Structural flexibilty of the dystrophin rod domain: Implications for gene therapy of Duchenne muscular dystrophy using adeno-associated viral vectors

Duchenne muscular dystrophy (DMD), the most common form of muscular dystrophy, is a degenerative, lethal disorder caused by mutations in the dystrophin gene. The full-length muscle isoform of dystrophin is composed of N-terminal actin-binding, central rod, cysteine rich, and C-terminal domains. Large deletions that remove portions of the rod domain can yield truncated, yet functional dystrophin proteins, leading to the milder Becker muscular dystrophy (BMD) in humans.; The feasibility of gene therapy for DMD was demonstrated by prevention of the development of dystrophy by expression of the muscle isoform of dystrophin in skeletal muscles of transgenic mdx mice, a model for DMD. However, available viral vectors that can carry the full-length dystrophin cDNA (14 kilobases) and regulatory elements are difficult to grow and may not persist after delivery to immunocompetent mice. Adeno-associated viral (AAV) vectors are promising shuttles for DMD gene therapy because they readily infect muscle, direct long-term gene expression, and do not elicit a detectable cellular immune response in normal muscle. However, the small (<5 kilobases) cloning capacity of AAV precludes packaging of large genes.; The primary goal of this dissertation research was to elucidate the minimal functional elements of the dystrophin rod domain to develop gene therapy for DMD using AAV vectors. We generated three different transgenic mdx mouse lines that expressed truncated ‘micro-dystrophins’ and examined their ability to prevent the development of dystrophic changes in skeletal muscle. One of the constructs completely prevented the development of morphological features associated with muscular dystrophy in mdx mice and afforded significant protection from contraction-induced injury to mdx muscles. When delivered to mdx muscle using AAV serotype 2 (AAV-2) vectors, this micro-dystrophin effected a reversal of the histopathological features associated with dystrophy. The efficiency of muscle transduction by AAV-2 indicated that enormous quantities of virus would be required to achieve a significant clinical benefit in patients. To improve transduction, we tested gene delivery to mouse muscle using AAV serotype 6 (AAV-6). We found that AAV-6 significantly increased the scope and magnitude of gene delivery. It is hoped that these studies may someday contribute to successful treatment for this devastating disease.