection system developed by Okayama University was used in the CRPC study. This system permits stable refrigerated storage of the test article, and accurate injection into the prostate [91].

The modes of action underlying the anti-cancer effects of non-replicating virus on cancer cells and immune cells have been analyzed. One major signaling pathway is the RIG-I/ MAVS pathway [54]. RIG-I is a cytosolic nucleic acid receptor that acts as a sensor that detects virus infection [82-84]. Similar to wild-type RNA viruses, the non-replicating virus (HVJ-E) is able to activate the RIG-I/MAVS pathway in DCs and induce both innate and adapted immunities. The non-replicating virus also activates the RIG-I/MAVS pathway in cancer cells and induces cancer cell-specific apoptosis. Genetic analyses suggest that differences in the expression of apoptosis-related genes define the sensitivity to the treatment with a non-replicating virus (HVJ-E) [54]. Furthermore, it is suggested that methylation of the respective enhancer/promoter regions underlies differences in transcrip‐

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)

http://dx.doi.org/10.5772/55014

169

RIG-I, TLR3, and MDA-5 signaling pathways are involved in the apoptosis of ovarian cancers [87-89] and melanoma [90]. Thus, the RIG-I/MAVS pathway is likely to emerge as a new target

The non-replicating virus (HVJ-E) activates DCs to produce IL-6, which suppress the function of Treg [45]. This effect is expected to maintain the induced cancer immunity because Treg is known to be a negative regulator of immune responses [92-94]. It has been reported that cancer cells escape cancer immunity by the recruitment and activation of Treg. Therefore, it will be important to control the function of Treg for long-term effec‐ tive induction and maintenance of cancer immunities. Suzuki and Kaneda *et al*. reported that the RIG-I/MAVS pathway was not required to induce the expression of IL-6 [95]. The attachment of the HVJ-E particle to the surface of DCs was sufficient for the production of IL-6, suggesting that the RNA fragments are unnecessary for the induction of this cyto‐ kine [95]. Detailed analyses identified that the F protein on the surface of HVJ-E is involved in the production of IL-6 [95]. Binding of the F protein to target cells requires expression of the HN protein [96]. Several gangliosides, such as GD1a and sialyl paragloboside, are implicated in the association of the HVJ-E particle and cancer cells because the HN protein binds the sialic acids of gangliosides. The receptor for the F protein remains unidentified to date. Taken together, the RIG-I/MAVS signal pathway, and a second pathway that induces the production of IL-6 may cooperate in the activation and sustainment of cancer

The development of a non-replicating oncolytic virus other than HVJ-E is possible because the manufacturing process for such a particle is similar to that of whole particle viral vaccines. In case of HVJ-E, the virus is inactivated by treatment with an alkylating agent and UV irradiation, a process used for the production of vaccines against viral diseases. Thus, the development of oncolytic viruses could be converted to the development of non-replicating oncolytic particles

A disadvantage associated with non-replicating oncolytic viruses may be the defect in transmission ability. Therefore, it is possible that a greater amount of non-replicating oncolytic virus may be required for effective treatment compared with a live oncolytic virus. Alterna‐ tively, more frequent injections may be necessary for complete tumor eradication compared with the use of live oncolytic viruses. However, it is important to achieve a balance between

for the development of drugs that induce cancer cell-specific apoptosis.

immunity induced by the non-replicating virus (HVJ-E).

by similar manufacturing processes.

tion of apoptosis-related genes [54].


**Table 2.** Design of investigational clinical studies

### **8. Discussion and conclusion**

The major disadvantages associated with oncolytic viruses are safety concerns because viral replication could theoretically cause the emergence of new pathogenic viruses [43]. The use of non-replicating oncolytic viruses is expected to resolve the safety issues associated with conventional oncolytic viruses because they are unable to replicate in target cells.

The modes of action underlying the anti-cancer effects of non-replicating virus on cancer cells and immune cells have been analyzed. One major signaling pathway is the RIG-I/ MAVS pathway [54]. RIG-I is a cytosolic nucleic acid receptor that acts as a sensor that detects virus infection [82-84]. Similar to wild-type RNA viruses, the non-replicating virus (HVJ-E) is able to activate the RIG-I/MAVS pathway in DCs and induce both innate and adapted immunities. The non-replicating virus also activates the RIG-I/MAVS pathway in cancer cells and induces cancer cell-specific apoptosis. Genetic analyses suggest that differences in the expression of apoptosis-related genes define the sensitivity to the treatment with a non-replicating virus (HVJ-E) [54]. Furthermore, it is suggested that methylation of the respective enhancer/promoter regions underlies differences in transcrip‐ tion of apoptosis-related genes [54].

study. This system permits stable refrigerated storage of the test article, and accurate injection

Study title Phase I/II investigational clinical study of inactivated HVJ-E administration for advanced

Secondary endpoint: Anti-tumor immunity and validity

URL https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type

Study title Phase I/II investigational clinical study to assess safety and efficacy of intratumoral and

Secondary endpoint: Anti-tumor immunity and validity

URL https://upload.umin.ac.jp/cgi-open-bin/ctr/ctr.cgi?function=brows&action=brows&type

The major disadvantages associated with oncolytic viruses are safety concerns because viral replication could theoretically cause the emergence of new pathogenic viruses [43]. The use of non-replicating oncolytic viruses is expected to resolve the safety issues associated with

conventional oncolytic viruses because they are unable to replicate in target cells.

subcutaneous injection of HVJ-E to castration-resistant prostate cancer patients

malignant melanoma patients Condition Malignant melanoma ( AJCC stage IIIC or stage IV)

Route: Intratumoral

Condition Castration resistant prostate cancer (CRPC)

Allocation: Non-Randomized

Target sample size: 6–12 patients

Primary endpoint: Safety and tolerability

Dose: 6 times in 2 weeks/cycle, 2 cycles Sponsor Osaka University Graduate School of Medicine (Osaka University Hospital)

=summary&recptno=R000002889&language=E

Allocation: Non-Randomized

Target sample size: 6–12 patients Route: Intratumoral × 1 then SC × 3 Dose: 4 times in 2 weeks/cycle, 2 cycles Sponsor Osaka University Graduate School of Medicine (Osaka University Hospital)

=summary&recptno=R000007153&language=E

Primary endpoint: Safety and tolerability

into the prostate [91].

168 Novel Gene Therapy Approaches

**a) Advanced melanoma**

Study design Masking: Open Label

**b) Castration-resistant prostate cancer**

Study design Masking: Open Label

**Table 2.** Design of investigational clinical studies

**8. Discussion and conclusion**

RIG-I, TLR3, and MDA-5 signaling pathways are involved in the apoptosis of ovarian cancers [87-89] and melanoma [90]. Thus, the RIG-I/MAVS pathway is likely to emerge as a new target for the development of drugs that induce cancer cell-specific apoptosis.

