**3.2 Molecular adaptors for AdV targeting**

Although genetic modification is the most direct form of vector targeting it has the disadvantage of not all peptides can be inserted into the vector capsid. An approach to overcome this difficulty is to use molecular adaptors consisting of a capsid-binding domain fused with a cell-binding domain (Fig. 2) (Campos & Barry, 2007). This strategy has the advantages of not requiring a correct processing of the cell-binding peptide, since it is not translated with the viral capsid proteins; and that virtually any cell-binding peptide can be fused with the capsid.

The primary adaptor molecules are bi-specific antibodies against a capsid protein and a cellular receptor. The use of a single-chain bi-specific antibody directed against human EGFR and fiber knob significantly increased gene transfer into primary glioma cells and organotypic glioma spheroids, while reducing natural tropism dramatically (Grill et al., 2001). The human epithelial cell adhesion molecule (EpCAM) is highly expressed in malignant lesions of the stomach and esophagus. By using bi-specific antibodies against adenovirus fiber knob protein and EpCAM a specific transduction of gastric and esophageal cancer cell lines was observed with reduced transduction in normal cells (Heideman et al., 2001). For other epithelial tumors, e.g. colon, lung and breast, it was possible to target vectors by using a bi-specific adapter protein, which fused the ectodomain of CAR with a single-chain anti-carcinoembryonic antigen (CEA) antibody. This adaptor molecule allowed the targeting of AdV vectors to CEA-positive epithelial tumor cells in cell culture, in subcutaneous tumor grafts, and in hepatic tumor grafts (Li et al., 2007). A similar strategy was used for vector targeting to pancreatic carcinoma cells by using a vector bearing an EGF peptide fused with CAR (Wesseling et al., 2001). The combination of this adaptor molecule with the insertion of an RGD-containing peptide into the fiber knob resulted in a significant enhancement of gene transfer.

## **3.3 Chemical modification of the AdV capsid**

The third strategy for AdV targeting is the chemical modification of the viral capsid by using primary-amine reactive groups (Fig. 2) (Campos & Barry, 2007). The capsid modification with polyethylene glycol (PEG - PEGylation) is a promising strategy to diminish vector toxicity and immune response. Although other polymers have also been used PEGylation can be employed to modify tropism. Polymer-coated vectors can be targeted to specific cells by incorporation onto the polymer molecules that bind to specific cell-surface proteins (Campos & Barry, 2007; Volpers & Kochanek, 2004).

PEG-modified AdV vectors were conjugated with an anti-HER2/neu monoclonal antibody leading to an enhanced and specific transduction of HER2/neu over-expressed breast cancer cells. Furthermore, a significant reduction of the innate immune response against the vector was accomplished (Jung et al., 2007; Kim et al., 2011). Conjugation of PEG-AdV vectors with

Although genetically modified AdV vectors have been widely studied, most of these modifications have been carried out only into fiber or hexo. Recently it was determined that protein IX in the capsid is a viable platform for the insertion of single-chain variablefragment antibodies (scFv) or single-domain antibodies (sdAb) for AdV vector retargeting. Even so, only sbAb enhanced virus infection of cells expressing the targeted receptor. Thus, proving that the nature of the ligand can significantly affect vector targeting as already had

Although genetic modification is the most direct form of vector targeting it has the disadvantage of not all peptides can be inserted into the vector capsid. An approach to overcome this difficulty is to use molecular adaptors consisting of a capsid-binding domain fused with a cell-binding domain (Fig. 2) (Campos & Barry, 2007). This strategy has the advantages of not requiring a correct processing of the cell-binding peptide, since it is not translated with the viral capsid proteins; and that virtually any cell-binding peptide can be

The primary adaptor molecules are bi-specific antibodies against a capsid protein and a cellular receptor. The use of a single-chain bi-specific antibody directed against human EGFR and fiber knob significantly increased gene transfer into primary glioma cells and organotypic glioma spheroids, while reducing natural tropism dramatically (Grill et al., 2001). The human epithelial cell adhesion molecule (EpCAM) is highly expressed in malignant lesions of the stomach and esophagus. By using bi-specific antibodies against adenovirus fiber knob protein and EpCAM a specific transduction of gastric and esophageal cancer cell lines was observed with reduced transduction in normal cells (Heideman et al., 2001). For other epithelial tumors, e.g. colon, lung and breast, it was possible to target vectors by using a bi-specific adapter protein, which fused the ectodomain of CAR with a single-chain anti-carcinoembryonic antigen (CEA) antibody. This adaptor molecule allowed the targeting of AdV vectors to CEA-positive epithelial tumor cells in cell culture, in subcutaneous tumor grafts, and in hepatic tumor grafts (Li et al., 2007). A similar strategy was used for vector targeting to pancreatic carcinoma cells by using a vector bearing an EGF peptide fused with CAR (Wesseling et al., 2001). The combination of this adaptor molecule with the insertion of an RGD-containing peptide into the fiber knob resulted in a significant

