Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD) are X-linked recessive diseases caused by mutations in the dystrophin gene. DMD is among the most common human genetic diseases, and there is no treatment or cure for this lethal disorder. Recent progress by this and other labs has demonstrated the feasibility of gene therapy for DMD, but many significant obstacles remain before a cure is available. One such obstacle is the enormous size of both the gene (>2.4 Mb) and gene product (14 kb mRNA, 427 Kd protein). This application proposes to further the development of gene therapy for DMD by exploring the structural basis of dystrophin functional domains and by designing truncated, but highly functional dystrophin mini-gene expression vectors suitable for viral delivery to muscle tissue. in addition to the relevance to gene therapy for DMD, a better understanding of the function of the dystrophin isoforms could lead to alternate approaches for therapy. Two major structural domains of dystrophin are the focus of this application: the 'central rod' and the 'C-terminal' domains. The rod domain is composed of 24 spectrin-like repeats, combinations of which are deleted from several patients with very mild cases of BMD. One patient missing almost 16 of 24 repeats is still walking in his late 60's, indicating that truncated dystrophins can retain considerable functional activity. In this study a variety of truncated mini-genes will be generated to identify the smallest construct with therapeutic potential. This work should lead to a mini-gene with increased function relative to the large deletion patient's own truncated gene, which we have recently tested in transgenic mice and found to be measurably less functional than the full length cDNA. Our assay system for these studies is mdx mice, a mouse model for DMD that displays a variety of morphological and functional impairments similar to the human disease, as well as human and mouse myogenic cell cultures. Analysis of the C-terminal domain of dystrophin might also identify regions not essential for function that could be excised from mini-gene vectors. However, our studies of the C- terminus will focus primarily on critical functional features required for interaction with the dystrophin associated proteins. This region is alternatively spliced, phosphorylated, contains a leucine zipper, and from patient and mouse studies appears to be the key functional domain of dystrophin. Transgenic mouse, cell culture, and in vitro assays will be used to explore the interactions of a variety of mutated and naturally occurring C-terminal isoforms with other proteins and to identify key structural features required for proper dystrophin function. Finally, promising dystrophin constructs will be tested in the context of adenovirus vectors following injection into mdx mice. These studies will lead to a greater understanding of the function of dystrophin and will enable the development of viral vectors for gene therapy of DMD.
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