**2.3 Indirect retroviral targeting**

This strategy is based on the finding that inclusion of certain peptides into the GP significantly impairs transduction of some cells. This prohibits gene delivering into cells that should not be transduced and extends viral tropism (Lavillette et al., 2001; Yu & Schaffer, 2005). First evidence of this approach was discovered while producing a MLV vector bearing a GP fused with epidermal growth factor (EGF). Although the chimeric GP was correctly expressed and processed, and the modified vector had the ability to bind to the EGF receptor, this manipulation completely inhibited vector transduction into cells expressing the EGF receptor (Cosset et al., 1995). This transduction inhibition was produced because of the modified vector was directed to the late endosome and destroyed by lysosomal enzymes.

A similar result was observed with a vector carrying the stem cell factor (SCF, a c-Kit receptor ligand), which selectively inhibited vector transduction on c-Kit-expressing cells (Fielding et al., 1998). When a mixture of EGF receptor-positive cancer cells and c-Kitpositive 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 MMPpositive cells (Peng et al., 1997).

#### **2.4 Non-genetic retroviral targeting**

54 Non-Viral Gene Therapy

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

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

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

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

This strategy is based on the finding that inclusion of certain peptides into the GP significantly impairs transduction of some cells. This prohibits gene delivering into cells that should not be transduced and extends viral tropism (Lavillette et al., 2001; Yu & Schaffer, 2005). First evidence of this approach was discovered while producing a MLV vector bearing a GP fused with epidermal growth factor (EGF). Although the chimeric GP was correctly expressed and processed, and the modified vector had the ability to bind to the EGF receptor, this manipulation completely inhibited vector transduction into cells expressing the EGF receptor (Cosset et al., 1995). This transduction inhibition was produced because of the modified vector was directed to the late endosome and destroyed by

A similar result was observed with a vector carrying the stem cell factor (SCF, a c-Kit receptor ligand), which selectively inhibited vector transduction on c-Kit-expressing cells (Fielding et al., 1998). When a mixture of EGF receptor-positive cancer cells and c-Kit-

pseudotyping to target vectors bearing these proteins.

et al., 2004).

vector (Kobinger et al., 2001).

**2.3 Indirect retroviral targeting** 

lysosomal enzymes.

VSV-G vector did not transduce these cell types.

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, infectious and immune diseases.

#### **3. Adenoviral vectors**

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

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 57

Fig. 2. Strategies for Adenoviral vectors targeting. Adv vectors can be modified by ligand or antibody fraction insertion (genetic modification), use of bi-specific antibodies (molecular adaptors), or conjugation with polymers bearing or not a ligand (chemical modification).

2007). A similar approach involves the pre-selection of peptides by using a phage display peptide library, from which peptides with high affinity for a certain cell type are selected and then inserted into the AdV vector capsid. This has allowed the targeting of AdV vectors to epithelium cells (Nicklin et al., 2001b), tumor cells (Rittner et al., 2007), and neurons

Most efforts to modify the AdV vector capsid have been on vector targeting to tumor cells (Bachtarzi et al., 2008). Some examples of this approach include: (i) production of an AdV vector bearing a CAR protein fused with EGF producing an enhanced gene transfer efficiency in pancreatic carcinoma cells (Wesseling et al., 2001); (ii) a vector with a polylysine motif in the fiber for a CAR-independent binding to HSPG for specific transduction of different breast cancer cells (Ranki et al., 2007); and (iii) vectors with high affinity for prostate adenocarcinoma

In addition to cancer cell, genetically modified AdV vectors have been used as tools for gene delivery to other cell types. The insertion of an RGD-modified AdV allowed the production of a vector that mediated cell entry via RGD binding to integrins. Administration into the synovial lining improved the outcome of gene therapy for arthritis (Bakker et al., 2001). Targeting to endothelial cells, an important target in vascular gene therapy, was possible after insertion of the SIGYPLP peptide into the fiber protein in combination with fiber mutations that block natural CAR binding (detargeting) (Nicklin et al., 2001b). For specific kidney gene delivery, the intravenous administration of HTTHREP- and HITSLLS-bearing AdV vectors resulted in selective renal targeting, specifically of tubular epithelium and

(Wu et al., 2010), and human colon carcinoma cells (Rittner et al., 2007).

(Schmidt et al., 2007).

glomeruli (Denby et al., 2007).

each of the twelve vertices. Seven polypeptides form this complex capsid: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII, and IX (Campos & Barry, 2007; Sharma et al., 2009). The cell receptor depends on the virus subgroup: A, E and F subgroups use the cell surface coxsackievirus B and adenovirus receptor (CAR); B1 and B2 subgroups use CD46, CD80/86, receptor X, or heparan sulfate proteoglycan (HSPG); C subgroup uses CAR, HSPG, MHC-I, vascular cell adhesion molecule-I (VCAM-I), or integrins; and D subgroup uses CAR, sialic acid, or CD46 (Arnberg, 2009; Campos & Barry, 2007; Sharma et al., 2009).