The third strategy for AdV targeting is the chemical modification of the viral capsid by using primary-amine reactive groups (Fig. 2) (Campos & Barry, 2007). The capsid modification with polyethylene glycol (PEG - PEGylation) is a promising strategy to diminish vector toxicity and immune response. Although other polymers have also been used PEGylation can be employed to modify tropism. Polymer-coated vectors can be targeted to specific cells by incorporation onto the polymer molecules that bind to specific

PEG-modified AdV vectors were conjugated with an anti-HER2/neu monoclonal antibody leading to an enhanced and specific transduction of HER2/neu over-expressed breast cancer cells. Furthermore, a significant reduction of the innate immune response against the vector was accomplished (Jung et al., 2007; Kim et al., 2011). Conjugation of PEG-AdV vectors with

cell-surface proteins (Campos & Barry, 2007; Volpers & Kochanek, 2004).

been observed with fiber and hexo (Poulin et al., 2010).

**3.2 Molecular adaptors for AdV targeting** 

fused with the capsid.

enhancement of gene transfer.

**3.3 Chemical modification of the AdV capsid** 

an RGD peptide led to a specific αvβ3 integrin vector cell entry. This resulted in a significant improvement in transduction and specificity of gene delivery into endothelial cells. These events have implications on the treatment of rheumatoid arthritis, inflammatory bowel disease and epithelial tumors (Eto et al., 2005; Ogawara et al., 2006). Transduction of bone marrow derived human mesenchymal stem cells (MSC) can be significantly improved by using a PEGylated AdV conjugated with a blocked poly-L-lysine. However, modification on vector tropism was not clearly reported in this study (Park et al., 2010).

Although a great part of systemically administrated vector particles is scavenged by Kupffer cells in the liver, the use of a PEG- or a dextran-coated vectors allowed the specific transduction of hepatocytes independent of the presence of Kupffer cells, emphasizing the potential for therapeutic liver-directed gene transfer (Prill et al., 2010). Similar result can be observed by using a multivalent hydrophilic polymer based on poly-[N-(2 hydroxypropyl)methacrylamide] conjugated with EGF or VCAM, producing a CARindependent binding and uptake into EGF- or VCAM-positive target cells selectively in mixed culture and also in xenografts *in-vivo* (Fisher et al., 2001).

### **4. Adeno-associated viral vectors**

Adeno-associated virus (AAV) are non-enveloped virus belonging to the *Parvoviridae* family that need a helper virus, such as AdV or herpes simplex, for efficient infection and replication (Flotte, 2004). AAV are formed by a single-stranded DNA genome of 4.7 kb that contains two open reading frames (ORFs): (i) *rep*, which encodes for proteins Rep78, Rep68, Rep52 and Rep40, involved in virus genome replication, packing and integration; and (ii) *cap*, which encodes for the capsid proteins VP1, VP2 and VP3 (Wu et al., 2006). These ORFs are flanked by two Inverted Terminal Repeats (ITRs), involved in complementary DNA synthesis, Rep binding proteins, and site-specific genome integration in human chromosome 19 (Wu et al., 2006).

AAV capsid is formed by 60 subunits of VP1, VP2 and VP3 in a 1:1:10 ratio (Michelfelder & Trepel, 2009). These proteins share the C-terminal sequence, while the N-terminal sequences differ according to the start codon, being VP1>VP2>VP3 (Wu et al., 2006). Capsid structure has been elucidated for AAV2, AAV3, AAV4, AAV8 and AAV9 serotypes (Govindasamy et al., 2006; Lerch et al., 2010; Mitchell et al., 2009; Nam et al., 2007; Xie et al., 2002). The difference within capsid proteins allows for each serotype to use a specific receptor: (i) AAV2 and AAV3 use the HSPG, (ii) AAV1, 4 and 5 use glycans with sialic acid ends, and (iii) AAV8 uses the 37/67 kDa laminin receptor (Nam et al., 2007; Summerford & Samulski, 1998; Wu et al., 2006). AAV can infect a wide number of tissues including liver, lung, central nervous system, muscle, and heart; although as a result of the differences in receptor used, each serotype has a characteristic tropism (Flotte, 2004; Verma & Weitzman, 2005).

