(Project 2) Development of U7snRNA Vectors for Dystrophinopathy

ABSTRACT Duchenne muscular dystrophy (DMD) typically results from mutations in the DMD gene that disrupt the open reading frame, resulting in no dystrophin protein, whereas the milder Becker muscular dystrophy (BMD) typically results from mutations that allow expression of a partially functional dystrophin protein. Current therapeutic approaches in trials are directed toward

expression of micro-dystrophin proteins. However, their success will likely ameliorate DMD into a BMD-like phenotype. We seek to develop a personalized medicine approach. Our long-term goal is to develop vectors that maximize RNA splice alteration efficiency to provide the best potential outcome for individual patients. Our central hypothesis is that this vectorized exon

skipping will provide a universally robust exon skipping response, leading to therapies with significant efficacy. For particularly rare mutations, such as single exon duplications within the rod domain of dystrophin, these may be considered personalized or bespoke therapies intended to alter the use of the existing exons within the DMD locus to express full-length dystrophin—the

best potential therapeutic outcome. For other more common mutations, such as certain out-of- frame rod domain deletions, these will be applicable to broader patient populations; based upon both our published studies in mice and our publicly presented data in the infant Dup2 patient, we can expect more robust exon skipping and protein expression than that seen with PMO therapies.

We also propose to fully characterize two new mouse models of utility to the muscular dystrophy community. Our rationale for this project is that a systematic approach to vector design, efficacy assessment, and evaluation of toxicity, along with early engagement with the FDA, will lead to a streamlined path toward vector development. The immediate impact of our work will be data to

support our ongoing engagement with the FDA in developing rapid approaches to personalized gene therapies based upon programmatic (U7snRNA) vector development in cell-line models, obviating the need for large animal studies.