The non-replicating virus (HVJ-E) activates DCs to produce IL-6, which suppress the function of Treg [45]. This effect is expected to maintain the induced cancer immunity because Treg is known to be a negative regulator of immune responses [92-94]. It has been reported that cancer cells escape cancer immunity by the recruitment and activation of Treg. Therefore, it will be important to control the function of Treg for long-term effec‐ tive induction and maintenance of cancer immunities. Suzuki and Kaneda *et al*. reported that the RIG-I/MAVS pathway was not required to induce the expression of IL-6 [95]. The attachment of the HVJ-E particle to the surface of DCs was sufficient for the production of IL-6, suggesting that the RNA fragments are unnecessary for the induction of this cyto‐ kine [95]. Detailed analyses identified that the F protein on the surface of HVJ-E is involved in the production of IL-6 [95]. Binding of the F protein to target cells requires expression of the HN protein [96]. Several gangliosides, such as GD1a and sialyl paragloboside, are implicated in the association of the HVJ-E particle and cancer cells because the HN protein binds the sialic acids of gangliosides. The receptor for the F protein remains unidentified to date. Taken together, the RIG-I/MAVS signal pathway, and a second pathway that induces the production of IL-6 may cooperate in the activation and sustainment of cancer immunity induced by the non-replicating virus (HVJ-E).

The development of a non-replicating oncolytic virus other than HVJ-E is possible because the manufacturing process for such a particle is similar to that of whole particle viral vaccines. In case of HVJ-E, the virus is inactivated by treatment with an alkylating agent and UV irradiation, a process used for the production of vaccines against viral diseases. Thus, the development of oncolytic viruses could be converted to the development of non-replicating oncolytic particles by similar manufacturing processes.

A disadvantage associated with non-replicating oncolytic viruses may be the defect in transmission ability. Therefore, it is possible that a greater amount of non-replicating oncolytic virus may be required for effective treatment compared with a live oncolytic virus. Alterna‐ tively, more frequent injections may be necessary for complete tumor eradication compared with the use of live oncolytic viruses. However, it is important to achieve a balance between the risks and benefits associated with the therapy. In our opinion, repeated administration of the non-replicating virus should be tolerable because no severe finding was observed during our safety studies.

**Summary**

cancer-specific cytotoxic T lymphocytes.

**Acknowledgements**

**Author details**

Toshihiro Nakajima1

oka, Suita, Osaka, Japan

Yasufumi Kaneda2

to become a novel concept for cancer therapy in the near future.

Ishikawa for their excellent administrative supports.

the National Institute of Biomedical Innovation (NIBIO).

, Toshimitsu Itai1

School of Medicine, Osaka University, Yamada-oka, Suita, Osaka, Japan

GenomIdea. The remaining authors have no conflicts of interest.

1 GenomIdea, Inc., Midorigaoka, Ikeda, Osaka, Japan

Conventional cancer therapies suffer from one paradox: although chemotherapeutic agents strongly kill cancer cells and decrease tumor volume, they simultaneously suppress the immune system. Chemotherapy frequently results in tumor relapse because residual cancer cells and cancer stem cells escape immune responses. In contrast, immune therapies including therapeutic cancer vaccines, effectively induce cancer immunity, but possess weak cytotoxic activity against cancer cells. Therefore, these treatments usually show weak efficacy. It has been reported that cancer is able to progress even after the activation and proliferation of

A Novel Therapy for Melanoma and Prostate Cancer Using a Non-Replicating Sendai Virus Particle (HVJ-E)

Virotherapy is predicted to become an alternative approach to obtain a model cancer therapy because it generally displays both oncolytic and immunostimulatory activities. However, the major drawback associated with current virotherapy is safety concerns. Virotherapy using a non-replicating virus is a new approach aimed at resolving safety issues. Thus, it is expected

We appreciate T. Yamazaki for helpful discussions. We also appreciate H. Ueda and S.

This study was supported by the advanced research for medical products Mining Program of

, Hiroshi Wada1

2 Division of Gene Therapy Science, Department of Molecular Therapeutics, Graduate

3 Department of Dermatology, Graduate School of Medicine, Osaka University, Yamada-

T. Nakajima is a CEO of GenomIdea. T. Itai, H. Wada, and T. Yamauchi are employees of

, Toshie Yamauchi1

, Eiji Kiyohara2,3 and

http://dx.doi.org/10.5772/55014

171

In conclusion, non-replicating virus particles such as HVJ-E may resolve the safety issue of conventional virotherapy and provide a new strategy in cancer treatment.

## **9. Future perspectives**

The first non-replicating oncolytic virus (HVJ-E) is currently under evaluation in clinical studies. Proof-of-concept data for non-replicating viruses in both clinical and non-clinical studies are necessary for further development of this approach. Osaka University Hospital is currently conducting two phase I/IIa studies: one for advanced melanoma and another for CRPC [55, 56]. The results of these studies will reveal the safety, efficacy, and optimal dosage regimen necessary for phase II study or randomized, double blind phase III study.

Combination treatment may be an effective approach to increase efficacy [97]. Indeed, an increase in therapeutic efficacy has been reported for virotherapies combined with photody‐ namic therapy [98, 99], radiotherapy [100], chemotherapy [101, 102], or gene therapy [103]. Kiyohara and Kaneda reported that combination of the non-replicating virus (HVJ-E) and gene therapy (IL-12) increased efficacy in a murine model of melanoma [104]. Furthermore, it was reported that a combination of non-replicating virus (HVJ-E) and chemotherapy [bleomycin or cis-diamminedichloroplatinum (CDDP)] increased efficacy in murine models of colon and bladder cancers [78, 105].

Technologies for systemic administration and targeting for HVJ-E are under development. The HN protein of HVJ-E has hemagglutinating activity and causes agglutination and lysis of erythrocytes *in vitro*. Currently, inactivation of the HN protein, decreased expression of the HN protein, and "masking" with platelets is being developed for intravenous injection of HVJ-E. Targeting after the intravenous injection is also important for systemic delivery. The addition of transferrin, a single chain antibody, or platelets have been suggested as suitable modifiers for HVJ-E.

The selection of viruses, or viral strains for the preparation of non-replicating oncolytic viruses is also important for obtaining higher efficacy because the level of immune response is dependent on the selection of virus strains [106]. A number of replicating oncolytic viruses are currently under clinical development [35, 97]. Therefore, it may be feasible to select a suitable virus for therapeutic application from the oncolytic viruses that have been developed [106, 107]. The tropism and complement resistance features of each virus should be considered for targeting and stabilization in serum.

HVJ-E is the only non-replicating oncolytic virus currently undergoing clinical investigation. These studies establish a new strategy for the virotherapy and gene therapy fields. The primary goal is to provide a novel approach for improving cancer therapy.

## **Summary**

the risks and benefits associated with the therapy. In our opinion, repeated administration of the non-replicating virus should be tolerable because no severe finding was observed during

In conclusion, non-replicating virus particles such as HVJ-E may resolve the safety issue of

The first non-replicating oncolytic virus (HVJ-E) is currently under evaluation in clinical studies. Proof-of-concept data for non-replicating viruses in both clinical and non-clinical studies are necessary for further development of this approach. Osaka University Hospital is currently conducting two phase I/IIa studies: one for advanced melanoma and another for CRPC [55, 56]. The results of these studies will reveal the safety, efficacy, and optimal dosage

Combination treatment may be an effective approach to increase efficacy [97]. Indeed, an increase in therapeutic efficacy has been reported for virotherapies combined with photody‐ namic therapy [98, 99], radiotherapy [100], chemotherapy [101, 102], or gene therapy [103]. Kiyohara and Kaneda reported that combination of the non-replicating virus (HVJ-E) and gene therapy (IL-12) increased efficacy in a murine model of melanoma [104]. Furthermore, it was reported that a combination of non-replicating virus (HVJ-E) and chemotherapy [bleomycin or cis-diamminedichloroplatinum (CDDP)] increased efficacy in murine models of colon and

