253. An AAV Vector-Mediated Gene Transfer into Canine Skeletal Muscle

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Duchenne muscular dystrophy (DMD) is an X-linked, lethal muscle disorder caused by mutations in the dystrophin gene (14 kb cDNA). An adeno-associated virus (AAV) vector-mediated gene transfer is one of attractive approaches to the treatment of DMD, but it has a limitation in insertion size up to 4.9 kb. We recently demonstrated that the AAV vector-mediated micro-dystrophin cDNA transfer could ameliorate dystrophic phenotypes in skeletal muscles of dystrophin-deficient mdx mice (Mol Ther 10: 821-828, 2004). For clinical application, it is important to examine therapeutic effects and the safety issue in larger animal models, such as dystrophic dogs. We established a colony of beagle-based canine X-linked muscular dystrophy in Japan (Exp Anim. 52: 93-97, 2003). To investigate transduction efficiency in canine skeletal muscle using an AAV vector, we injected the AAV vector encoding the LacZ gene driven by a CMV promoter (1.0-2.0 |[times]| 1013 vg/ml, 100-500 |[mu]|l/muscle) into skeletal muscles of normal dogs. |[beta]|-galactosidase (|[beta]|-gal) was expressed only in few fibers at 2 weeks after the injection, and not detected at 4 or 8 weeks after the injection. Instead, large numbers of mononuclear cells appeared around |[beta]|-gal-expressing fibers. To clarify mechanisms of low transduction and cellular infiltration in canine muscle after transfer of the AAV vector, we examined viral infectivity in vitro, cytotoxicity and immune responses of AAV vector transduction in vivo. First, we infected the AAV vector into canine primary myotubes. This in vitro study showed that the AAV vector could allow higher transgene expression in canine myotubes than in murine ones. Second, we tested whether injection of AAV particles elicit cytotoxicity or not. When a promoter-less AAV vector expressing no transgene (5 |[times]| 1012 vg/muscle) was injected into canine muscle, almost no infiltrating cells was observed in injected muscle. Third, we investigated immune responses. A lot of CD4- or CD8-positive cells were detected in clusters of infiltrating cells, together with elevated serum level of anti-|[beta]|-gal IgG. To confirm low transduction depending on immune response, dogs received daily oral administration of cyclosporine (20 mg/kg/day) from |[ndash]|5 day of the introduction of the AAV vector. Immunosuppression largely but not completely improves transduction efficiency of the AAV vector. These results suggest that AAV vector-mediated gene transfer elicits stronger immune responses in canine muscle, but immune responses against transgene products can not thoroughly explain the phenomenon. Cellular toxicity of transgene products might also participate in these infiltrations, while cytotoxicity and immunity of the AAV particles themselves can be negligible based on the result of a promoter-less AAV vectors. It is indispensable to know the molecular background of excess immune responses and cellular toxicity in canine models to establish AAV vector-mediated gene transfer in dystrophic patients.