**5.1 Lipoplexes**

62 Non-Viral Gene Therapy

capsid AAV is a vector produced by the insertion of a sequence from another wild-type AAV into the ORF of the capsid gene (Fig. 3) (Choi et al., 2005; Michelfelder & Trepel, 2009). AAV vectors produced by the mixture of capsid proteins of AAV serotypes 1 to 5 led to high-titer viral particles with mixtures of serotype 1, 2, or 3; whereas intermediate titers were observed from AAV5 mixtures (Rabinowitz et al., 2004). Transduction levels varied depending on capsid mixture, producing a synergistic effect in transduction when AAV1 capsids were combined with AAV2 or AAV3 (Rabinowitz et al., 2004). Similar results were observed for a vector produced by the mixture of AAV1 and AAV2 capsid proteins, which exhibited similar titers to those observed for native vectors. The biodistribution profile combined transduction characteristics of both parent vectors (i.e. muscle and liver

A variant of this strategy uses capsid proteins bearing foreign peptides mixed with wildtype capsids. An AAV2 vector containing wild-type and immunoglobulin-binding Z34C fragment of protein A-bearing capsid proteins; made possible the production of a vector with high virus particle and transduction titers, and the capacity to transduce selectively and efficiently MO7e (human megakaryoblastic leukemia cell line) and Jurkat cells (T lymphocyte cells line) (Gigout et al., 2005). A mosaic vector produced by the mixture of wild-type and integrin-targeted capsid proteins resulted in a vector with a 50 - 100-fold enhancement in endothelial cell gene transfer. This is higher compared to the vector

This strategy involves the binding of molecules onto the viral capsid to alter natural tropism of AAV vectors (Fig. 3). Initial approaches used bi-specific antibodies consisting of an anti-AAV antibody cross-linked with another antibody that binds specifically to a cellular receptor (Choi et al., 2005). In the first approach, an anti-AAV antibody was chemically cross-linked to Fab arms of the αIIβ3 integrin binding monoclonal AP-2 antibody, allowing the specific transduction of megakaryocyte cells expressing the

In a second approach, a high-affinity biotin-avidin interaction was used as a molecular bridge for the adsorption of streptavidin-fused ligands (Ponnazhagan et al., 2002). This strategy did not affect virus production and allowed the vector to be specifically targeted to EGFR- or fibroblast growth factor receptor (FGFR)-positive cells after conjugation of EGF or FGF onto the biotinilated capsid. Recently the exogenous glycation of the viral capsid was presented as a potential alternative to redirect vectors from liver to skeletal and cardiac

Non-viral vectors for gene delivery offer several advantages over viral vectors, since these include lack of an immune response, simple synthesis, and low scale-up production cost (Niidome & Huang, 2002). Non-viral vectors may include lipoplexes (DNA-liposome complex), polyplexes (DNA-polymer complex), lipopolyplexes (DNA-liposome-polymer complex) and peptide-based complexes (Douglas, 2008); which can be mixed with physical methods (e.g. electroporation, ultrasound, gene gun or hydrodynamic infusion) to improve the gene delivery (Niidome & Huang, 2002). In addition, most used non-viral vectors do not integrate into the host genome reducing the risk of mutagenic events in the transfected cells

produced only with integrin-targeted capsid (Stachler & Bartlett, 2006).

muscle after systemic administration in mice (Horowitz et al., 2011).

transduction) (Hauck et al., 2003).

**4.3 Adsorption of receptor ligands** 

integrin (Ponnazhagan et al., 1996).

**5. Non-viral vectors** 

Lipoplexes are the result of a complex formation between cationic liposomes (artificial closed vesicles of lipid bilayer membranes) and genetic material. They can be considered a synthetic means of encapsulating genetic material until it reaches its cellular target (Tros et al., 2010). The main strategies for targeting of lipoplexes involve use of chelator lipids that bind metal-bearing ligands, or the modification with polymers bearing a ligand (Fig. 4).