Technologies for systemic administration and targeting for HVJ-E are under development. The HN protein of HVJ-E has hemagglutinating activity and causes agglutination and lysis of erythrocytes *in vitro*. Currently, inactivation of the HN protein, decreased expression of the HN protein, and "masking" with platelets is being developed for intravenous injection of HVJ-E. Targeting after the intravenous injection is also important for systemic delivery. The addition of transferrin, a single chain antibody, or platelets have been suggested as suitable

The selection of viruses, or viral strains for the preparation of non-replicating oncolytic viruses is also important for obtaining higher efficacy because the level of immune response is dependent on the selection of virus strains [106]. A number of replicating oncolytic viruses are currently under clinical development [35, 97]. Therefore, it may be feasible to select a suitable virus for therapeutic application from the oncolytic viruses that have been developed [106, 107]. The tropism and complement resistance features of each virus should be considered for

HVJ-E is the only non-replicating oncolytic virus currently undergoing clinical investigation. These studies establish a new strategy for the virotherapy and gene therapy fields. The primary

goal is to provide a novel approach for improving cancer therapy.

regimen necessary for phase II study or randomized, double blind phase III study.

conventional virotherapy and provide a new strategy in cancer treatment.

our safety studies.

170 Novel Gene Therapy Approaches

**9. Future perspectives**

bladder cancers [78, 105].

modifiers for HVJ-E.

targeting and stabilization in serum.

Conventional cancer therapies suffer from one paradox: although chemotherapeutic agents strongly kill cancer cells and decrease tumor volume, they simultaneously suppress the immune system. Chemotherapy frequently results in tumor relapse because residual cancer cells and cancer stem cells escape immune responses. In contrast, immune therapies including therapeutic cancer vaccines, effectively induce cancer immunity, but possess weak cytotoxic activity against cancer cells. Therefore, these treatments usually show weak efficacy. It has been reported that cancer is able to progress even after the activation and proliferation of cancer-specific cytotoxic T lymphocytes.

Virotherapy is predicted to become an alternative approach to obtain a model cancer therapy because it generally displays both oncolytic and immunostimulatory activities. However, the major drawback associated with current virotherapy is safety concerns. Virotherapy using a non-replicating virus is a new approach aimed at resolving safety issues. Thus, it is expected to become a novel concept for cancer therapy in the near future.

## **Acknowledgements**

We appreciate T. Yamazaki for helpful discussions. We also appreciate H. Ueda and S. Ishikawa for their excellent administrative supports.

This study was supported by the advanced research for medical products Mining Program of the National Institute of Biomedical Innovation (NIBIO).

## **Author details**

Toshihiro Nakajima1 , Toshimitsu Itai1 , Hiroshi Wada1 , Toshie Yamauchi1 , Eiji Kiyohara2,3 and Yasufumi Kaneda2

1 GenomIdea, Inc., Midorigaoka, Ikeda, Osaka, Japan

2 Division of Gene Therapy Science, Department of Molecular Therapeutics, Graduate School of Medicine, Osaka University, Yamada-oka, Suita, Osaka, Japan

3 Department of Dermatology, Graduate School of Medicine, Osaka University, Yamadaoka, Suita, Osaka, Japan

T. Nakajima is a CEO of GenomIdea. T. Itai, H. Wada, and T. Yamauchi are employees of GenomIdea. The remaining authors have no conflicts of interest.

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[103] Hwang TH., Moon A., Burke J., Ribas A., Stephenson J., Breitbach CJ., Daneshmand M., De Silva N., Parato K., Diallo JS., Lee YS., Liu TC., Bell JC. and Kirn DH., A mech‐ anistic proof-of-concept clinical trial with JX-594, a targeted multi-mechanistic onco‐ lytic poxvirus, in patients with metastatic melanoma., Mol Ther. 2011 Oct;19(10): 1913-1922. doi: 10.1038/mt.2011.132. Epub 2011 Jul 19.

[92] Nishikawa H. and Sakaguchi S., Regulatory T cells in tumor immunity., Int J Cancer.

[93] Jacobs JF., Nierkens S., Figdor CG., de Vries IJ., Adema GJ., Regulatory T cells in mel‐ anoma: the final hurdle towards effective immunotherapy?, Lancet Oncol. 2012 Jan;

[94] Le DT. and Jaffee EM., Regulatory T-cell modulation using cyclophosphamide in vaccine approaches: a current perspective., Cancer Res. 2012 Jul 15;72(14):3439-3444.

[95] Suzuki H., Kurooka M., Hiroaki Y., Fujiyoshi Y., Kaneda Y., Sendai virus F glycopro‐ tein induces IL-6 production in dendritic cells in a fusion-independent manner.,

[96] Chang A. and Dutch RE., Paramyxovirus fusion and entry: multiple paths to a com‐

[97] Galanis E., Cancer: Tumour-fighting virus homes in., Nature. 2011 Aug 31;477(7362):

[98] Fujii H., Matsuyama A., Komoda H., Sasai M., Suzuki M., Asano T., Doki Y., Kirihata M., Ono K., Tabata Y., Kaneda Y., Sawa Y. and Lee CM., Cationized gelatin-HVJ en‐ velope with sodium borocaptate improved the BNCT efficacy for liver tumors in

[99] Sakai M., Fujimoto N., Ishii K., Nakamura H., Kaneda Y. and Awazu K., In vitro in‐ vestigation of efficient photodynamic therapy using a nonviral vector; hemaggluti‐

[100] Harrington KJ., Karapanagiotou EM., Roulstone V., Twigger KR., White CL., Vidal L., Beirne D., Prestwich R., Newbold K., Ahmed M., Thway K., Nutting CM., Coffey M., Harris D., Vile RG., Pandha HS., Debono JS. and Melcher AA., Two-stage phase I dose-escalation study of intratumoral reovirus type 3 dearing and palliative radio‐ therapy in patients with advanced cancers., Clin Cancer Res. 2010 Jun 1;16(11):

[101] Lolkema MP., Arkenau HT., Harrington K., Roxburgh P., Morrison R., Roulstone V., Twigger K., Coffey M., Mettinger K., Gill G., Evans TR. and de Bono JS., A phase I study of the combination of intravenous reovirus type 3 Dearing and gemcitabine in patients with advanced cancer., Clin Cancer Res. 2011 Feb 1;17(3):581-588. Epub 2010

[102] Kanai R., Rabkin SD., Yip S., Sgubin D., Zaupa CM., Hirose Y., Louis DN., Wakimoto H. and Martuza RL., Oncolytic virus-mediated manipulation of DNA damage re‐ sponses: synergy with chemotherapy in killing glioblastoma stem cells. J Natl Cancer

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180 Novel Gene Therapy Approaches

Epub 2012 Jul 3.


