**2. Retroviral vectors**

Retroviruses are a family of enveloped, diploid, and positive-stranded RNA virus. This family is formed by alpharetrovirus, betaretrovirus, deltaretrovirus epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus (foamy virus), from which only the last three have been used as gene therapy vectors (Verma & Weitzman, 2005). The genome basically consists of three genes: (i) *gag*, which encodes for viral matrix, capsid and nucleocapsid proteins, (ii) *pol*, which encodes for protease, reverse transcriptase and integrase, and (iii) *env*, which encodes a bipartite membrane-anchored surface (SF) protein (Escors & Breckpot, 2010). In addition, complex retroviruses (e.g. lentivirus and foamy virus) have accessory genes for the regulation of gene expression, assembly and replication (Escors & Breckpot, 2010). In the viral life cycle, glycoproteins (GPs) play an important role in viral tropism, receptor recognition and cell entry (Liang et al., 2009). The binding of the viral GP to the cellular surface protein induces a conformational change into the viral GP and leads to fusion and insertion of the capsid into the cytoplasm (Yu & Schaffer, 2005). Most of the advantages and limitations of retrovirus targeting are related with this cell entry mechanism.

Retroviral vectors used in gene therapy have properties that allow the transduction of a broad array of cells (Cronin et al., 2005; Frecha et al., 2008). However, the use of targeted retroviral vectors allowing the delivery of the transgene to cells which are not normally transduced, or limiting the delivery to cells which should not be transduced would be advantageous (Cronin et al., 2005; Frecha et al., 2008; Lavillette et al., 2001). In general, retroviral targeting can be achieved by: (i) engineering the GP to produce a vector that recognizes a specific cell surface protein (direct targeting), (ii) pseudotyping with GPs derived from other retrovirus and that recognize specific receptors, (iii) using ligands that block the cell entry of the vector to the cells expressing the receptor for the ligand, while allow the transduction of others cell types (indirect targeting), or (iv) using biospecific antibodies, chemically crosslinking ligands and grafting polymers (Fig. 1) (Lavillette et al., 2001; Verhoeyen & Cosset, 2004; Yu & Schaffer, 2005).

#### **2.1 Retroviral targeting by engineering the glycoproteins**

Direct targeting consists of the insertion of ligands or antibody fragments into the viral GP (Fig. 1), which allows recognition of cell surface proteins different from those naturally used by the vector (Lavillette et al., 2001). Direct modification of a GP was first reported in 1990, with the successful fusion of CD4 with the GP of a gammaretroviral vector (Young et al., 1990). Later, a functional murine leukemia virus (MLV) vector bearing a GP protein fused with an antibody fragment against a hapten (4-hidroxy-5-iodo-3-nitrophenacetyl caproate) demonstrated binding of the modified retroviral vector to hapten, and the ability to package a transgene and transduce culture cells (Russell et al., 1993).

transfected gene product. In addition, to date gene therapy still encounters two main problems: (i) gene delivery to the central nervous system (CNS), as the gene or its product are naturally blocked by the blood brain barrier; and (ii) gene delivery into poorly circulated tissues such as bone, where up until now, it has only been achieved with low transfection efficiencies. In this chapter, we will explore the modifications carried out in viral and non-viral vectors to enhance their natural tropism. Regardless of the vector, modification objectives can be summarized in: (i) tropism expansion to permit gene delivery to cells not easily transfected with non-modified vector, (ii) cell-type specific transfection reducing side effects, and (iii) stealth vector improvement to reduce protein interactions or cells that limit its therapeutic

Retroviruses are a family of enveloped, diploid, and positive-stranded RNA virus. This family is formed by alpharetrovirus, betaretrovirus, deltaretrovirus epsilonretrovirus, gammaretrovirus, lentivirus, and spumavirus (foamy virus), from which only the last three have been used as gene therapy vectors (Verma & Weitzman, 2005). The genome basically consists of three genes: (i) *gag*, which encodes for viral matrix, capsid and nucleocapsid proteins, (ii) *pol*, which encodes for protease, reverse transcriptase and integrase, and (iii) *env*, which encodes a bipartite membrane-anchored surface (SF) protein (Escors & Breckpot, 2010). In addition, complex retroviruses (e.g. lentivirus and foamy virus) have accessory genes for the regulation of gene expression, assembly and replication (Escors & Breckpot, 2010). In the viral life cycle, glycoproteins (GPs) play an important role in viral tropism, receptor recognition and cell entry (Liang et al., 2009). The binding of the viral GP to the cellular surface protein induces a conformational change into the viral GP and leads to fusion and insertion of the capsid into the cytoplasm (Yu & Schaffer, 2005). Most of the advantages and limitations of retrovirus

