Advances in recombinant adeno-associated virus technology and implications for the treatment of Duchenne muscular dystrophy

Recombinant adeno-associated virus (rAAV) based vectors have emerged as widely used gene transfer vehicles in a variety of gene therapy protocols. With recent engineering of truncated, yet highly functional dystrophin proteins (micro-dystrophins), rAAV vectors have begun to be widely exploited for the development of gene therapies for the treatment of Duchenne muscular dystrophy (DMD). Currently gene therapy protocols for the treatment of DMD could benefit from the characterization of rAAV vectors that more efficiently transduce the striated musculature as well as development of a simple method to deliver genes to the entire striated musculature, a target comprising approximately 40% of the biomass. This dissertation describes the characterization of muscle transduction using rAAV vectors pseudo-typed with the serotype 6 capsid (rAAV6). We demonstrate that rAAV6 vectors transduce striated muscles with a faster onset of expression and up to 500 fold more efficiently than the commonly used rAAV2 based vectors. This high efficiency allows for transduction of the murine diaphragm following intra-thoracic delivery and body-wide transduction following intra-peritoneal delivery into neonate mouse pups. We also describe the first reported method for simultaneous transduction of all striated muscles in the mouse, including the heart and diaphragm, following a single intravenous injection of rAAV6 vectors. This method of systemic gene delivery via rAAV6 vectors leads to amelioration of several aspects of the mdx mouse dystrophy, a model of dystrophin deficient muscular dystrophy, when used to deliver a micro-dystrophin transgene. Treated mdx mice demonstrate reduced susceptibility to contraction induced injury as well as lowered serum creatine kinase levels. Finally, we describe the construction of a highly truncated utrophin (micro-utrophin) protein. This protein fails to prevent the onset of muscular dystrophy when expressed in transgenic mdx mice and restores the proteins of the dystroglycan complex (DGC) at a lower efficiency than full length utrophin or truncated micro-dystrophins. Though this particular micro-utrophin does not prevent the dystrophy of the mdx mouse, it provides guidance for future structure/function studies with the aim of creating a functional micro-utrophin.