**Chapter 9**

**Sendai Virus-Based Oncolytic Gene Therapy**

The first gene therapy that was used on humans was performed for adenosine deaminase (ADA) deficiency in 1990 [1]. In the early days of gene therapy, it was thought to be a break‐ through in the treatment of a number of human diseases including cancer, cardiovascular disease, genetic disease, and so on. However, two severe adverse events in gene therapy served as triggers for the rethinking of the safety of gene therapy. The use of adenoviral gene therapy in an 18-year-old with an inherited enzyme deficiency at the University of Pennsylvania's Institute for Human Gene Therapy resulted in the death of the patient 4 days after the injection of the vectors into the liver in 1999 [2]. The second accident involved derived carcinogenesis that was caused by gene therapy that was performed to treat severe combined immunodefi‐ ciency-X1 in 1999 [3]. On the other hand, nonviral vectors (e.g., plasmids, liposomes, polymers, and so on.) have been developed because of these safety concerns. However, the low effec‐

The key to gene therapy is safety and effectiveness. Sendai virus (SeV) vectors are able to overcome many of these problems related to gene therapy. The advantages of SeV in terms of gene therapy are the following: 1) it is nonpathogenic to human, 2) it has a high efficiency of

First, we would like to discuss the nonpathogenicity of SeV. The vector that is most used in gene therapy clinical trials is the Adenovirus, which is followed by the Retrovirus [4]. Ade‐ novirus infections in humans cause pneumonia, bronchitis, croup, and so on. Retrovirus infections are one of the causes of human carcinogenesis or immunodeficiency. These vectors are pathogenic to human beings. Moreover, the infection of these vectors to human cells is associated with the risk of viral gene integration into the human genome, which contributes

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

© 2013 Morodomi 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.

© 2013 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

tiveness of nonviral vectors in gene transduction remains a serious problem.

infection, and 3) it results in high levels of gene expression.

Yosuke Morodomi, Makoto Inoue,

http://dx.doi.org/10.5772/55328

**1. Introduction**

Mamoru Hasegawa, Tatsuro Okamoto,

Yoshihiko Maehara and Yoshikazu Yonemitsu

Additional information is available at the end of the chapter

## **Sendai Virus-Based Oncolytic Gene Therapy**

Yosuke Morodomi, Makoto Inoue, Mamoru Hasegawa, Tatsuro Okamoto, Yoshihiko Maehara and Yoshikazu Yonemitsu

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/55328

## **1. Introduction**

The first gene therapy that was used on humans was performed for adenosine deaminase (ADA) deficiency in 1990 [1]. In the early days of gene therapy, it was thought to be a break‐ through in the treatment of a number of human diseases including cancer, cardiovascular disease, genetic disease, and so on. However, two severe adverse events in gene therapy served as triggers for the rethinking of the safety of gene therapy. The use of adenoviral gene therapy in an 18-year-old with an inherited enzyme deficiency at the University of Pennsylvania's Institute for Human Gene Therapy resulted in the death of the patient 4 days after the injection of the vectors into the liver in 1999 [2]. The second accident involved derived carcinogenesis that was caused by gene therapy that was performed to treat severe combined immunodefi‐ ciency-X1 in 1999 [3]. On the other hand, nonviral vectors (e.g., plasmids, liposomes, polymers, and so on.) have been developed because of these safety concerns. However, the low effec‐ tiveness of nonviral vectors in gene transduction remains a serious problem.

The key to gene therapy is safety and effectiveness. Sendai virus (SeV) vectors are able to overcome many of these problems related to gene therapy. The advantages of SeV in terms of gene therapy are the following: 1) it is nonpathogenic to human, 2) it has a high efficiency of infection, and 3) it results in high levels of gene expression.

First, we would like to discuss the nonpathogenicity of SeV. The vector that is most used in gene therapy clinical trials is the Adenovirus, which is followed by the Retrovirus [4]. Ade‐ novirus infections in humans cause pneumonia, bronchitis, croup, and so on. Retrovirus infections are one of the causes of human carcinogenesis or immunodeficiency. These vectors are pathogenic to human beings. Moreover, the infection of these vectors to human cells is associated with the risk of viral gene integration into the human genome, which contributes

© 2013 Morodomi 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. © 2013 The Author(s). Licensee InTech. This chapter is 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.

to gene mutations or structural changes to chromosomes. On the other hand, SeV was originally discovered as the cause of pneumonia in rodents. Because the SeV gene exists as RNA in the cell cytoplasm throughout the life cycle of the virus from infection of the target cells to viral budding, no genetic toxicities have been confirmed (Figure 1). For these reasons, the risk of pathogenicity to humans is surprisingly low, and its safety has already been assured when it is used as a gene therapy drug.

**2. Structure of SeV**

SeV is a negative-sense, single-stranded RNA virus of the Paramyxoviridae family. SeV genome consists of 15,384 base pairs and encodes the following 6 genes: nucleocapsid protein (N), which binds to RNA; phosphoprotein (P), which forms a small subunit of RNA polymer‐ ase; matrix protein (M), which lines the inside of viral particles; fusion protein (F), which is important for host cell penetration; HN, which is involved in the attachment to the host cells; and large protein (L), which forms a big subunit of RNA polymerase. The HN protein serves in the attachment to target cells by recognizing sialic acid on the cell surface. The F protein is cleaved through conformational change into F1 and F2, and this is triggered by local enzymatic activity, particularly that of trypsin. The cleaved F protein penetrates into the cellular mem‐

Sendai Virus-Based Oncolytic Gene Therapy http://dx.doi.org/10.5772/55328 185

Yoshio Okada discovered the phenomenon that SeV causes the fusion of Ehrlich's tumor cells [11]. Paramixoviridae family members including SeV have the property of cell-to-cell fusion. The fusion process, which occurs between the viral envelope and cells, may also occur between adjacent viral-infected cells when the Fusion protein is expressed on the cell surface, thus causing extensive membrane fusion and the formation of a syncytium. Cell-to-cell fusion

We have applied these fusogenic activity characteristics of SeV to cancer therapy. It is impor‐ tant in gene therapy for the treatment of cancer that 1) tumor-specific infections are enhanced

brane, which induces the membrane and the viral envelope to merge [10].

**Figure 2.** Schematic model and electron microscope photograph of SeV.

induces apoptotic signals, resulting in cell death [12,13].

**3. Fusogenic activity of SeV**

**3.1. Oncolytic virotherapy with SeV**

Second, we would like to discuss the features of SeV with respect to its high efficiency of infection. Hemagglutinin-neuraminidase (HN) proteins recognize sialic acid, which is expressed as a glycoprotein or glycolipid on the cell surface. Sialic acid is widely expressed in the cells of mammals or other species, and its expression enables a large variety of SeV infections, such as those in the airway epithelium [5], saphenous vein [6], or in a variety of tumors [7,8]. In contrast, adenoviruses require the coxsackievirus–adenovirus receptor (CAR) to attach to cells, and the CAR is selectively expressed among cells, which limits its infection. In addition, adenovirus-mediated gene transfer requires a relatively long exposure time to reach maximum gene transfer efficiency, and this is a common characteristic of other currently available vectors. In contrast, SeV can infect cells in a minute or less [6]. Moreover, SeV can infect dividing cells or nondividing cells.

The third feature of SeV is its high level of gene expression. SeV has a dramatically high gene transfer efficiency compared with adenovirus vectors [6,9]. Namely, SeV can efficiently load a therapeutic gene, and gene therapy using SeV is able to decrease the amount of administra‐ tion vectors in the clinical setting, resulting in a lower risk of gene therapy.

We examinedwhetherwe canapplytheseoutstandingcharacteristicsofSeVtothegene therapy of cancer. In this chapter, we describe the history of the investigations of oncolytic gene therapy using SeV, the present developmental status of this therapy, and the future of this therapy.

**Figure 1.** Redrawn with permission from Kinoh H et al., Front Biosci. 2008 Jan 1;13:2327-34. The life cycle of the Sen‐ dai Virus (SeV) and other vectors. SeV, Measles, and the Newcastle Disease Virus (NDV) do not result in chromosomal integration, whereas other existing vectors do. AAV, adeno-associated virus.