Retroviral vectors used in gene therapy have properties that allow the transduction of a broad array of cells (Cronin et al., 2005; Frecha et al., 2008). However, the use of targeted retroviral vectors allowing the delivery of the transgene to cells which are not normally transduced, or limiting the delivery to cells which should not be transduced would be advantageous (Cronin et al., 2005; Frecha et al., 2008; Lavillette et al., 2001). In general, retroviral targeting can be achieved by: (i) engineering the GP to produce a vector that recognizes a specific cell surface protein (direct targeting), (ii) pseudotyping with GPs derived from other retrovirus and that recognize specific receptors, (iii) using ligands that block the cell entry of the vector to the cells expressing the receptor for the ligand, while allow the transduction of others cell types (indirect targeting), or (iv) using biospecific antibodies, chemically crosslinking ligands and grafting polymers (Fig. 1) (Lavillette et al., 2001; Verhoeyen & Cosset, 2004; Yu & Schaffer, 2005).

Direct targeting consists of the insertion of ligands or antibody fragments into the viral GP (Fig. 1), which allows recognition of cell surface proteins different from those naturally used by the vector (Lavillette et al., 2001). Direct modification of a GP was first reported in 1990, with the successful fusion of CD4 with the GP of a gammaretroviral vector (Young et al., 1990). Later, a functional murine leukemia virus (MLV) vector bearing a GP protein fused with an antibody fragment against a hapten (4-hidroxy-5-iodo-3-nitrophenacetyl caproate) demonstrated binding of the modified retroviral vector to hapten, and the ability to package

activity, e.g. immune system (Yu & Schaffer, 2005).

targeting are related with this cell entry mechanism.

**2.1 Retroviral targeting by engineering the glycoproteins** 

a transgene and transduce culture cells (Russell et al., 1993).

**2. Retroviral vectors** 

After these pioneer works revealed the feasibility of GP engineering, different authors studied the real potential of this targeting approach. The fusion of the erythropoietin polypeptide with the GP of a MLV vector showed for the first time that a chimeric GP was not only correctly incorporated into the virus envelope but also allowed the specific transduction of cells expressing the EPO receptor (Kasahara et al., 1994). A similar result was reported with a gammaretrovirus bearing a GP fused with a single-chain antigen-binding site against a cell surface expressed on human carcinoma cells (Kasahara et al., 1994).

Nevertheless, different studies have exposed the limitations of this approach. The first evidence of this limitation was observed with vectors carrying the Ram-1 phosphate transporter or the epidermal growth factor (EFG) into the GP, which showed a reduced transduction efficiency although the chimeric GPs were correctly expressed, processed, and exposed in the viral envelope (Cosset et al., 1995). A similar observation was reported for a gammaretroviral vector with a GP bearing a peptide binding to the αvβ3 integrin (Wu et al., 2000a), or with the stromal cell derived factor 1-alpha (Katane et al., 2002). These low transductions efficiencies were associated with loss or impairment of viral and cell surface proteins binding and subsequent membrane fusion and penetration of the viral core into the cytosol (Frecha et al., 2008; Lavillette et al., 2001; Yu & Schaffer, 2005). In addition, these results strongly suggest that only few set of peptides sequences can be inserted into the GPs without altering the mechanism of retrovirus cell entry (Verhoeyen & Cosset, 2004). The insertion of spacer sequences between the inserted peptide and the GP has proved to overcome this issue, allowing a significant improvement in the transduction efficiency of the modified vectors (Kayman et al., 1999).

#### **2.2 Retroviral targeting by pseudotyping**

Pseudotyping involves the production of viral particles bearing GP derived from other enveloped viruses (Fig. 1). This strategy was developed due to the narrow cellular transduction profile observed in vectors with native GPs (Cronin et al., 2005; Yu & Schaffer,

Fig. 1. Strategies for retroviral targeting. Retroviral vectors can be modified by ligand or antibody fraction insertion (engineered glycoproteins), use of glycoprotein (GP) from other retrovirus (pseudotyping), or the use of bi-specific antibodies (non-genetic targeting).

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 55

positive hematopoietic cells were exposed to the above mentioned vectors, cancer cells were selectively transduced by the SCF-displaying vector, whereas hematopoietic cells were selectively transduced by the EGF-displaying vector. *In-vivo* proof-of-concept of this strategy was established after intravenous infusion of an EGF-displaying HIV-1 vector, showing preferable transduction of spleen with very low levels on EGF-receptor rich tissues (e.g. liver). Furthermore, the VSV-G vector transduced heart, skeletal muscle, lung, brain, kidney, ovaries and bone-marrow (Peng et al., 2001). Transduction inhibition of human and canine cells was also observed for a spleen necrosis virus bearing EFG, which was restored after cleavage via factor Xa at a site located between EFG and GP (Merten et al., 2003). A variant of this strategy takes advantage of protease-activatable gene delivery vehicles. Matrix metalloproteinases (MMPs) are commonly overexpressed in angiogenesis, inflammation, and cancer invasion. The inclusion of a MMPs-cleavable peptide within an EGF-retroviral vector permitted that in the presence of exogenous MMPs, the infectivity of the MMP-EGF vector but not of the EGF-vector, could be restored. This MMPss-sensitive vector could efficiently discriminate between two different cell types, infecting only MMP-

