**2.4 Nuclear translocation**

602 Non-Viral Gene Therapy

functional domains (see Table 1), being one of them the monoclonal antibody MC192 against p75NTR, the low affinity neurotrophin receptor (Berhanu and Rush 2008). By injecting it intracerebroventricularly coupled to siRNA against TrkA, one of the high affinity neurotrophin receptors, Berhanu and Rush were able to down-regulate TrkA expression in p75NTR expressing cells and correlate this with functional alterations like impaired spatial

A limiting step for receptor-mediated gene delivery is the escape from endosomes, as the vector needs to gain access to the cytosol to enter the nuclei. Fusogenic peptides are reported to strongly enhance *in vitro* gene transfer after being incorporated into carrier systems by chemical linkage (Box *et al.* 2003; Fisher and Wilson 1997; Navarro-Quiroga *et al.* 2002; Nishikawa *et al.* 2000b; Ogris *et al.* 2001; Wagner *et al.* 1992) or by ionic interaction (Gottschalk *et al.* 1996; Plank *et al.* 1994), but co-treatment of the cells with the vector and the fusogenic peptide may also be effective (Read *et al.* 2005). The most widely used method for endosome escape is based on the amino-terminal motif of inuenza virus hemagglutinin subunit HA2 (Plank *et al.* 1994; Wagner *et al.* 1992). For example, Nishikawa and colleagues showed that when an acid-sensitive fusogenic peptide derived from HA2 was incubated with mouse erythrocytes at pH 5.0, it induced hemolysis while it did not show any signicant hemolytic activity at pH 7.4 (Nishikawa *et al.* 2000b). Interestingly, the same study showed that the *in vivo* liver transgene expression obtained after intravenous injection of the vector DNA/Gal-pOrn-mHA2 was 300 fold higher than that obtained with the same

Other domains used for DNA condensation or vector purification as polylysine or polyhistidine have shown endosome disrupting activities (Read *et al.* 2005; Zauner *et al.* 1997). The most effective ones were histidine rich polyplexes formed by the condensation of approximately 50 monomers of Cys-His6-Lys3-His6-Cys and DNA (Read *et al.* 2005). In this study, the endosomolytic agent chloroquine, which normally enhance the transfection capacity of most non-viral vectors, did not enhance the transfection with the histidine rich polyplexes while it enhanced transfection with other non-viral vectors, suggesting that the poly-his domains are in fact endosomolytic. Though polycations like polylysine may be toxic to cells, especially if they have membrane-disrupting activity, this polyhistidine vector showed no toxicity. Histidine becomes positively charged when the pH decrease to less than 7 and thus becomes useful for the permeabilization of the endosomal membrane induced by acidification of endosomes, increasing cell transfection (Midoux *et al.* 1998). *In vivo*, many of these endosomal escape systems have shown success (see Table 1). For an extensive review on different strategies and domains used for endosomal escape please refer to Ferrer-

For the introduction of siRNA and DNA into cells, several cationic peptide transduction domains or also called cell-penetrating domains have been used. TAT, 8xArg, Hph-1or Antp domains can deliver a wide variety of cargo into primary cells, to most tissues, and are in addition being evaluated in clinical trials (Gump and Dowdy 2007). For instance, when the Tat-domain was combined with a poly-His domain and the (ds)RNA-binding domain DRBD, the vector coupled to siRNA could successfully down-regulate Luciferase expression in the nasal and tracheal passages for 4 days after intranasal administration (Eguchi *et al.* 2009). An important characteristic of these systems using cell-penetrating peptides is that they are not cell-specific, and thus should be used for general non-selective transfection. A

memory.

**2.3 Endosomal escape** 

vector lacking the HA2 domain (Nishikawa *et al.* 2000b).

Miralles et al. (Ferrer-Miralles *et al.* 2008).

