**2. Functional domains available**

#### **2.1 Nucleic acid attachment and condensation**

Trans-membrane transport of DNA is an inefficient process, and thus the successful introduction of a transgene into a target cell must include two important steps regarding the plasmid or oligonucleotid DNA that is included in the vector. First, the extended DNA needs to be condensed into an ordered compact nano-particle, and second, once inside the nucleus, the DNA must be de-condensed and thus accessible to transcription. Basic peptides or polycations have been exploited for the interaction with the DNA backbone due to their electrostatic interactions (Bloomfield 1996; Saccardo *et al.* 2009)(see Table 1). When DNA is mixed with these condensing agents, smaller molecules of different shapes are formed mainly depending on DNA size (Vijayanathan *et al.* 2002). For instance, in the absence of DNA, the HNRK modular vector that uses poly-lysine for DNA condensation, self-organize as amorphous, polydisperse particulate entities ranging from a few nanometres up to around one micron. However, in the presence of DNA, protein-DNA complexes appear as tight and rather monodisperse spherical-like nanoparticles of around 80 nm in diameter (Domingo-Espín *et al.* 2011). The most widely used condensing agent is a poly-lysine chain (Saccardo *et al.* 2009). Poly-lysine polymers containing at least 6 lysines will efficiently condense DNA, however, additional 4 to 9 lysines are needed to fully condensate the DNA into smaller particles of 50-100nm, increasing in this way many folds the transfection capacity (Wadhwa *et al.* 1997). Other basic peptides used are the poly-arginine peptides, which not only induce DNA condensation, but also show membrane translocation potential (Futaki *et al.* 2001) and nuclear translocation capacity, determining in this way transgene expression (Kim *et al.* 2003; Vazquez *et al.*).

The condensed DNA by these peptides is partially protected from cellular acid nucleases of the lysosomal compartment (Krishnamoorthy *et al.* 2003; Ross *et al.* 1998; Wolfert and Seymour 1998) and serum nucleases, inducing an extended half-life in serum (Kumar *et al.* 2007) and in the circulation, making tissue targeting possible (Kawabata *et al.* 1995; Nishikawa *et al.* 2000a). For example, the addition of the acid nuclease inhibitor DMI-2 induced a 10-fold increase in receptor-mediated transfection in cultured cells exposed to a

magnetic fields have been used to concentrate suitable engineered vectors to a given area

An interesting type of non-viral vectors is the one based on multifunctional proteins (Aris and Villaverde 2004; Mastrobattista *et al.* 2006). The combination of functional domains in a single polypeptide is a simple yet powerful approach for the development of vectors suitable for gene therapy. In fact, this approach has generated the first prototypes of modular protein gene therapy vectors. Three general methods have been used for the engineering of these molecules: i) production of a recombinant protein by the direct fusion of the functional domains; ii) production of a recombinant protein by combining a known scaffold protein and several functional domains inserted into exposed regions of the scaffold protein; and iii) chemical conjugation of functional domains and proteins. Many of these vectors can be produced recombinantly, generating reproducible and stable stocks appropriate for the formulation of clinically usable drugs. Moreover, the modular nature of these versatile vectors enables the combination of different domains to fulfil the changing

Trans-membrane transport of DNA is an inefficient process, and thus the successful introduction of a transgene into a target cell must include two important steps regarding the plasmid or oligonucleotid DNA that is included in the vector. First, the extended DNA needs to be condensed into an ordered compact nano-particle, and second, once inside the nucleus, the DNA must be de-condensed and thus accessible to transcription. Basic peptides or polycations have been exploited for the interaction with the DNA backbone due to their electrostatic interactions (Bloomfield 1996; Saccardo *et al.* 2009)(see Table 1). When DNA is mixed with these condensing agents, smaller molecules of different shapes are formed mainly depending on DNA size (Vijayanathan *et al.* 2002). For instance, in the absence of DNA, the HNRK modular vector that uses poly-lysine for DNA condensation, self-organize as amorphous, polydisperse particulate entities ranging from a few nanometres up to around one micron. However, in the presence of DNA, protein-DNA complexes appear as tight and rather monodisperse spherical-like nanoparticles of around 80 nm in diameter (Domingo-Espín *et al.* 2011). The most widely used condensing agent is a poly-lysine chain (Saccardo *et al.* 2009). Poly-lysine polymers containing at least 6 lysines will efficiently condense DNA, however, additional 4 to 9 lysines are needed to fully condensate the DNA into smaller particles of 50-100nm, increasing in this way many folds the transfection capacity (Wadhwa *et al.* 1997). Other basic peptides used are the poly-arginine peptides, which not only induce DNA condensation, but also show membrane translocation potential (Futaki *et al.* 2001) and nuclear translocation capacity, determining in this way transgene

The condensed DNA by these peptides is partially protected from cellular acid nucleases of the lysosomal compartment (Krishnamoorthy *et al.* 2003; Ross *et al.* 1998; Wolfert and Seymour 1998) and serum nucleases, inducing an extended half-life in serum (Kumar *et al.* 2007) and in the circulation, making tissue targeting possible (Kawabata *et al.* 1995; Nishikawa *et al.* 2000a). For example, the addition of the acid nuclease inhibitor DMI-2 induced a 10-fold increase in receptor-mediated transfection in cultured cells exposed to a

(Corchero and Villaverde 2009).

requirements of pathological end experimental situations.