Adenoviral vectors have been used as tools for gene therapy since the late 80s (Friedmann, 1992), and the first clinical trial was started in 1993 (Douglas, 2007). Since then, over 392 clinical trials using AdV vectors have been carried out. Currently AdV are the most used vectors for gene therapy and represent 24% of clinical trials total (Edelstein, 2007). In addition, the first commercially approved gene therapy product, Gendicine, is based on an AdV vector engineered to express p53 for treatment of patients with head and neck squamous cell carcinoma (Peng, 2005).

AdV vectors are mainly derived from serotypes 2 (AdV2) and 5 (AdV5). They have the advantage of inducing high levels of gene expression, are able to infect both diving and nondividing cells, and can be purified to high titers. Furthermore, AdV2 and AdV5 have high bloodstream stability with a reduced risk of insertional mutagenesis, since their genome remains extrachromosomal (Douglas, 2007; Edelstein, 2007; Volpers & Kochanek, 2004). Nonetheless, several drawbacks have been identified during *in-vivo* evaluation of these vectors: (i) presence of pre-existing antibodies that rapidly neutralize the vector, (ii) after intravenous administration the vector is mainly taken up by liver cells, limiting the vector to reach its target tissue in adequate concentrations, and (iii) use of high doses in an effort to overcome these problems has not proven to be an adequate and safe approach (Arnberg, 2009). Three strategies can be used to solve these issues: (i) use of vectors from other subgroups different from AdV2 and AdV5 (subgroup C), (ii) use of non-human serotypes, and (iii) retargeting of the vector to cells or tissues of interest (Arnberg, 2009). In this section we will explore different strategies for AdV retargeting that include genetic modification of the capsid, use of molecular adaptors and chemical modification of the capsid (Fig. 2) (Campos & Barry, 2007).

#### **3.1 Genetic modification of the AdV capsid**

Genetic modification of the viral capsid involves incorporation of foreign peptides into exposed regions of the capsid. In this manner the gene encoding for the peptide is inserted into the vector's genome (Fig. 2) (Campos & Barry, 2007). The possibility of altering the AdV vector capsid was first evaluated by the insertion of octapeptides into the hexon (Crompton et al., 1994) or the fiber knob (Krasnykh et al., 1998). This established the possibility of producing viable vectors with foreign proteins present on the vector capsid surface. Further experiments revealed the possibility of expanding the vector tropism by inserting a heparinbinding domain (Wickham et al., 1996), or an Arg-Gly-Asp (RGD)-containing peptide into the fiber knob (Dmitriev et al., 1998).

Although different studies indicated the feasibility to target vectors to specific cell types, genetic modification often resulted in failure to rescue viable viruses, or in an impaired virus packing, peptide exposure, and vector transduction (Leissner et al., 2001; Wu et al., 2005). Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, use of random peptide libraries displayed directly on the AdV capsid allows isolation of viable vectors with high affinity for specific tissues or cells (Miura et al.,

each of the twelve vertices. Seven polypeptides form this complex capsid: hexon (II), penton base (III), fiber (IV), IIIa, VI, VIII, and IX (Campos & Barry, 2007; Sharma et al., 2009). The cell receptor depends on the virus subgroup: A, E and F subgroups use the cell surface coxsackievirus B and adenovirus receptor (CAR); B1 and B2 subgroups use CD46, CD80/86, receptor X, or heparan sulfate proteoglycan (HSPG); C subgroup uses CAR, HSPG, MHC-I, vascular cell adhesion molecule-I (VCAM-I), or integrins; and D subgroup uses CAR, sialic acid, or CD46 (Arnberg, 2009; Campos & Barry, 2007; Sharma

Adenoviral vectors have been used as tools for gene therapy since the late 80s (Friedmann, 1992), and the first clinical trial was started in 1993 (Douglas, 2007). Since then, over 392 clinical trials using AdV vectors have been carried out. Currently AdV are the most used vectors for gene therapy and represent 24% of clinical trials total (Edelstein, 2007). In addition, the first commercially approved gene therapy product, Gendicine, is based on an AdV vector engineered to express p53 for treatment of patients with head and neck