The transgene expression levels obtained after plasmid DNA injection into the cytoplasm or the nucleus showed that de nuclear double membrane and its pores are important barriers for naked DNA (Liu *et al.* 2003; Pollard *et al.* 1998). The selection of macromolecules that will be actively imported into the nucleus occurs at the nuclear pore complex, which is composed of more than 50 different proteins. The pore complex will recognise importin proteins bound to short (normally 4-8 amino acids) nuclear localization signals which can be located almost anywhere in the amino acid sequence of the protein, and which are rich in the positively charged amino acids lysine and arginine and usually contains proline (Pouton 1998). This mechanism has been exploited for the design of modular protein vectors, introducing nuclear localization sequences like the SV40 NLS peptide from the T antigen (Aris and Villaverde 2003; Fritz *et al.* 1996). For instance, Aris and co-workers introduced this nuclear localization sequence into the 249AL modular vector (see Table 1) and they observed an enhanced transgene expression with the resulting vector termed NLSCt (Aris and Villaverde 2003). However, studies performed in cells in culture show that even in the presence of nuclear localizations sequences, complexes of more than 60nm seem to be excluded (Chan *et al.* 2000). This data are in contrast to the high transfection efficiency obtained, even *in vivo*, with different modular protein vectors that exceeds this size, reaching 200nm (see Table 1). One can speculate that in fact some molecules of up to 200nm can be imported into the nucleus by being flexible, or that during the interaction of the vector with the nuclear import machinery the vector is disassembled and only the DNA is imported.

Another important step for efficient transgene expression may be the release of the nucleic acid from the vector once in the nucleus. Several studies have addressed the possible enhancement of the release of the DNA by the cellular reducing conditions. For example, histidine rich polyplexes were able to release the complexed DNA when exposed to the reducing agent Dithiothreitol (DTT), suggesting that in cells a similar mechanism would occur (Read *et al.* 2005). In fact, the increase in the cellular antioxidant and reducing agent glutathione, induced an important 200 fold increase in the transfection observed with the histidine rich polyplexes, but only a 3fold increase was observed with the PEI/DNA vector, another non-viral vector with no reduction-dependent release of DNA. Though this is an interesting phenomenon, it is difficult to understand why the cytosol reducing conditions do not disassemble the vector too early, determining that the DNA is released into the cytosol instead of inside the nucleus, not favouring the transfection process.

### **2.5 Trophic vectors/functional vectors**

An attractive possibility is the combination of the effects mediated by the overexpression of a transgene and the direct effects of the vector per se. In fact, as modular vectors normally take advantage of a cell attaching motif for receptor mediated endocytosis, they tend to display intrinsic activities. More importantly, the use of trophic factors or toxin domains for cell attachment and internalization is ideal, as their natural mechanism of action includes the attachment to high affinity cell surface receptors, the endocytosis to early endosomes,

Modular Multifunctional Protein Vectors for Gene Therapy 605

excitotoxic injury, an increase in macrophage/microglia number and in the levels of IL1β and Cox2 enzyme were observed in the lesion (Gonzalez *et al.* 2011). Most interesting, the same set of studies discovered that this vector, with or without accomplished control DNA, besides inducing an inflammatory response, also induced a decrease in the brain lesion volume and in the number of degenerating neurons (Peluffo *et al.* 2006; Peluffo *et al.* 2007), an effect that was mediated by the prototypic RGD-integrin interacting motif of the vector (Peluffo *et al.* 2007). These data may suggest that the modulation of the inflammation by the vector may be beneficial under some circumstances. Another vector termed Tat-PTD-DRBD (see Table 1) did not induce interferon (IFN)-α or tumour necrosis factor (TNF)-α responses when incubated with primary human peripheral blood mononuclear cells (Eguchi *et al.* 2009). Thus, the overall data suggests that these types of vectors are less immunogenic and pro-inflammatory than

If these types of vectors are useful for gene therapy applications is still an open question, and adequate testing of these vectors in preclinical and actual clinical studies need to be performed. In fact, it has been well established that there is no ideal vector for all gene therapy applications, being the characteristics of each vector critical for each pathological paradigm. The use of modular protein vectors is limited to pathologies accepting an acute treatment, but would be ineffective for chronic ones as the transgene expression that they determine is normally short lived. The time of transgene expression varies from a few days to more than two months, depending on the doses and the method of administration. For instance, multifunctional recombinant vectors can induce the *in vivo* brain expression of a reporter gene after direct injection into the bran in a model of acute brain injury, lasting the transgenic protein in the brain for 3 days (Peluffo *et al.* 2003), but another vector was able to determine expression in normal brain for two months after intracerebral injection (Navarro-Quiroga *et al.* 2002). In the case of other administration routes and pathologies, as for example the intravenous administration of these vectors, the time for transgenic protein expression in the liver may range from a few days to more that 4 months (Perales *et al.* 1994). In another study, the liver-selective and transient overexpression of the therapeutic protein human coagulation factor IX could be achieved using a synthetic modular glycoprotein vector, and secreted factor IX into the serum could be detected for 30 days (Ferkol *et al.* 1993). This same paradigm could be used for vaccination, overexpressing transiently the desired immunogenic protein (Chen and Huang 2005). Even the use of modular vectors coupled to plasmids producing shRNA show potent downregulation of an endogenous gene during 20 days when infused with osmotic pumps into the nervous system (Berhanu and Rush 2008). In all this approaches, the transient expression of a protein by means of multifunctional vectors would be desirable when compared to viral vector inoculation, which present higher risks of oncogenic and inflammatory complications, may produce very high levels of transgenic protein, and will