The insertion of the chelator lipid 3 (nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) in a lipoplex preparation allowed the engraftment of a His-tagged vascular epidermal growth factor (VEGF) ligand and specific transfection of cells expressing the VEGF receptor (Fig. 4) (Herringson et al., 2009). A similar approach was used to target small iRNAs to B lymphocytes by using a lipoplex bearing the NTA(3)-DTDA chelator lipid engrafted with a His-tagged CD4 or to cells expressing the receptor for tumor necrosis factor alpha (TNFα) by engrafting with a His-tagged TNFα ligand (Herringson & Altin, 2009).

Fig. 4. Modification of lipoplexes and polyplexes. Ligands can be bound to non-viral vectors through direct binding, spacer (polymer)-mediated binding, or chelator lipid-mediated binding (ligand should be fused with a metal).

To improve hepatic specific delivery of a lipoplex after systemic administration, PEG molecules were added to the complex and galactose molecules were covalently affixed at the distal end of the PEG chain to allow an active targeting to the asialoglycoprotein-receptor present on hepatocytes. This resulted in an 18-fold increase in hepatic gene expression (Morille et al., 2009). Inclusion of a PEG conjugated with a nuclear localization signal (NLS) and added to a lipoplex preparation permitted transfection of endothelial cells in the

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 65

cell surface adherence) was conjugated with an ICAM-1 ligand by using a PEG spacer allowing an efficient and specific transfection of ICAM-1 expressing cells (Khondee et al., 2011). Similarly, a PEI-derived polyplex was engineered for enhanced and specific delivery of VEGF iRNA in prostate cancer cells. The conjugation with prostate cancer-binding peptide via a PEG spacer, showed a significantly higher VEGF inhibition than with the unmodified PEI polyplex in human prostate carcinoma cells (Kim et al., 2009). Anionic liposomes and cationic polymers do not display an efficient targeting to liver or tumors cells. Thus, a lipopolyplex was constructed with a liposome. A PEI polymer and a PEG polymer bearing a monoclonal antibody permitted an *in-vitro* and *in-vivo* transfection of tumor cells (Hu et al., 2010). A similar strategy was used to target an iRNA to tumor cells expressing the EGFR by using this lipopolyplex bearing the EGFR ligand (Hu et al., 2011). Direct binding of ligands in other polyplexes has also been evaluated. Conjugation of folic acid with the aminomethacrylate-phosphoryl-choline based copolymer, allowed transfection of cells overexpressing the receptor for folic acid in some human tumors cells (Lam et al., 2009). A PEI-derived polyplex conjugated with an Arg-Gly-Asp peptide, which binds to the αvβ3 integrin receptor, resulted in efficient transfection of HeLa cells with low and high densities of αvβ3 integrin (Ng et al., 2009). As observed in viral vectors and lipoplexes, antibodies can be also conjugated with polyplexes to target genetic material to specific cell types. A successful overexpression of the human epidermal growth factor receptor-2 (HER2) by transfection with a polyplex conjugated with an anti-HER2 monoclonal antibody has been reported for a breast cancer cell line (Chiu et al., 2004). Another approach in adult MSCs used a modification of a PLL polymer with a palmitic acid substitution to target bone marrow stromal cells. A transfection efficiency similar to that of adenoviral vectors was obtained, opening the possibly to efficiently transfecting these cells without the side-effects

*In-vivo* evaluations of these modified polyplexes include use of a PEI-derived complex bearing an FGFR ligand for transfection of tumor cell lines. It demonstrated *in-vitro* and *invivo* specific transduction of tumor cells overexpressing the FGFR (Li & Huang, 2006), and the use of a poly[N-(2-hydroxypropyl)methacrylamide]-derived polyplex modified with an anti-P-selectin antibody. *In-vivo* specific and enhanced gene delivery to inflamed cremasteric venules was observed (Newman et al., 2009). A significant retargeting to tumor cells was also observed with a PEI polyplex carrying an antibody against a tumor-specific protein via