AdV vectors are mainly derived from serotypes 2 (AdV2) and 5 (AdV5). They have the advantage of inducing high levels of gene expression, are able to infect both diving and nondividing cells, and can be purified to high titers. Furthermore, AdV2 and AdV5 have high bloodstream stability with a reduced risk of insertional mutagenesis, since their genome remains extrachromosomal (Douglas, 2007; Edelstein, 2007; Volpers & Kochanek, 2004). Nonetheless, several drawbacks have been identified during *in-vivo* evaluation of these vectors: (i) presence of pre-existing antibodies that rapidly neutralize the vector, (ii) after intravenous administration the vector is mainly taken up by liver cells, limiting the vector to reach its target tissue in adequate concentrations, and (iii) use of high doses in an effort to overcome these problems has not proven to be an adequate and safe approach (Arnberg, 2009). Three strategies can be used to solve these issues: (i) use of vectors from other subgroups different from AdV2 and AdV5 (subgroup C), (ii) use of non-human serotypes, and (iii) retargeting of the vector to cells or tissues of interest (Arnberg, 2009). In this section we will explore different strategies for AdV retargeting that include genetic modification of the capsid, use of molecular adaptors and chemical modification of the capsid (Fig. 2)

Genetic modification of the viral capsid involves incorporation of foreign peptides into exposed regions of the capsid. In this manner the gene encoding for the peptide is inserted into the vector's genome (Fig. 2) (Campos & Barry, 2007). The possibility of altering the AdV vector capsid was first evaluated by the insertion of octapeptides into the hexon (Crompton et al., 1994) or the fiber knob (Krasnykh et al., 1998). This established the possibility of producing viable vectors with foreign proteins present on the vector capsid surface. Further experiments revealed the possibility of expanding the vector tropism by inserting a heparinbinding domain (Wickham et al., 1996), or an Arg-Gly-Asp (RGD)-containing peptide into

Although different studies indicated the feasibility to target vectors to specific cell types, genetic modification often resulted in failure to rescue viable viruses, or in an impaired virus packing, peptide exposure, and vector transduction (Leissner et al., 2001; Wu et al., 2005). Because the insertion of a pre-selected peptide into a fiber knob often fails to generate an adenovirus vector, use of random peptide libraries displayed directly on the AdV capsid allows isolation of viable vectors with high affinity for specific tissues or cells (Miura et al.,

et al., 2009).

squamous cell carcinoma (Peng, 2005).

(Campos & Barry, 2007).

**3.1 Genetic modification of the AdV capsid** 

the fiber knob (Dmitriev et al., 1998).

Fig. 2. Strategies for Adenoviral vectors targeting. Adv vectors can be modified by ligand or antibody fraction insertion (genetic modification), use of bi-specific antibodies (molecular adaptors), or conjugation with polymers bearing or not a ligand (chemical modification).

2007). A similar approach involves the pre-selection of peptides by using a phage display peptide library, from which peptides with high affinity for a certain cell type are selected and then inserted into the AdV vector capsid. This has allowed the targeting of AdV vectors to epithelium cells (Nicklin et al., 2001b), tumor cells (Rittner et al., 2007), and neurons (Schmidt et al., 2007).

Most efforts to modify the AdV vector capsid have been on vector targeting to tumor cells (Bachtarzi et al., 2008). Some examples of this approach include: (i) production of an AdV vector bearing a CAR protein fused with EGF producing an enhanced gene transfer efficiency in pancreatic carcinoma cells (Wesseling et al., 2001); (ii) a vector with a polylysine motif in the fiber for a CAR-independent binding to HSPG for specific transduction of different breast cancer cells (Ranki et al., 2007); and (iii) vectors with high affinity for prostate adenocarcinoma (Wu et al., 2010), and human colon carcinoma cells (Rittner et al., 2007).

In addition to cancer cell, genetically modified AdV vectors have been used as tools for gene delivery to other cell types. The insertion of an RGD-modified AdV allowed the production of a vector that mediated cell entry via RGD binding to integrins. Administration into the synovial lining improved the outcome of gene therapy for arthritis (Bakker et al., 2001). Targeting to endothelial cells, an important target in vascular gene therapy, was possible after insertion of the SIGYPLP peptide into the fiber protein in combination with fiber mutations that block natural CAR binding (detargeting) (Nicklin et al., 2001b). For specific kidney gene delivery, the intravenous administration of HTTHREP- and HITSLLS-bearing AdV vectors resulted in selective renal targeting, specifically of tubular epithelium and glomeruli (Denby et al., 2007).

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 59

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

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

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

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

vector tropism was not clearly reported in this study (Park et al., 2010).

mixed culture and also in xenografts *in-vivo* (Fisher et al., 2001).

**4. Adeno-associated viral vectors** 

19 (Wu et al., 2006).

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