From the first clinical trial for cystic fibrosis patients with AAV vectors in 1995, over 75 clinical trials have been conducted. Pathologies such as α1-antitrypsin deficiency, Alzheimer's disease, Canavan's disease, hemophilia B, Leber congenital amaurosis, Parkinson's disease and muscular dystrophy; have attained promising results and no direct side effects have been associated with the vector (Warrington & Herzog, 2006). Although AAV vectors have shown to be promising tools for therapeutic gene delivery, they cannot transduce all cell types and could be useful to restrict its transduction to specific cell types. Strategies to modify the natural tropism of AAV vectors include: (i) the insertion of ligands into the viral capsid, (ii) use of chimeric or mosaic capsids, and (iii) conjugation with ligands

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 61

of binding to HSPG (Büning et al., 2003; Perabo et al., 2006). AAV vectors designed for vascular tissue targeting were constructed by insertion after position 587 of peptides MSLTTPPAVARP and MTPFPTSNEANL, which showed increased transduction and specificity for venous endothelial cells and reduction of hepatocytes transduction (White et al., 2004). *In-vivo* experiments with these modified AAV vectors demostrated a reduction in liver and kidney transduction, while a significantly higher and specific targeting towards vena cava cells was observed (White et al., 2004). Recently, two new peptides (CAPGPSKSC and CNHRYMQMC) were evaluated for AAV retargeting to atherosclerotic lesions (White et al., 2007). The modified vectors showed higher levels of *in vitro* transduction than those observed for the untagged vector in human, murine, and rat endothelial cells. *In vivo* experiments showed that substantial higher levels of both peptide-modified AAV2 vectors were detected in the brachiocephalic artery (the site of advanced atherosclerotic plaques) and aorta, whereas reduced levels of modified vector were detected in all other organs examined (White et al., 2007). Serpin receptor ligand inserted after position 138 increased by 15-fold the transduction of vector on a lung epithelial cell line expressing the serpin receptor (Wu et al., 2000b). Insertion of a peptide that binds to the luteinizing hormone receptor after position 139 allowed production of a modified vector able to specifically transduce ovarian cancer cells (a luteinizing hormone receptor-bearing cell line) in an HSPG-independent manner (Shi et al., 2001). Insertion after position 138 of the 28-amino acid ApoE-derived ligand led to a 90-fold increase in pancreatic islet cells *in-vitro* transduction. Additionally, a four-fold increase of human antitrypsin expression was observed compared to unmodified AAV vector transduction (Loiler et al., 2003). Viral dose can be reduced 100-fold with similar transduction results as a consequence of ApoE-derived ligand insertion with a critical impact on side effect reduction. Functional AAV-modified vectors with long peptides up to 30 kDa have been reported, yet a laborious producing protocol is necessary to increase yield

Although these studies have demonstrated the feasibility of AAV retargeting, there is only one study evaluating the therapeutic effect of a modified AAV vector. In the study, AAV vectors were designed for brain-endothelial targeting, using peptides identified by phage display (Chen et al., 2009). After intravenous administration of modified vectors in MPS VII mice, there was extensive expression of enzyme in brain, leading to correction of lysosomal

During the last years we have worked in the development of a gene therapy strategy for the Mucopolysaccharidosis IV A (Morquio A disease) by using AAV vectors (Alméciga-Díaz et al., 2010; Alméciga-Díaz et al., 2009; Gutierrez et al., 2008). Morquio A disease, as well as other mucopolysaccharidosis, has a marked bone involvement, and an effective therapy should focus on the treatment in these manifestations. Recently, we designed a bone-targeting AAV vector bearing an aspartic acid octapeptide inserted immediately after the N-terminal of the VP2 capsid protein. The vector was designed to interact with hydroxyapatite (HA), a main component of bone (Tomatsu et al., 2010). We observed that the unmodified AAV vector had low bone affinity, while the bone-targeting vector had significantly higher HA affinity with up to 36.6-fold higher vector genome copies in cortical bone compared with the unmodified vector matrix (Alméciga-Díaz CJ, Montaño A, Barrera L, Tomatsu S., unpublished data).

A mosaic capsid AAV is a virion that is composed of a mixture of viral capsid proteins from different serotypes, which are mixed during viral assembly. On the other hand, a chimeric

storage and reversal of established behavioral deficits (Chen et al., 2009).

