Duchenne muscular dystrophy, DMD, is a severe X-linked recessive disease that kills boys in their second or third decade of life. A related... Show moreDuchenne muscular dystrophy, DMD, is a severe X-linked recessive disease that kills boys in their second or third decade of life. A related condition Becker Muscular Dystrophy, BMD, is very heterogeneous with clinical severity ranging from nearly as bad as DMD, to nearly benign. Both of these are caused by defects in the dystrophin gene, which encodes a 427 kDa cytoskeletal protein, dystrophin, which can provide a mechanical link between the cytoskeleton and the muscle membrane and stables muscle tissue. In general, DMD results from defects that eliminate all dystrophin expression, whereas BMD patients carry mutated products: modified and malfunctioned dystrophin proteins. Since DMD is invariably fatal, monogenic, has a high incidence (1:3500 male births) and afflicts a charismatic patient profile, it has been an attractive target for gene therapy. However, the huge size of the dystrophin gene (2.4 Mbp) and more importantly its processed mRNA (14.2 kb) poses a problem for viral gene therapy which is limited to payloads of about 8 kb. This has prompted efforts to reduce the size of the dystrophin protein while still maintaining functionality. A major inspiration for such efforts was the identification of a very unusual, very large deletion serendipitously found in an extremely mild BMD case. The patient identified with this defect was ambulatory at 65 year of ages. This edit removes more than half the central rod region, from exon 17 to 48, Δe17-48, and, fortunately, the size of this BMD type edit protein is available for viral vector loading. In this project, edited dystrophin with central rod region deletion (D2:22 Δe17-48) and other two related edits, D2:22 Δe17-47 (exon 48 is added in D2:22 Δe17-48) and D2:22 Δe17-49 (exon 49 is deleted from D2:22 Δe17-48), were studied. According to these results, D2:22 Δe17-47 demonstrated the best biophysical and biochemical properties, but D2:22 Δe17-48 displayed higher to protease sensitivity, and D2:22 Δe17-49 had the worst thermal stability. This work will help us to understand the factors that result in benign large scale edits, and facilitate the development of effective viral gene therapy vectors. M.S. in Biology, December 2014 Show less