**2. Functional domains available** 

expression (Kim *et al.* 2003; Vazquez *et al.*).

**2.1 Nucleic acid attachment and condensation**

Surfactant Protein A-poly-lysine modular vector or a transferrin-poly-lysine modular vector (Ross *et al.* 1998). Naturally DNA condensing proteins have also been used for the construction of modular vectors. For instance, Histones condense plasmid DNA and protects it from endonucleases, being the lysine-rich H1 Histone the most effective one (Pyhtila *et al.* 1976). Moreover, some nuclear localization signals like the NLS peptide from SV40 virus large T-antigen are lysine-rich peptides that when used as a tetramer can efficiently condense DNA without loosing its nuclear localization properties (Ritter *et al.* 2003).

#### **2.2 Cell attachment and cell targeting**

When a viral or non-viral gene therapy vector is injected intravenously, most of the vectors will localize mainly in the liver but also in the kidneys, lungs and spleen. While this is normally a problem to circumvent for most gene therapy applications, it constitutes an advantage for the expression of molecules in the liver. There are many fetal metabolic diseases resulting from a defect or a deciency of hepatocyte-derived proteins. Moreover, the liver can be considered as a platform to produce various proteins secreted into the blood. Therefore, many pioneer studies focused on the development of more efficient gene delivery systems for the introduction of therapeutic genes selectively into hepatocytes (Wu and Wu 1988). Intravenously injected plasmids are cleared from the circulation by the liver non-parenchymal cells by a scavenging receptor mediated mechanism (Kawabata *et al.* 1995). When Nishikawa and colleagues administered naked 32PDNA into the tail vein of mice, about 40% and 10% of the radioactivity rapidly accumulated in the liver and kidneys, respectively (Nishikawa *et al.* 2000b). Again, the main cell-types targeted were the liver nonparenchymal cells: Kupffer cells and endothelial cells. When they injected a vector composed of 32PDNA/polyornithine, little effect on the distribution of the DNA was observed. However, the injection of the 32PDNA/Gal-pOrn galactose-mediated hepatocytetargeting vector induced a 60% hepatic accumulation of radioactivity, but more interestingly, most of the targeted cells were now hepatocytes instead of Kupffer or endothelial cells. The same effect was observed at the level of luciferase transgene expression, indicating that the DNA/Gal-pOrn vector was not only able to adhere and enter preferentially into hepatocytes, but it could also transfect them.

Many different domains of known proteins and sugars have been used for cell targeting of modular vectors, like galactose (Wu and Wu 1987), transferrin (Wagner *et al.* 1990), foot-andmouth disease virus integrin interacting peptide (Aris *et al.* 2000; Aris and Villaverde 2003; Domingo-Espín *et al.* 2011), nerve growth factor (Ma *et al.* 2004; Zeng *et al.* 2004), surfactant protein A (Ross *et al.* 1995), rabies virus glycoprotein (Kumar *et al.* 2007), tetanus toxin fragment Hc (Box *et al.* 2003; Knight *et al.* 1999), cholera toxin b chain (Barrett *et al.* 2004), and neurotensin (Navarro-Quiroga *et al.* 2002). In an interesting study, Arango-Rodríguez and colleagues showed that they could target only substantia nigra neurotensin high affinity receptor positive neurons by means of a modular vector that displayed neurotensin, while no other neurons were transfected (Arango-Rodriguez *et al.* 2006). *In vivo*, many of these targeting systems have shown success (see Table 1). An additional interesting targeting strategy is the use of antibodies (Berhanu and Rush 2008; Buschle *et al.* 1995; Thurnher *et al.* 1994). For instance, the use of the 1E3 antibody against the Tn antigen expressed on many carcinomas coupled to polylysine induced an important increase in the transfection of a cancer cell line (Thurnher *et al.* 1994). Another vector, named fkAbp75-ipr, possess several


Table 1. Modular protein vectors effective in vivo



Modular Multifunctional Protein Vectors for Gene Therapy 603

careful evaluation of the toxicity of this cell penetrating domains in *in vivo* settings has to be performed as toxicity of Tat protein has been reported (Cardozo *et al.* 2007), specially for the CNS (Bonavia *et al.* 2001; Nath *et al.* 1996). In addition, toxicity of the Antp domain has also

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

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

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,

instead of inside the nucleus, not favouring the transfection process.

**2.5 Trophic vectors/functional vectors** 

been reported for many cell types (Cardozo *et al.* 2007).

**2.4 Nuclear translocation** 

DNA is imported.

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 memory.

#### **2.3 Endosomal escape**

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

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

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 careful evaluation of the toxicity of this cell penetrating domains in *in vivo* settings has to be performed as toxicity of Tat protein has been reported (Cardozo *et al.* 2007), specially for the CNS (Bonavia *et al.* 2001; Nath *et al.* 1996). In addition, toxicity of the Antp domain has also been reported for many cell types (Cardozo *et al.* 2007).
