Adeno Associated Viral Vector

Recently, AAV vectors have been utilized most extensively in preclinical and clinical muscle gene therapy studies. AAV is a nonenveloped, single-stranded DNA virus with a gene insert size capacity of about 4.7 kb for AAV serotype 2 (Srivastava et al. 1983). The discovery of different AAV serotypes has led to AAV vectors that have differing tissue tropisms and transduction efficiencies. AAV infect dividing and non-dividing cells and appear to have lower tendency to induce immunity compared to many other vectors. AAV vectors have been studied in utero in various animal models such as mice (Bilbao et al. 2005a), rats (Garrett et al. 2003), rabbits (Boyle et al. 2001), and primates (Garrett et al. 2003; Larson et al. 2000; Nathwani et al. 2007).

The first AAV vector muscle gene transfer studies in utero were done with direct intramuscular injections into the fetus. Mitchell et al. showed that the intramuscular injection of AAV2 vector with a transgene driven by the HCMV promoter produced persistent expression in muscles up to 3 months after birth (Mitchell et al. 2000). Similarly, Schneider et al. showed b-galactosidase expression in muscle tissues up to 6 weeks after in utero intramuscular injections of AAV2 vector carrying a CMV-driven lacZ transgene (Schneider et al. 2002). In addition, another study by Schneider et al. using AAV2 and AAV5 vectors by intramuscular injection demonstrated muscle transgene expression up to 18 weeks (Schneider et al. 2002). Furthermore, intramuscular injection studies of AAV1 (Bilbao et al. 2005a), and AAV2 vectors into fetal muscle demonstrated good transgene expression (Mitchell et al. 2000; Yang et al. 1999). Moreover Bilbao et al. showed that intramuscular injection of AAV1 vector transduction to be 20 times higher compared to AAV2 vector transduction of skeletal muscles 4 weeks after in utero treatment (Bilbao et al. 2005a). This high transgene expression from an intramuscular administration of AAV1 vector in utero compared to AAV2 vector was further corroborated by Sabatino et al (Sabatino et al. 2007).

There is limited experience with systemic AAV vector gene delivery to muscle in utero. Intraperitoneal injection of AAV2 vector carrying an EF1a promoter-driven luciferase transgene demonstrated transgene expression in various tissues including heart (Lipshutz et al. 2001). This study demonstrated the absence of germ line integration, hepatotoxicity, and antibodies against the vector or the transgene (Lipshutz et al. 2001). Bilbao et al. demonstrated that systemic delivery of AAV1 vector in utero transduced diaphragm significantly better than the limb muscle tissue (Bilbao et al. 2005a). The recently utilized serotypes of AAV vector, such as AAV8 vector carrying a lacZ reporter gene, showed a high level of transduction in the diaphragm and a more moderate and mosaic pattern of transduction of multiple limb muscles and heart (BMK and PRC, in press). Furthermore, recently in utero gene delivery of an AAV8 vector carrying a truncated dystrophin cDNA not only restored the dystrophin associated glycoprotein complex proteins, a-sarcoglycan and b-dystroglycan, but also provided a functional benefit in mdx diaphragm (BMK and PRC, unpublished data).

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