## **2. Structure of SeV**

to gene mutations or structural changes to chromosomes. On the other hand, SeV was originally discovered as the cause of pneumonia in rodents. Because the SeV gene exists as RNA in the cell cytoplasm throughout the life cycle of the virus from infection of the target cells to viral budding, no genetic toxicities have been confirmed (Figure 1). For these reasons, the risk of pathogenicity to humans is surprisingly low, and its safety has already been assured

Second, we would like to discuss the features of SeV with respect to its high efficiency of infection. Hemagglutinin-neuraminidase (HN) proteins recognize sialic acid, which is expressed as a glycoprotein or glycolipid on the cell surface. Sialic acid is widely expressed in the cells of mammals or other species, and its expression enables a large variety of SeV infections, such as those in the airway epithelium [5], saphenous vein [6], or in a variety of tumors [7,8]. In contrast, adenoviruses require the coxsackievirus–adenovirus receptor (CAR) to attach to cells, and the CAR is selectively expressed among cells, which limits its infection. In addition, adenovirus-mediated gene transfer requires a relatively long exposure time to reach maximum gene transfer efficiency, and this is a common characteristic of other currently available vectors. In contrast, SeV can infect cells in a minute or less [6]. Moreover, SeV can

The third feature of SeV is its high level of gene expression. SeV has a dramatically high gene transfer efficiency compared with adenovirus vectors [6,9]. Namely, SeV can efficiently load a therapeutic gene, and gene therapy using SeV is able to decrease the amount of administra‐

We examinedwhetherwe canapplytheseoutstandingcharacteristicsofSeVtothegene therapy of cancer. In this chapter, we describe the history of the investigations of oncolytic gene therapy using SeV, the present developmental status of this therapy, and the future of this therapy.

**Figure 1.** Redrawn with permission from Kinoh H et al., Front Biosci. 2008 Jan 1;13:2327-34. The life cycle of the Sen‐ dai Virus (SeV) and other vectors. SeV, Measles, and the Newcastle Disease Virus (NDV) do not result in chromosomal

integration, whereas other existing vectors do. AAV, adeno-associated virus.

tion vectors in the clinical setting, resulting in a lower risk of gene therapy.

when it is used as a gene therapy drug.

184 Novel Gene Therapy Approaches

infect dividing cells or nondividing cells.

SeV is a negative-sense, single-stranded RNA virus of the Paramyxoviridae family. SeV genome consists of 15,384 base pairs and encodes the following 6 genes: nucleocapsid protein (N), which binds to RNA; phosphoprotein (P), which forms a small subunit of RNA polymer‐ ase; matrix protein (M), which lines the inside of viral particles; fusion protein (F), which is important for host cell penetration; HN, which is involved in the attachment to the host cells; and large protein (L), which forms a big subunit of RNA polymerase. The HN protein serves in the attachment to target cells by recognizing sialic acid on the cell surface. The F protein is cleaved through conformational change into F1 and F2, and this is triggered by local enzymatic activity, particularly that of trypsin. The cleaved F protein penetrates into the cellular mem‐ brane, which induces the membrane and the viral envelope to merge [10].

**Figure 2.** Schematic model and electron microscope photograph of SeV.

## **3. Fusogenic activity of SeV**

Yoshio Okada discovered the phenomenon that SeV causes the fusion of Ehrlich's tumor cells [11]. Paramixoviridae family members including SeV have the property of cell-to-cell fusion. The fusion process, which occurs between the viral envelope and cells, may also occur between adjacent viral-infected cells when the Fusion protein is expressed on the cell surface, thus causing extensive membrane fusion and the formation of a syncytium. Cell-to-cell fusion induces apoptotic signals, resulting in cell death [12,13].

#### **3.1. Oncolytic virotherapy with SeV**

We have applied these fusogenic activity characteristics of SeV to cancer therapy. It is impor‐ tant in gene therapy for the treatment of cancer that 1) tumor-specific infections are enhanced and 2) secondary infection is prevented. In order to obtain these properties, we modified the SeV gene by altering the F gene and deleting the M gene.

First, we would like to describe the background of the tumor-specificity abilities of BioknifeTM. We focused our attention on the urokinase-type plasminogen activator (uPA). uPA is a trypsinlike serine protease that is synthesized and secreted as pro-uPA, which has little or no proteolytic activity [14]. The urokinase-type plasminogen activator receptor (uPAR) is a 55– 60-kD glycoprotein that is anchored on the cell surface by a glycosyl-phosphatidylinositol linkage [15,16]. uPA binds to uPAR with high affinity. uPAR anchors uPA to the cell membrane and converts pro-uPA to active uPA, thereby localizing the proteolytic activity around the cell surface [17]. Activated uPA plays an important role in extracellular matrix degradation and results in tumor invasion and metastasis [18]. A wide variety of cancers overexpress uPAR and are associated with poor prognosis [19-23]. However, uPAR is expressed less in normal tissue except for in unusual circumstances such as inflammation [21,22,24].

protein is cleaved, and the contiguous cells go into chain fusion reaction. These completely inhibited secondary viral particles served not only to promote fusion efficiency, but also to

**Figure 4.** Modification of the cleavage site of the SeV-F protein, which is sensitive to the urokinase-type plasminogen

Sendai Virus-Based Oncolytic Gene Therapy http://dx.doi.org/10.5772/55328 187

activator, and truncation of the cytoplasmic tail resulted in optimization of the cell-fusion activity.

I would like to emphasize that the oncolysis that is mediated by BioKnifeTM is entirely different from conventional oncolysis. Oncolytic viruses, which is a term used to describe most viruses such as adenovirus or herpes simplex viruses, provoke the disruption of infected cells with a large number of secondary viral particles. However, the production of secondary viruses by these oncolytic viruses may limit gene therapy with respect to safety. Thus, a large number of viruses may evoke viremia and induce uncontrollable inflammatory reactions. In contrast, the oncolysis that is caused by BioKnifeTM is cell death that is mediated by caspase-dependent apoptosis [13,25]. There is no need to worry about viremia, even if an explosive spread of

**Figure 5.** Gene structure of recombinant SeV. Wild type SeV is pictured at the top of the figure, which is followed by the Mgene deleted SeV with a substitutive load of the Green fluorescent protein (GFP) gene (rSeV/dM-GFP) in the middle. Final‐

To test the cytotoxicity of BioknifeTM against tumor cells, we conducted an *in vitro* infection experiment in many types of tumor cells. Cell fusion and cell death were observed in many tumor types, and this was dependent on the uPA activity of the tumor cells. As expected,

ly, at the bottom, the F gene of rSeV/dM-GFP is transformed to a uPA-sensitive sequence (BioKnifeTM-GFP).

improve gene therapy safety.

infection is observed.

**4. The potential of BioKnifeTM**

**Figure 3.** Schematic model of the urokinase activation system.

Bound and inactive pro-urokinase-type plasminogen activator (uPA) is converted to active uPA, inducing extracellular matrix (ECM) degradation. As a result, tumor invasion and metastasis are promoted.

Given that uPA activity is high around tumor cells and low around nontumor cells, we converted the F gene, which is specific to Trypsin in the wild type SeV, to a uPA-specific sequence (Figure 4). As a result, we succeeded in fusing infected cells to tumor cells only. Moreover, to optimize the fusion ability, the F gene was given an additional change, which truncated the cytoplasmic domain of the F protein (Figure 4). This genetic modification resulted in more efficient fusogenic abilities [8].