This strategy involves the use of antibodies or adapter molecules bound to the GP (Fig. 1) (Lavillette et al., 2001; Yu & Schaffer, 2005). This was the first approach used for cell targeting of a gammaretrovirus by using two biotinilated antibodies bridged by streptavidin: one against a GP and the other one against a specific cell membrane marker (Roux et al., 1989). This strategy allowed the specific transduction of cells expressing class I or II major histocompatibility complex (MHC) molecules by using monoclonal antibodies against these MHC molecules and against GP from the retroviral vector (Roux et al., 1989). For targeting of a retroviral vector to folate receptor-expressing epidermoid carcinoma cells, a myeloproliferative sarcoma retrovirus was modified with folic acid (Reddy et al., 2001). Although receptor binding was observed, this vector was not able to induce gene expression. Similar results were observed for a MLV vector bearing a single chain anti-folate receptor antibody to produce vectors targeting ovarian cancer cells (Pizzato et al., 2001), manifesting the limitations of folate receptor targeting. A modification of this approach combines genetic modification of the GP by inclusion of an IgG-binding domain and the conjugation with an antibody that reacts with specific cell surface molecules expressing the antigen (Ohno et al., 1997). By changing the monoclonal antibody it was possible to transduce efficiently and specifically a variety of cell lines. The potential of this approach was confirmed by *in-vivo* evaluation of modified vectors conjugated with antibodies against surface proteins expressed in cancer cells, indicating that after intravenous infusion the vector preferentially transduced these cells (Liang et al., 2009). This strategy has been used both with gammaretroviral and lentiviral vectors. It represents a powerful tool that can be easily modified to obtain a site-specific gene expression for the treatment of cancers, genetic,

An adenovirus (AdV) is a non-enveloped virus formed by an icosahedral protein capsid surrounding a lineal double-stranded DNA of 36 kb (Douglas, 2007). The AdV capsid is characterized by the presence of 252 different capsomers and long fibers protruding from

positive cells (Peng et al., 1997).

infectious and immune diseases.

**3. Adenoviral vectors** 

**2.4 Non-genetic retroviral targeting** 

2005). Production of pseudotyped retroviral vectors is a well-established protocol (Bischof & Cornetta, 2010), which in most cases is used to allow virus purification, improve the cellular transduction, extend the range of transduced cells, and reduce cell toxicity (Cronin et al., 2005). However, it should be possible to employ natural tropism in GPs used for pseudotyping to target vectors bearing these proteins.

A large set of GPs has been evaluated for vector pseudotyping, among which vesicular stomatitis virus glycoprotein (VSV-G) is the most used [reviewed in (Bischof & Cornetta, 2010; Cronin et al., 2005)]. However, only a few GPs have allowed the transduction of different cell types compared to those observed VSV-G pseudotyped vectors. The use of a GP derived from rabies virus (RV) for pseudotyping an HIV-1 lentiviral vector allowed the transduction of neurons usually non-transduced with VSV-G vectors, since the RV glycoprotein induced a retrograde transport along axons (Mazarakis et al., 2001). The infusion of an animal model of familial amyotrophic lateral sclerosis (ALS) with a RV-HIV-1 vector carrying the gene of vascular endothelial growth factor (VEGF) delayed the onset of the disease and slowed progression in treated animals. Although treatment was initiated at the onset of paralysis CNS tropism of the vector was still significantly improved (Azzouz et al., 2004).

An interesting tropism was also observed for an HIV-1 vector pseudotyped with a GP from the lymphocytic choriomeningitis virus (LCMV) (Miletic et al., 2004). *In-vitro* and *in-vivo* evaluation of a vector pseudotyped with a LCMV GP showed that it transduced, almost exclusively, astrocytes; while VSV-G pseudotypes vectors infected neurons as well as astrocytes. In addition, LCMV-HIV-1 vector presented a specific transduction of infiltrating tumor cells, while VSV-G-HIV-1 vectors transduced mostly normal brain cells in infiltrating tumor areas. An HIV-1 vector pseudotyped with an Ebola Zaire virus-derived GP appeared useful in the treatment of airway diseases (e.g. cystic fibrosis). This vector allowed *in-vitro* and *in-vivo* transduction of airway epithelial cells, which was not observed with a VSV-G vector (Kobinger et al., 2001).

A recent modification of this approach used an HIV-1 vector pseudotyped with the GP and the fusion protein from a measles virus. The GP had an epidermal growth factor (EGF) or a single chain antibody against CD20 (Funke et al., 2008). The CD20-bearing vector was able to transduce primary and CD20-positive B cells both alone or within a cell mixture, while a VSV-G vector did not transduce these cell types.
