**Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues**

Carlos J. Alméciga-Díaz, Rocio Cuaspa and Luis A. Barrera *Institute for the Study of Inborn Errors of Metabolism Pontificia Universidad Javeriana, Bogotá D.C. Colombia* 

### **1. Introduction**

50 Non-Viral Gene Therapy

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S.D.L. (2006). Effect of different nuclear localization sequences on the immune responses induced by a MIDGE vector encoding bovine herpesvirus-1 glycoprotein The gene therapy concept was developed more than forty years ago. Edward Tatum in a historic lecture affirmed: "*We can anticipate that viruses will be used effectively for man's benefit, in theoretical studies concerning somatic cell genetics and possibly in genetic therapy... We even can be somewhat optimistic about the long-range possibility of therapy based on the isolation or design, synthesis, and introduction of new genes into defective cells of particular organs*". Two years later Marshall Niremberg predicted that: "*in 25 years it would be possible to program the cells with synthetic messages*", but called to attention that "*it should be postponed until having the sufficient wisdom to use this knowledge for the benefit of mankind*".

Initial efforts to construct vectors were achieved by Rogers and Pfudere in 1968. However, it was not until 1990 that the first gene therapy clinical trial was carried-out in a patient with adenosine deaminase (ADA) deficiency (Friedmann, 1992). Within the last several years, significant advances have been attained in therapies for cancer, AIDS, Parkinson's disease, Xlinked severe combined immunedeficiency (X-SCID), hemophilias, cystic fibrosis, Leber's congenital amaurosi, and β-thalassemia, among others. Nonetheless, an adolescent patient suffering from a urea cycle defect died of an anaphylactic reaction a few hours after being injected with an adenoviral vector. Soon after, five children participating in a clinical trial to correct a X-SCID developed a leukemia-like condition due to the activation of an oncogene (Edelstein, 2007). Recently, the tragic death of a volunteer in an clinical trial for rheumatoid arthritis using an adenoassociated virus vector was initially associated to a side effect of the vector (Williams, 2007). However, further studies showed that this was caused by an infection with *Histoplasma capsulatum*, which was produced by an immunosuppression condition induced for a simultaneous systemic anti-TNF-alpha therapy in the form of the drug adalimumab (Williams, 2007). All these failures halted the progress of gene therapy and seriously questioned the efficacy of the procedure. It also prompted the need for more basic studies on the immunogenic aspects and how to make safer, and more efficient vectors.

During the last 35 years a long list of viral and non-viral vectors have been developed to design a vector that allows gene delivery to specific cell types, has a high gene transfer efficiency, produces therapeutic levels of gene expression during long-term periods, and minimizes the generation of side effects (Verma & Weitzman, 2005). Although the field has made great strides in producing an ideal vector, one of the main challenges remains to aim for a cell-specific vector. Most probably a vector left in the general circulation without a specific targeting signal, will be sequestered by the liver or may end-up in cells with no need of the

Gene Delivery Systems: Tailoring Vectors to Reach Specific Tissues 53

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

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

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).

surface expressed on human carcinoma cells (Kasahara et al., 1994).

modified vectors (Kayman et al., 1999).

**2.2 Retroviral targeting by pseudotyping** 

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