Second, we would like to describe the background of the deleting of the M gene (Figure 5). Deletion of the M gene resulted in avoidance of the budding of secondary viral particles because the M protein is indispensable for the budding of SeV. Consequently, the F proteins and HN proteins, which are expected to be the second particles in the viral spike, accumulate on the infected cell surface. If uPA is activated around the cell surface, the recombinant F


**Figure 4.** Modification of the cleavage site of the SeV-F protein, which is sensitive to the urokinase-type plasminogen activator, and truncation of the cytoplasmic tail resulted in optimization of the cell-fusion activity.

protein is cleaved, and the contiguous cells go into chain fusion reaction. These completely inhibited secondary viral particles served not only to promote fusion efficiency, but also to improve gene therapy safety.

I would like to emphasize that the oncolysis that is mediated by BioKnifeTM is entirely different from conventional oncolysis. Oncolytic viruses, which is a term used to describe most viruses such as adenovirus or herpes simplex viruses, provoke the disruption of infected cells with a large number of secondary viral particles. However, the production of secondary viruses by these oncolytic viruses may limit gene therapy with respect to safety. Thus, a large number of viruses may evoke viremia and induce uncontrollable inflammatory reactions. In contrast, the oncolysis that is caused by BioKnifeTM is cell death that is mediated by caspase-dependent apoptosis [13,25]. There is no need to worry about viremia, even if an explosive spread of infection is observed.

**Figure 5.** Gene structure of recombinant SeV. Wild type SeV is pictured at the top of the figure, which is followed by the Mgene deleted SeV with a substitutive load of the Green fluorescent protein (GFP) gene (rSeV/dM-GFP) in the middle. Final‐ ly, at the bottom, the F gene of rSeV/dM-GFP is transformed to a uPA-sensitive sequence (BioKnifeTM-GFP).

## **4. The potential of BioKnifeTM**

and 2) secondary infection is prevented. In order to obtain these properties, we modified the

First, we would like to describe the background of the tumor-specificity abilities of BioknifeTM. We focused our attention on the urokinase-type plasminogen activator (uPA). uPA is a trypsinlike serine protease that is synthesized and secreted as pro-uPA, which has little or no proteolytic activity [14]. The urokinase-type plasminogen activator receptor (uPAR) is a 55– 60-kD glycoprotein that is anchored on the cell surface by a glycosyl-phosphatidylinositol linkage [15,16]. uPA binds to uPAR with high affinity. uPAR anchors uPA to the cell membrane and converts pro-uPA to active uPA, thereby localizing the proteolytic activity around the cell surface [17]. Activated uPA plays an important role in extracellular matrix degradation and results in tumor invasion and metastasis [18]. A wide variety of cancers overexpress uPAR and are associated with poor prognosis [19-23]. However, uPAR is expressed less in normal

Bound and inactive pro-urokinase-type plasminogen activator (uPA) is converted to active uPA, inducing extracellular matrix (ECM) degradation. As a result, tumor invasion and

Given that uPA activity is high around tumor cells and low around nontumor cells, we converted the F gene, which is specific to Trypsin in the wild type SeV, to a uPA-specific sequence (Figure 4). As a result, we succeeded in fusing infected cells to tumor cells only. Moreover, to optimize the fusion ability, the F gene was given an additional change, which truncated the cytoplasmic domain of the F protein (Figure 4). This genetic modification

Second, we would like to describe the background of the deleting of the M gene (Figure 5). Deletion of the M gene resulted in avoidance of the budding of secondary viral particles because the M protein is indispensable for the budding of SeV. Consequently, the F proteins and HN proteins, which are expected to be the second particles in the viral spike, accumulate on the infected cell surface. If uPA is activated around the cell surface, the recombinant F

tissue except for in unusual circumstances such as inflammation [21,22,24].

SeV gene by altering the F gene and deleting the M gene.

186 Novel Gene Therapy Approaches

**Figure 3.** Schematic model of the urokinase activation system.

resulted in more efficient fusogenic abilities [8].

metastasis are promoted.

To test the cytotoxicity of BioknifeTM against tumor cells, we conducted an *in vitro* infection experiment in many types of tumor cells. Cell fusion and cell death were observed in many tumor types, and this was dependent on the uPA activity of the tumor cells. As expected, nontumor cells were not injured [8]. Next, we tested the antitumor efficacy of BioKnifeTM *in vivo*. Cells of the human prostate tumor cell line, PC3, were implanted into a nude mouse, and then BioKnifeTM was injected into the tumor. BioKnifeTM-infected tumor presented GFP fluorescence from day 1 with the maximum GFP intensity on day 7. A microscopic examination of the subcutaneous tumor on day 16 showed that the tumor cells had been eradicated.

that uPAR is necessary for the cell fusion that is mediated by BioKnifeTM, even if uPA is not

Sendai Virus-Based Oncolytic Gene Therapy http://dx.doi.org/10.5772/55328 189

**Figure 7.** A schematic model of the induction of uPA expression through BioknifeTM infection. Retinoic acid-inducible

In summary, we have demonstrated the potential and the mechanisms of BioknifeTM with numerous fundamental experiments. Admittedly, BioKnifeTM has no ability to infect distant tumor lesions or metastatic lesions through intravascular routes because of its instability in the blood. However, the ability of local infections of the tumor cells and the killing power are outstanding. Next, we explored diseases in which gene therapy using BioKnifeTM can be

MPM is a malignancy that arises from the pleural cavity. Because MPM has a long latency period after the inhalation of asbestos [26], the number of deaths by MPM is expected to increase in the next several decades, reflecting the past usage of asbestos [27]. MPM is highly malignant due to its intractableness to treatment. Although a large number of studies have examined approaches to MPM therapy, no marked progress has appeared to overcome this disease. The median overall survival rate is less than 30 months, even if it is treated with multimodality therapy [28,29]. Thus, novel therapeutics are highly desired. MPM spreads widely throughout the pleural cavity and rarely metastasizes to distant sites in the earlier stage. In addition, MPM expresses high levels of uPAR. These characteristics suggest favorable conditions for gene therapy with BioknifeTM. Thus, we explored the possibility of treatment

applied. We examined malignant mesothelioma (MPM) in particular.

gene-1 (RIG-I) activation promotes the fusion cascade.

with BioknifeTM in this disease.

expressed in tumor cells, and BioKnifeTM infection itself facilitates the fusion activity.

**Figure 6.** A time-course analysis of BioKnifeTM infection of subcutaneously inoculated PC3 tumor cells in a nude mouse. Photomicrographs of tissue specimens on day 16 are presented in the bottom two panels.

## **5. BioKnifeTM infections create a positive feedback loop of cell-to-cell fusion**

The cell-to-cell fusion that is mediated by BioknifeTM provided another effect. We demonstrat‐ ed that BioknifeTM infections induce simultaneous activation of the uPA expression. In addition, we found that the induction of uPA is mediated by the retinoic acid-inducible gene-1 (RIG-I), which is a viral RNA sensor that is activated by BioknifeTM infection and which activates the nuclear factor-kappa B (NF-κB) signaling pathway [25]. Activated RIG-I upre‐ gulates levels of uPA expression through the downstream protein, NFκB. Extracellularly secreted uPA binds uPAR on the tumor surface, which increases the activity of uPA. As a result, the F protein on BioKnifeTM-infected cells is activated and cleaved, resulting in cell fusion. It is possible that BioKnifeTM results in self-induced fusion (Figure 7). This phenomenon suggests that uPAR is necessary for the cell fusion that is mediated by BioKnifeTM, even if uPA is not expressed in tumor cells, and BioKnifeTM infection itself facilitates the fusion activity.

nontumor cells were not injured [8]. Next, we tested the antitumor efficacy of BioKnifeTM *in vivo*. Cells of the human prostate tumor cell line, PC3, were implanted into a nude mouse, and then BioKnifeTM was injected into the tumor. BioKnifeTM-infected tumor presented GFP fluorescence from day 1 with the maximum GFP intensity on day 7. A microscopic examination of the subcutaneous tumor on day 16 showed that the tumor cells had been eradicated.