Various approaches have been undertaken to overcome the interaction of vectors with blood components to avoid aggregation as well as embolisms. Moreover for most strategies, the phagocytic clearing system of the organism must be eluded. Pharmacokinetic analysis has

most viral and other non-viral vectors.

**4. Administration routes and transgene expression** 

produce the transgenic protein for life or for extended periods.

**5. Pharmacokinetics and biodistribution** 

and even being transported to the cell soma in the case of neurons (Lalli and Schiavo 2002). An interesting modular vector was produced combining a polylysine tail with the loop 4 of the nerve growth factor (NGF) (Zeng *et al.* 2004; Zeng and Wang 2005). This "trophic vector" maintained the trophic effects of NGF, was able to condensate DNA, and when combined with polyethylenimine (PEI600), transfected cells in culture that expressed NGF receptors but not cells without these receptors. Interestingly, the DNA-PEI600 showed a size of 445nm and an zeta potential of 6,2mV, but the addition of the NGF loop4 poly-lysine peptide to the complex induced the formation of smaller 180nm particles with a zeta potential of 23,2mV (Zeng *et al.* 2007). This shows that the addition of targeting peptides to non-specific DNA/condensing products complexes may in fact contribute to enhance not only the targeted delivery but also to decrease the particle size and charge of the resulting vector. A somehow more complex trophic vector including NGF loops was also produced. It combined the loops 1 and 2 of NGF and the SPKR4 domain derived from histone H1 DNA binding motif, linked together by a α-helical linker (Ma *et al.* 2004). Both NGF-loop derived vectors could even transfer a transgene *in vivo* preferentially to dorsal root ganglia neurons (which express NGF receptors) after intrathecal spinal cord injection (Ma *et al.* 2004; Zeng *et al.* 2007). Several toxins have been used as cell attachment motifs (Andreu *et al.* 2008; Box *et al.* 2003; Knight *et al.* 1999), and some motifs of these toxins can in fact display trophic effects (Chaib-Oukadour *et al.* 2004), and have thus been used to design trophic vectors.

An important consideration regarding many acute injuries is that the therapeutic time window is short. In those cases, a direct trophic or functional effect of the vector per se could extend the therapeutic window, giving time for the transgene to be expressed and mediate its own effects. For example, the neuroprotection observed after an acute brain injury using the vector termed NLSCt was partially mediated by the transgene overexpressed, but also partially mediated by the RGD integrin-interacting motif of the vector itself (Peluffo *et al.* 2006; Peluffo *et al.* 2007). In this experimental setting the direct injection of the vector into the lesioned brain area was performed 4 hours after the lesion. The CNS is a tissue that tolerates injures very badly due to its high dependence on blood flow and oxygen consumption, and its poor regeneration capacity. This determines that the therapeutic window for the treatment of acute injuries is very short. Interestingly, even in this experimental paradigm, the modular recombinant NLSCt vector overexpressing the anti-oxidant enzyme Cu/Zn superoxide dismutase (SOD) could mediate neuroprotection (Peluffo *et al.* 2006). These studies shows the wide possibilities of combining the vectors themselves with active protein domains like trophic factors, which will exert rapid direct effects, which in turn may increase the therapeutic window or the potency of the effect of the transgene used.

#### **3. Immunogenicity and inflammation**

The introduction of modular protein vectors into the organism may be accompanied by a humoral or cell-mediated immune response against the inserted motifs, which in many cases are derived from viral molecules. However, when injected intravenously, the Rabies virus glycoprotein (29aa)-Poly-Arg vector (RVG-9R) (see Table 1) did not induce an antibody response or an increase in several pro-inflammatory cytokines evaluated (Kumar *et al.* 2007). In another example, when the recombinant 249AL vector (see Table 1) was injected into the normal postnatal brain, no changes were observed in glial activation, demyelination, recruitment of cytotoxic CD8 lymphocytes, or expression of IL1β. Interestingly, when a very similar vector termed NLSCt (see Table 1) was injected into the postnatal brain after an