Modification of gene therapy vectors for specific cell type gene delivery can have a significant impact on vector tropism limitations, side effects, and consolidation of gene therapy as a viable treatment. Vector modifications can be grouped in three basic strategies regardless of vector type: genetic modification (ligand insertion within the capsid), chemical modification (polymers or ligand cross-linking), or the use of adaptor molecules (bi-specific antibodies). Most studies have focused on vector design and proof-of-concept of specific gene delivery, while only few of them have evaluated the therapeutic impact of these modifications. In that sense, future studies should focus on assessing the potential of these modified vectors to improve gene therapy trials in an effort to advance from the bench science to the clinic trials. Special attention should be placed in the development of modified vectors for gene delivery to CNS and bone. Since, under normal conditions they show a

and cell toxicity observed with viral vectors (Incani et al., 2007).

a PEG spacer, with low expression in non-tumor cells (Duan et al., 2010).

**6. Conclusions** 

presence of serum. This method was used as a model for transfection of blood-brain barrier cells considered difficult targets for non-modified viral and non-viral vectors (Zhang et al., 2009). A similar strategy was used for targeting of a lipoplex to tumor cells expressing the tumor-associated glycoprotein (TAG)-72 (Kim et al., 2008). In this work, a Fab' fragment from an anti-TAG-72 monoclonal antibody was conjugated with PEG and included within the lipoplex, providing specific gene delivery to TAG-expressing tumor cells both *in-vitro* and *in-vivo*, while non-targeted lipoploxes did not produce the transfection of those cells.

Ligands can be also directly conjugated with liposome molecules (Fig. 4). A hyaluronic acidliposome conjugate resulted in an increased gene delivery to breast cancer cells with high CD44 levels. Conversely, cells with low CD44 levels were not transfected (Surace et al., 2009). Conjugation of the peptide 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(Lcitrulline)-PYR-(L-citrulline)-CR with a liposome permitted targeting of a lipoplex to CXCR4-expressing cells, with transfection efficiency depending upon CXCR4 expression levels (Driessen et al., 2008). Conjugation of a liposome with transferrin presented a significant increment in iRNA delivery to hepatocarcinoma cells, with reduced toxicity and enhanced specific gene silencing compared to non-modified lipoplexes (Cardoso et al., 2007). Transferin-conjugated lipoplexes were also used to evaluate specific gene delivery to a metastatic mammary carcinoma cell line, although the results did not demonstrate a significant increase in cytotoxicity with modified lipoplexes in comparison with nonmodified lipoplex (Lopez-Barcons et al., 2005).

Another strategy based on *ex-vivo* gene therapy assessed the challenge of nuclear entry by using plasmids transfected with a cationic lipid vector in MSC (Hoare et al., 2010). To enhance gene expression in MSC, they used NLS peptides to direct the plasmid to the nucleus. A significant increase was observed in reporter gene delivery compared to nonmodified plasmid. They also noticed the lipid had a protective impact on the plasmid-NLS complex. Differentiation potential was not affected by NLS peptides.

#### **5.2 Polyplexes**

Polyplexes are non-viral vectors formed by genetic material coupled with cationic polymers including polylysine (PLL), polyarginine, PEG, polyethylenimine (PEI), polyamidoamine (PAMAM), dendrimers, and chitosan. The mechanism involves interaction between negatively charged DNA or iRNA molecules with positively charged molecules within the polymer (Tros et al., 2010). To target polyplexes to specific cells, they can be modified by incorporation of ligands as small molecules, vitamins, carbohydrates, peptides, or proteins (Wagner et al., 2005).