(Warrington et al., 2004).

**4.2 Chimeric or mosaic capsids** 

through non-genetic modifications (Fig. 3) (Choi et al., 2005; Kwon & Schaffer, 2008; Michelfelder & Trepel, 2009).

Fig. 3. Strategies for AAV vectors retargeting. AAV vectors can be modified by insertion of ligands into the capsid's proteins (ligand insertion), mixing capsid proteins from different serotypes (mosaic or chimeric capsids), or absorption of molecules onto the capsid using bispecific antibodies or ligands fused with molecular bridges (non-genetic modification).

## **4.1 Targeting by ligand insertion**

Mutagenesis analysis have permitted the identification of positions within the viral capsid that allow peptide insertion with little or no effect on DNA packaging and virus trafficking. For AAV2-derived vectors, peptides inserted in positions 1, 34, 138, 161, 459, 584, 587 and 588 (relative to VP1 start codon) are displayed on the vector surface, and allow production of vectors with similar viral titers to those observed for unmodified AAV2 vectors (Shi et al., 2001; Wu et al., 2000b). Most of the studies have used positions 138 (VP2 N-terminal), 587 and 588 (HSPG binding domain) to insert peptides ranging from 5 to 272 amino acids (Loiler et al., 2003; Michelfelder et al., 2009; Nicklin et al., 2001a; Perabo et al., 2006; Shi et al., 2001; White et al., 2007; White et al., 2004; Wu et al., 2000b; Yu et al., 2009). Capsid protein modifications have improved gene delivery to lung (Kwon & Schaffer, 2008), endothelial cells (Nicklin et al., 2001a), pancreatic islets (Loiler et al., 2003), vascular tissue (White et al., 2004), atherosclerotic lesions (White et al., 2007), muscle (Yu et al., 2009), myocardium (Yang et al., 2009), and cancer cells (Michelfelder et al., 2009).

Although the therapeutic effect of these vectors still remains to be seen, there is a long list of reports showing different advantages of peptide insertion within different positions of the viral capsid. Insertion after position 588 provided a complete AAV2 retargeting with inhibition

through non-genetic modifications (Fig. 3) (Choi et al., 2005; Kwon & Schaffer, 2008;

Fig. 3. Strategies for AAV vectors retargeting. AAV vectors can be modified by insertion of ligands into the capsid's proteins (ligand insertion), mixing capsid proteins from different serotypes (mosaic or chimeric capsids), or absorption of molecules onto the capsid using bispecific antibodies or ligands fused with molecular bridges (non-genetic modification).

Mutagenesis analysis have permitted the identification of positions within the viral capsid that allow peptide insertion with little or no effect on DNA packaging and virus trafficking. For AAV2-derived vectors, peptides inserted in positions 1, 34, 138, 161, 459, 584, 587 and 588 (relative to VP1 start codon) are displayed on the vector surface, and allow production of vectors with similar viral titers to those observed for unmodified AAV2 vectors (Shi et al., 2001; Wu et al., 2000b). Most of the studies have used positions 138 (VP2 N-terminal), 587 and 588 (HSPG binding domain) to insert peptides ranging from 5 to 272 amino acids (Loiler et al., 2003; Michelfelder et al., 2009; Nicklin et al., 2001a; Perabo et al., 2006; Shi et al., 2001; White et al., 2007; White et al., 2004; Wu et al., 2000b; Yu et al., 2009). Capsid protein modifications have improved gene delivery to lung (Kwon & Schaffer, 2008), endothelial cells (Nicklin et al., 2001a), pancreatic islets (Loiler et al., 2003), vascular tissue (White et al., 2004), atherosclerotic lesions (White et al., 2007), muscle (Yu et al., 2009), myocardium (Yang

Although the therapeutic effect of these vectors still remains to be seen, there is a long list of reports showing different advantages of peptide insertion within different positions of the viral capsid. Insertion after position 588 provided a complete AAV2 retargeting with inhibition

Michelfelder & Trepel, 2009).