**Figure 6.** A time-course analysis of BioKnifeTM infection of subcutaneously inoculated PC3 tumor cells in a nude

**5. BioKnifeTM infections create a positive feedback loop of cell-to-cell**

The cell-to-cell fusion that is mediated by BioknifeTM provided another effect. We demonstrat‐ ed that BioknifeTM infections induce simultaneous activation of the uPA expression. In addition, we found that the induction of uPA is mediated by the retinoic acid-inducible gene-1 (RIG-I), which is a viral RNA sensor that is activated by BioknifeTM infection and which activates the nuclear factor-kappa B (NF-κB) signaling pathway [25]. Activated RIG-I upre‐ gulates levels of uPA expression through the downstream protein, NFκB. Extracellularly secreted uPA binds uPAR on the tumor surface, which increases the activity of uPA. As a result, the F protein on BioKnifeTM-infected cells is activated and cleaved, resulting in cell fusion. It is possible that BioKnifeTM results in self-induced fusion (Figure 7). This phenomenon suggests

mouse. Photomicrographs of tissue specimens on day 16 are presented in the bottom two panels.

**fusion**

188 Novel Gene Therapy Approaches

**Figure 7.** A schematic model of the induction of uPA expression through BioknifeTM infection. Retinoic acid-inducible gene-1 (RIG-I) activation promotes the fusion cascade.

In summary, we have demonstrated the potential and the mechanisms of BioknifeTM with numerous fundamental experiments. Admittedly, BioKnifeTM has no ability to infect distant tumor lesions or metastatic lesions through intravascular routes because of its instability in the blood. However, the ability of local infections of the tumor cells and the killing power are outstanding. Next, we explored diseases in which gene therapy using BioKnifeTM can be applied. We examined malignant mesothelioma (MPM) in particular.

MPM is a malignancy that arises from the pleural cavity. Because MPM has a long latency period after the inhalation of asbestos [26], the number of deaths by MPM is expected to increase in the next several decades, reflecting the past usage of asbestos [27]. MPM is highly malignant due to its intractableness to treatment. Although a large number of studies have examined approaches to MPM therapy, no marked progress has appeared to overcome this disease. The median overall survival rate is less than 30 months, even if it is treated with multimodality therapy [28,29]. Thus, novel therapeutics are highly desired. MPM spreads widely throughout the pleural cavity and rarely metastasizes to distant sites in the earlier stage. In addition, MPM expresses high levels of uPAR. These characteristics suggest favorable conditions for gene therapy with BioknifeTM. Thus, we explored the possibility of treatment with BioknifeTM in this disease.

### **6. Antitumor effects of BioknifeTM in a MPM orthotopic murine model**

cells, and local control is its primary advantage. Moreover, we would like to emphasize the accessibility of BioKnifeTM in treatments of MPM. We suggest that video-assisted thoracoscopic surgery (VATS) and chest tubes are the best way to administer BioKnifeTM. MPM often forms nodular lesions on the pleural surface. For these targets, it is best to inject BioKnifeTM intratu‐ morally with VATS. In addition, because MPM frequently produces malignant pleural effusion [30], most cases need chest tubes. In these cases, it is convenient to administer BioKnifeTM intrapleurally through the chest tube. This access route enables us to administer BioKnifeTM repeatedly and safely because multiple cycles of the administration of BioKnifeTM are more effective (Figure 8). Based on these results, we are planning a clinical trial using BioKnifeTM to

Sendai Virus-Based Oncolytic Gene Therapy http://dx.doi.org/10.5772/55328 191

We described above the developmental history and the usefulness of BioKnifeTM. It should be noted that BioKnifeTM has the ability to load other treatment genes, cytokines, tumor sup‐ pressing genes, or cancer antigens. Amazingly, the cytotoxicity of BioKnifeTM depends solely on its fusion ability. In other words, there is still considerable room for improvements of this treatment modality. Moreover, there is room for further examination of the relationship between BioKnifeTM and cancer immunity. Viral oncolysate is applied as a cancer vaccine in cancer immunotherapy. BioKnifeTM-lysed tumor cells make an extract of tumor cells. The extract contains both cancer cell proteins and virus proteins. This extract may facilitate the antigen presentation activity to dendritic cells or activate natural killer cells. Further studies

We developed BioKnifeTM, which is a uPA activity-dependent oncolytic SeV vector. This promising oncolytic vector, BioKnifeTM, may overcome the limitations of current gene therapy vectors. Further studies are needed to examine whether this new modality is effective in the

We thank Chie Arimatsu, Aki Furuya, and Ryoko Nakamura for their assistance with the animal experiments, and Drs. Tagawa, Kanaya, Ban, and Hironaka for their excellent technical assistance in the vector construction and large-scale production. This work was supported in part by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to Y.M. and Y.Y.). Y.Y. is a member of the Scientific Advisory Board of DNAVEC

clinical setting as a therapeutic alternative for this intractable disease.

Corporation. The other authors declare no conflicts of interest.

treat MPM.

**7. BioKnifeTM in the future**

are necessary to confirm this fact.

**8. Conclusion**

**Acknowledgements**

To confirm the antitumor effects in MPM, we first established two independent human orthotopic murine models. The human MPM cell lines, MSTO-211H (biphasic subtype) and H226 (epithelioid subtype), were injected into the thoracic cavity of Balb/c nu/nu mice. The tumor cells spread and formed multiple nodules in the thoracic cavity, which is similar to the pathology observed during the clinical course of human MPM. Untreated mice eventually died due to MPM progression. We assessed the performance of BioknifeTM in these MPM murine models. MPM-bearing mice were treated with BioKnifeTM at the following frequencies: once, three times, or six times. The result was that, in both murine models, BioKnifeTM-treated cohort exhibited a significantly prolonged survival compared with the control group. The greater the number of BioKnifeTM injection times, the higher the survival rate. In the group receiving 6 injections of BioKnifeTM, long-term survivors were observed.

**Figure 8.** Kaplan–Meier survival plot of BALB/c nude mice bearing H226 or MSTO-211H tumors that were left untreat‐ ed or treated with phosphate-buffered saline or BioKnifeTM in multicycle treatments. No treat indicates no treatment, PBS indicates phosphate- buffered saline treatment, and BK indicates BioKnifeTM treatment.