PEG-derived vectors are widely used as gene therapy tools as a result of their well-known capacity to enhance gene delivery and induce a longer DNA half-life time (Midoux et al., 2008). Due to the possibility to bind ligands in the terminal ends of the polymer (Fig. 4), PEG-derived vectors are commonly used for cell specific gene delivery using non-viral vectors (Hughes & Rao, 2005). PEG polyplexes have been conjugated with a transferrin peptide to target vectors to receptor-expressing cells in two prostate cancer cell lines (Nie et al., 2011). In addition they have been conjugated with EGFR ligand or lactose for specific transfection of human hepatocarcinoma cell lines (Klutz et al., 2011; Oishi et al., 2007).

PEG has also been used as a spacer to bind ligands to polyplexex (Fig. 4), although it has been associated in some cases with reduced transfection efficiencies. A complex of DNA and the HIV-derived TAT protein (which has important translocation abilities due to its strong cell surface adherence) was conjugated with an ICAM-1 ligand by using a PEG spacer allowing an efficient and specific transfection of ICAM-1 expressing cells (Khondee et al., 2011). Similarly, a PEI-derived polyplex was engineered for enhanced and specific delivery of VEGF iRNA in prostate cancer cells. The conjugation with prostate cancer-binding peptide via a PEG spacer, showed a significantly higher VEGF inhibition than with the unmodified PEI polyplex in human prostate carcinoma cells (Kim et al., 2009). Anionic liposomes and cationic polymers do not display an efficient targeting to liver or tumors cells. Thus, a lipopolyplex was constructed with a liposome. A PEI polymer and a PEG polymer bearing a monoclonal antibody permitted an *in-vitro* and *in-vivo* transfection of tumor cells (Hu et al., 2010). A similar strategy was used to target an iRNA to tumor cells expressing the EGFR by using this lipopolyplex bearing the EGFR ligand (Hu et al., 2011).

Direct binding of ligands in other polyplexes has also been evaluated. Conjugation of folic acid with the aminomethacrylate-phosphoryl-choline based copolymer, allowed transfection of cells overexpressing the receptor for folic acid in some human tumors cells (Lam et al., 2009). A PEI-derived polyplex conjugated with an Arg-Gly-Asp peptide, which binds to the αvβ3 integrin receptor, resulted in efficient transfection of HeLa cells with low and high densities of αvβ3 integrin (Ng et al., 2009). As observed in viral vectors and lipoplexes, antibodies can be also conjugated with polyplexes to target genetic material to specific cell types. A successful overexpression of the human epidermal growth factor receptor-2 (HER2) by transfection with a polyplex conjugated with an anti-HER2 monoclonal antibody has been reported for a breast cancer cell line (Chiu et al., 2004). Another approach in adult MSCs used a modification of a PLL polymer with a palmitic acid substitution to target bone marrow stromal cells. A transfection efficiency similar to that of adenoviral vectors was obtained, opening the possibly to efficiently transfecting these cells without the side-effects and cell toxicity observed with viral vectors (Incani et al., 2007).

*In-vivo* evaluations of these modified polyplexes include use of a PEI-derived complex bearing an FGFR ligand for transfection of tumor cell lines. It demonstrated *in-vitro* and *invivo* specific transduction of tumor cells overexpressing the FGFR (Li & Huang, 2006), and the use of a poly[N-(2-hydroxypropyl)methacrylamide]-derived polyplex modified with an anti-P-selectin antibody. *In-vivo* specific and enhanced gene delivery to inflamed cremasteric venules was observed (Newman et al., 2009). A significant retargeting to tumor cells was also observed with a PEI polyplex carrying an antibody against a tumor-specific protein via a PEG spacer, with low expression in non-tumor cells (Duan et al., 2010).

