Allele Specific Knockdown for LGMD-D1

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PROJECT SUMMARY Gene-based therapies are becoming a reality for disabling neuromuscular disorders. However, the limb girdle muscular dystrophy (LGMD) gene therapy field is focused almost entirely on recessively inherited disorders, leaving dominantly inherited LGMDs far behind in the path towards a cure. Recently mutations in DNAJB6, an HSP-40 co-chaperone, were discovered to cause LGMD-D1, an adult onset, dominantly inherited myopathy. The disease mechanism behind LGMD-D1 is incompletely understood, preventing therapeutic strategy development. Addressing this unmet medical need will help us better understand not only how to treat LGMD- D1, but also how to efficiently approach therapeutic strategy development for other dominantly inherited myopathies. Dominant myopathies are known to have complex, heterogenous disease mechanisms, including toxic gain of function, dominant negative, and haploinsufficiency. Each requires a unique therapeutic approach, such as global knockdown, allele specific knockdown (ASKD), knockdown and replace, or simply gene replacement in the case of haploinsufficiency. Several lines of evidence indicate mutations in DNAJB6 exert a dominant effect through a toxic gain of function. Additionally, DNAJB6 knockout (KO) mice are embryonic lethal due to aggregation of client proteins, indicating a global knockdown treatment may be deleterious. However, haploinsufficiency appears to be tolerated as heterozygous KO mice are viable and able to breed. Targeted knockdown of the mutant allele is therefore an ideal LGMD-D1 treatment strategy to address the toxic gain of function mechanism, while avoiding the damaging effects of complete DNAJB6 knockout. This proposal aims to validate ASKD has therapeutic potential for LGMD-D1. As several siRNAs are now FDA approved, this project will optimize siRNAs to achieve ASKD of mutant DNAJB6 (aim 1), and validate whether ASKD improves disease phenotypes in mouse and human LGMD-D1 models (aim 2). Foundational to these studies, is a knock-in LGMD-D1 mouse model containing a flag tag on the wild type allele, allowing for size- based detection of allele specific changes. Successful completion of these studies will identify a therapeutic target for LGMD-D1, which will in turn, also provide additional insights into disease mechanism. LGMD-D1 is only one of several disorders that may be amenable to the strategies described in this proposal.