Considering these findings, MPM is a good target for BioKnifeTM treatment because the biological characteristics of MPM match the characteristics of BioKnifeTM. MPM spreads in the thoracic cavity and rarely develops distant metastasis. BioKnifeTM can spread to adjacent tumor cells, and local control is its primary advantage. Moreover, we would like to emphasize the accessibility of BioKnifeTM in treatments of MPM. We suggest that video-assisted thoracoscopic surgery (VATS) and chest tubes are the best way to administer BioKnifeTM. MPM often forms nodular lesions on the pleural surface. For these targets, it is best to inject BioKnifeTM intratu‐ morally with VATS. In addition, because MPM frequently produces malignant pleural effusion [30], most cases need chest tubes. In these cases, it is convenient to administer BioKnifeTM intrapleurally through the chest tube. This access route enables us to administer BioKnifeTM repeatedly and safely because multiple cycles of the administration of BioKnifeTM are more effective (Figure 8). Based on these results, we are planning a clinical trial using BioKnifeTM to treat MPM.

## **7. BioKnifeTM in the future**

**6. Antitumor effects of BioknifeTM in a MPM orthotopic murine model**

injections of BioKnifeTM, long-term survivors were observed.

190 Novel Gene Therapy Approaches

To confirm the antitumor effects in MPM, we first established two independent human orthotopic murine models. The human MPM cell lines, MSTO-211H (biphasic subtype) and H226 (epithelioid subtype), were injected into the thoracic cavity of Balb/c nu/nu mice. The tumor cells spread and formed multiple nodules in the thoracic cavity, which is similar to the pathology observed during the clinical course of human MPM. Untreated mice eventually died due to MPM progression. We assessed the performance of BioknifeTM in these MPM murine models. MPM-bearing mice were treated with BioKnifeTM at the following frequencies: once, three times, or six times. The result was that, in both murine models, BioKnifeTM-treated cohort exhibited a significantly prolonged survival compared with the control group. The greater the number of BioKnifeTM injection times, the higher the survival rate. In the group receiving 6

**Figure 8.** Kaplan–Meier survival plot of BALB/c nude mice bearing H226 or MSTO-211H tumors that were left untreat‐ ed or treated with phosphate-buffered saline or BioKnifeTM in multicycle treatments. No treat indicates no treatment,

Considering these findings, MPM is a good target for BioKnifeTM treatment because the biological characteristics of MPM match the characteristics of BioKnifeTM. MPM spreads in the thoracic cavity and rarely develops distant metastasis. BioKnifeTM can spread to adjacent tumor

PBS indicates phosphate- buffered saline treatment, and BK indicates BioKnifeTM treatment.

We described above the developmental history and the usefulness of BioKnifeTM. It should be noted that BioKnifeTM has the ability to load other treatment genes, cytokines, tumor sup‐ pressing genes, or cancer antigens. Amazingly, the cytotoxicity of BioKnifeTM depends solely on its fusion ability. In other words, there is still considerable room for improvements of this treatment modality. Moreover, there is room for further examination of the relationship between BioKnifeTM and cancer immunity. Viral oncolysate is applied as a cancer vaccine in cancer immunotherapy. BioKnifeTM-lysed tumor cells make an extract of tumor cells. The extract contains both cancer cell proteins and virus proteins. This extract may facilitate the antigen presentation activity to dendritic cells or activate natural killer cells. Further studies are necessary to confirm this fact.

## **8. Conclusion**

We developed BioKnifeTM, which is a uPA activity-dependent oncolytic SeV vector. This promising oncolytic vector, BioKnifeTM, may overcome the limitations of current gene therapy vectors. Further studies are needed to examine whether this new modality is effective in the clinical setting as a therapeutic alternative for this intractable disease.

## **Acknowledgements**

We thank Chie Arimatsu, Aki Furuya, and Ryoko Nakamura for their assistance with the animal experiments, and Drs. Tagawa, Kanaya, Ban, and Hironaka for their excellent technical assistance in the vector construction and large-scale production. This work was supported in part by a grant from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (to Y.M. and Y.Y.). Y.Y. is a member of the Scientific Advisory Board of DNAVEC Corporation. The other authors declare no conflicts of interest.

## **Author details**

Yosuke Morodomi1 , Makoto Inoue2 , Mamoru Hasegawa2 , Tatsuro Okamoto3 , Yoshihiko Maehara3 and Yoshikazu Yonemitsu4

1 Department of Thoracic Oncology, National Kyushu Cancer Center, Japan

2 DNAVEC Corporation, Japan

3 Department of Surgery and Science, Graduate School of Medicine, Kyushu University, Japan

http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?

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[9] Tokusumi, T, Iida, A, Hirata, T, Kato, A, Nagai, Y, & Hasegawa, M. Recombinant Sendai viruses expressing different levels of a foreign reporter gene. Virus Res.

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[11] Okada, Y, & Tadokoro, J. Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells. II. Quantitative analysis of giant polynuclear

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[15] Nielsen, L. S, Kellerman, G. M, Behrendt, N, Picone, R, Danø, K, & Blasi, F. A. Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J. Biol. Chem. (1988). Feb. 15;, 263(5),

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[19] Alpà zar-Alpà zar WNielsen BS, Sierra R, Illemann M, Ramà rez JA, Arias A, et al. Urokinase plasminogen activator receptor is expressed in invasive cells in gastric car‐ cinomas from high- and low-risk countries. Int. J. Cancer. (2010). Jan. 15;, 126(2),

[20] Pedersen, H, Brünner, N, Francis, D, Osterlind, K, Rønne, E, Hansen, H. H, et al. Prognostic impact of urokinase, urokinase receptor, and type 1 plasminogen activa‐

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4 R&D Laboratory for Innovative Biotherapeutics, Graduate School of Pharmaceutical Scien‐ ces, Kyushu University, Japan

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Yosuke Morodomi1

Yoshihiko Maehara3

2 DNAVEC Corporation, Japan

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**Gene Therapy for Cancer**

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**Chapter 10**

**Challenges in Advancing the Field**

**the Multi-Functional Nanocarriers**

Additional information is available at the end of the chapter

Azam Bolhassani and Tayebeh Saleh

tissue at an efficient dose [Scanlon, 2004].

http://dx.doi.org/10.5772/54862

**1. Introduction**

**of Cancer Gene Therapy: An Overview of**

Recent developments in molecular biology and cell biology have led to the discovery of novel genes and proteins having therapeutic potentials for various diseases including cancers. Based on these findings, novel categories of therapeutic biomacromolecules in‐ cluding genes, small interfering RNA (siRNAs), antisense oligonucleic acids, bioactive proteins and peptides have been developed. These macromolecules can be more advanta‐ geous than small-molecular-weight therapeutic agents in terms of their specificity and high potency to the target molecules [Nakase et al., 2010]. Gene therapy is the newest therapeutic strategy for treating human diseases. The basic idea of gene therapy is a gene or gene product that can be selectively delivered to a specific cell/tissue with mini‐ mal toxicity. This product can inhibit the expression of a specific defective gene or ex‐ press a normal gene. Efficient and safe delivery is one of the key issues for the clinical application of nucleic acids as therapeutic agents [Du et al., 2010]. The goal of the Phar‐ maceutical Industry is to have a gene therapy medical product that can be delivered sys‐ temically. *In vivo* gene therapies have focused on viral vectors for gene delivery and have had marginal clinical successes. Major disadvantage of these delivery systems is the integration of some viral vectors into human chromosomes of normal tissue. There are four issues to be solved before cancer gene therapy will be successful: 1) Identification of key target genes critical for the disease pathology and progression; 2) Determination of the correct therapeutic gene to inhibit disease progression; 3) Optimal trans-gene expres‐ sion for suppressing the target gene; and 4) Delivery of therapeutic product to the target

> © 2013 Bolhassani and Saleh; 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.

© 2013 The Author(s). Licensee InTech. This chapter is 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.

**Chapter 10**