### **6. Conclusions**

64 Non-Viral Gene Therapy

presence of serum. This method was used as a model for transfection of blood-brain barrier cells considered difficult targets for non-modified viral and non-viral vectors (Zhang et al., 2009). A similar strategy was used for targeting of a lipoplex to tumor cells expressing the tumor-associated glycoprotein (TAG)-72 (Kim et al., 2008). In this work, a Fab' fragment from an anti-TAG-72 monoclonal antibody was conjugated with PEG and included within the lipoplex, providing specific gene delivery to TAG-expressing tumor cells both *in-vitro* and *in-vivo*, while non-targeted lipoploxes did not produce the transfection of those cells. Ligands can be also directly conjugated with liposome molecules (Fig. 4). A hyaluronic acidliposome conjugate resulted in an increased gene delivery to breast cancer cells with high CD44 levels. Conversely, cells with low CD44 levels were not transfected (Surace et al., 2009). Conjugation of the peptide 4-fluorobenzoyl-RR-(L-3-(2-naphthyl)alanine)-CYEK-(Lcitrulline)-PYR-(L-citrulline)-CR with a liposome permitted targeting of a lipoplex to CXCR4-expressing cells, with transfection efficiency depending upon CXCR4 expression levels (Driessen et al., 2008). Conjugation of a liposome with transferrin presented a significant increment in iRNA delivery to hepatocarcinoma cells, with reduced toxicity and enhanced specific gene silencing compared to non-modified lipoplexes (Cardoso et al., 2007). Transferin-conjugated lipoplexes were also used to evaluate specific gene delivery to a metastatic mammary carcinoma cell line, although the results did not demonstrate a significant increase in cytotoxicity with modified lipoplexes in comparison with non-

Another strategy based on *ex-vivo* gene therapy assessed the challenge of nuclear entry by using plasmids transfected with a cationic lipid vector in MSC (Hoare et al., 2010). To enhance gene expression in MSC, they used NLS peptides to direct the plasmid to the nucleus. A significant increase was observed in reporter gene delivery compared to nonmodified plasmid. They also noticed the lipid had a protective impact on the plasmid-NLS

Polyplexes are non-viral vectors formed by genetic material coupled with cationic polymers including polylysine (PLL), polyarginine, PEG, polyethylenimine (PEI), polyamidoamine (PAMAM), dendrimers, and chitosan. The mechanism involves interaction between negatively charged DNA or iRNA molecules with positively charged molecules within the polymer (Tros et al., 2010). To target polyplexes to specific cells, they can be modified by incorporation of ligands as small molecules, vitamins, carbohydrates, peptides, or proteins

PEG-derived vectors are widely used as gene therapy tools as a result of their well-known capacity to enhance gene delivery and induce a longer DNA half-life time (Midoux et al., 2008). Due to the possibility to bind ligands in the terminal ends of the polymer (Fig. 4), PEG-derived vectors are commonly used for cell specific gene delivery using non-viral vectors (Hughes & Rao, 2005). PEG polyplexes have been conjugated with a transferrin peptide to target vectors to receptor-expressing cells in two prostate cancer cell lines (Nie et al., 2011). In addition they have been conjugated with EGFR ligand or lactose for specific transfection of human hepatocarcinoma cell lines (Klutz et al., 2011; Oishi et al., 2007). PEG has also been used as a spacer to bind ligands to polyplexex (Fig. 4), although it has been associated in some cases with reduced transfection efficiencies. A complex of DNA and the HIV-derived TAT protein (which has important translocation abilities due to its strong

modified lipoplex (Lopez-Barcons et al., 2005).

**5.2 Polyplexes** 

(Wagner et al., 2005).

complex. Differentiation potential was not affected by NLS peptides.

Modification of gene therapy vectors for specific cell type gene delivery can have a significant impact on vector tropism limitations, side effects, and consolidation of gene therapy as a viable treatment. Vector modifications can be grouped in three basic strategies regardless of vector type: genetic modification (ligand insertion within the capsid), chemical modification (polymers or ligand cross-linking), or the use of adaptor molecules (bi-specific antibodies). Most studies have focused on vector design and proof-of-concept of specific gene delivery, while only few of them have evaluated the therapeutic impact of these modifications. In that sense, future studies should focus on assessing the potential of these modified vectors to improve gene therapy trials in an effort to advance from the bench science to the clinic trials. Special attention should be placed in the development of modified vectors for gene delivery to CNS and bone. Since, under normal conditions they show a

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