**4.1 Targeting by ligand insertion** 

et al., 2009), and cancer cells (Michelfelder et al., 2009).

of binding to HSPG (Büning et al., 2003; Perabo et al., 2006). AAV vectors designed for vascular tissue targeting were constructed by insertion after position 587 of peptides MSLTTPPAVARP and MTPFPTSNEANL, which showed increased transduction and specificity for venous endothelial cells and reduction of hepatocytes transduction (White et al., 2004). *In-vivo* experiments with these modified AAV vectors demostrated a reduction in liver and kidney transduction, while a significantly higher and specific targeting towards vena cava cells was observed (White et al., 2004). Recently, two new peptides (CAPGPSKSC and CNHRYMQMC) were evaluated for AAV retargeting to atherosclerotic lesions (White et al., 2007). The modified vectors showed higher levels of *in vitro* transduction than those observed for the untagged vector in human, murine, and rat endothelial cells. *In vivo* experiments showed that substantial higher levels of both peptide-modified AAV2 vectors were detected in the brachiocephalic artery (the site of advanced atherosclerotic plaques) and aorta, whereas reduced levels of modified vector were detected in all other organs examined (White et al., 2007). Serpin receptor ligand inserted after position 138 increased by 15-fold the transduction of vector on a lung epithelial cell line expressing the serpin receptor (Wu et al., 2000b). Insertion of a peptide that binds to the luteinizing hormone receptor after position 139 allowed production of a modified vector able to specifically transduce ovarian cancer cells (a luteinizing hormone receptor-bearing cell line) in an HSPG-independent manner (Shi et al., 2001). Insertion after position 138 of the 28-amino acid ApoE-derived ligand led to a 90-fold increase in pancreatic islet cells *in-vitro* transduction. Additionally, a four-fold increase of human antitrypsin expression was observed compared to unmodified AAV vector transduction (Loiler et al., 2003). Viral dose can be reduced 100-fold with similar transduction results as a consequence of ApoE-derived ligand insertion with a critical impact on side effect reduction. Functional AAV-modified vectors with long peptides up to 30 kDa have been reported, yet a laborious producing protocol is necessary to increase yield (Warrington et al., 2004).

Although these studies have demonstrated the feasibility of AAV retargeting, there is only one study evaluating the therapeutic effect of a modified AAV vector. In the study, AAV vectors were designed for brain-endothelial targeting, using peptides identified by phage display (Chen et al., 2009). After intravenous administration of modified vectors in MPS VII mice, there was extensive expression of enzyme in brain, leading to correction of lysosomal storage and reversal of established behavioral deficits (Chen et al., 2009).

During the last years we have worked in the development of a gene therapy strategy for the Mucopolysaccharidosis IV A (Morquio A disease) by using AAV vectors (Alméciga-Díaz et al., 2010; Alméciga-Díaz et al., 2009; Gutierrez et al., 2008). Morquio A disease, as well as other mucopolysaccharidosis, has a marked bone involvement, and an effective therapy should focus on the treatment in these manifestations. Recently, we designed a bone-targeting AAV vector bearing an aspartic acid octapeptide inserted immediately after the N-terminal of the VP2 capsid protein. The vector was designed to interact with hydroxyapatite (HA), a main component of bone (Tomatsu et al., 2010). We observed that the unmodified AAV vector had low bone affinity, while the bone-targeting vector had significantly higher HA affinity with up to 36.6-fold higher vector genome copies in cortical bone compared with the unmodified vector matrix (Alméciga-Díaz CJ, Montaño A, Barrera L, Tomatsu S., unpublished data).

#### **4.2 Chimeric or mosaic capsids**

A mosaic capsid AAV is a virion that is composed of a mixture of viral capsid proteins from different serotypes, which are mixed during viral assembly. On the other hand, a chimeric

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 63

(Kim et al., 2010). However, transduction efficiencies are significantly lower than that of viral vectors (Douglas, 2008). The reader is referred to other chapters in this book edition for

Although some physical methods allow gene delivery to a specific cell type, we will only consider some strategies developed to modify vector tropism. It is important to note that although most of these studies have been evaluated exclusively *in-vitro*, they constitute

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)

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

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

by engrafting with a His-tagged TNFα ligand (Herringson & Altin, 2009).

more thorough treatises of non-viral vectors.

binding (ligand should be fused with a metal).

proof-of-concept.

**5.1 Lipoplexes** 

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 transduction) (Hauck et al., 2003).

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

#### **4.3 Adsorption of receptor ligands**

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 integrin (Ponnazhagan et al., 1996).

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

#### **5. Non-viral vectors**

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 (Kim et al., 2010). However, transduction efficiencies are significantly lower than that of viral vectors (Douglas, 2008). The reader is referred to other chapters in this book edition for more thorough treatises of non-viral vectors.

Although some physical methods allow gene delivery to a specific cell type, we will only consider some strategies developed to modify vector tropism. It is important to note that although most of these studies have been evaluated exclusively *in-vitro*, they constitute proof-of-concept.