and even being transported to the cell soma in the case of neurons (Lalli and Schiavo 2002). An interesting modular vector was produced combining a polylysine tail with the loop 4 of the nerve growth factor (NGF) (Zeng *et al.* 2004; Zeng and Wang 2005). This "trophic vector" maintained the trophic effects of NGF, was able to condensate DNA, and when combined with polyethylenimine (PEI600), transfected cells in culture that expressed NGF receptors but not cells without these receptors. Interestingly, the DNA-PEI600 showed a size of 445nm and an zeta potential of 6,2mV, but the addition of the NGF loop4 poly-lysine peptide to the complex induced the formation of smaller 180nm particles with a zeta potential of 23,2mV (Zeng *et al.* 2007). This shows that the addition of targeting peptides to non-specific DNA/condensing products complexes may in fact contribute to enhance not only the targeted delivery but also to decrease the particle size and charge of the resulting vector. A somehow more complex trophic vector including NGF loops was also produced. It combined the loops 1 and 2 of NGF and the SPKR4 domain derived from histone H1 DNA binding motif, linked together by a α-helical linker (Ma *et al.* 2004). Both NGF-loop derived vectors could even transfer a transgene *in vivo* preferentially to dorsal root ganglia neurons (which express NGF receptors) after intrathecal spinal cord injection (Ma *et al.* 2004; Zeng *et al.* 2007). Several toxins have been used as cell attachment motifs (Andreu *et al.* 2008; Box *et al.* 2003; Knight *et al.* 1999), and some motifs of these toxins can in fact display trophic effects

(Chaib-Oukadour *et al.* 2004), and have thus been used to design trophic vectors.

increase the therapeutic window or the potency of the effect of the transgene used.

The introduction of modular protein vectors into the organism may be accompanied by a humoral or cell-mediated immune response against the inserted motifs, which in many cases are derived from viral molecules. However, when injected intravenously, the Rabies virus glycoprotein (29aa)-Poly-Arg vector (RVG-9R) (see Table 1) did not induce an antibody response or an increase in several pro-inflammatory cytokines evaluated (Kumar *et al.* 2007). In another example, when the recombinant 249AL vector (see Table 1) was injected into the normal postnatal brain, no changes were observed in glial activation, demyelination, recruitment of cytotoxic CD8 lymphocytes, or expression of IL1β. Interestingly, when a very similar vector termed NLSCt (see Table 1) was injected into the postnatal brain after an

**3. Immunogenicity and inflammation** 

An important consideration regarding many acute injuries is that the therapeutic time window is short. In those cases, a direct trophic or functional effect of the vector per se could extend the therapeutic window, giving time for the transgene to be expressed and mediate its own effects. For example, the neuroprotection observed after an acute brain injury using the vector termed NLSCt was partially mediated by the transgene overexpressed, but also partially mediated by the RGD integrin-interacting motif of the vector itself (Peluffo *et al.* 2006; Peluffo *et al.* 2007). In this experimental setting the direct injection of the vector into the lesioned brain area was performed 4 hours after the lesion. The CNS is a tissue that tolerates injures very badly due to its high dependence on blood flow and oxygen consumption, and its poor regeneration capacity. This determines that the therapeutic window for the treatment of acute injuries is very short. Interestingly, even in this experimental paradigm, the modular recombinant NLSCt vector overexpressing the anti-oxidant enzyme Cu/Zn superoxide dismutase (SOD) could mediate neuroprotection (Peluffo *et al.* 2006). These studies shows the wide possibilities of combining the vectors themselves with active protein domains like trophic factors, which will exert rapid direct effects, which in turn may

excitotoxic injury, an increase in macrophage/microglia number and in the levels of IL1β and Cox2 enzyme were observed in the lesion (Gonzalez *et al.* 2011). Most interesting, the same set of studies discovered that this vector, with or without accomplished control DNA, besides inducing an inflammatory response, also induced a decrease in the brain lesion volume and in the number of degenerating neurons (Peluffo *et al.* 2006; Peluffo *et al.* 2007), an effect that was mediated by the prototypic RGD-integrin interacting motif of the vector (Peluffo *et al.* 2007). These data may suggest that the modulation of the inflammation by the vector may be beneficial under some circumstances. Another vector termed Tat-PTD-DRBD (see Table 1) did not induce interferon (IFN)-α or tumour necrosis factor (TNF)-α responses when incubated with primary human peripheral blood mononuclear cells (Eguchi *et al.* 2009). Thus, the overall data suggests that these types of vectors are less immunogenic and pro-inflammatory than most viral and other non-viral vectors.
