Project 3 (Flanigan) 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. Among the exceptions to this rule are people with mutations in the first few exons of the gene would be predicted to result in DMD but instead are very mild BMD or are even asymptomatic. We have recently shown that this is due to translation of an N- truncated dystrophin which we call ?CH1, and which results from a cap-independent translational process mediated by a newly described internal ribosome entry site (IRES) within exon 5. We have recently shown that we can activate the IRES by skipping exon 2 using an adeno-associate virus (AAV)-U7snRNA approach, or by antisense oligomer-mediated exon skipping. Our long-term goal is to develop exon 2 skipping as a therapy for patients who carry a duplication of exon 2, which is the most common single exon duplication in DMD patients, as well as for other mutations within the first few exons of the gene. To study this, we have developed a new mouse model of DMD that contains an exon 2 duplication (the Dup2 mouse) and have used it to generate extensive preclinical data showing that AAV-mediated delivery of a modified U7snRNA targeting exon 2 works very well. Our objective in this project is to broaden the applicability of this approach, and to test other ways to stimulated the IRES, as inducing expression would be a meaningful therapeutic approach for up to 5% of dystrophinopathy patients.
Under Aim 1, we will assess exon 2 skipping and IRES activation using phosphorodiamidate morpholino oligomers (PMOs) provided by Sarepta Therapeutics.
Under Aim 2, we will seek to validate small molecule activators of the dystrophin IRES identified in a high-throughput screen at PTC Therapeutics.
Under Aim 3, we will develop a develop a muscle-specific AAV-mediated CRISPR/Cas9 approach to induce somatic IRES activation. As we have already demonstrated the proof-of-concept of IRES activation using an AAV9.U7snRNA approach to exon 2 skipping, the expected outcome will be to demonstrate the broader applicability of our approach to 5' mutations using complementary methods directed at both splicing and translational modification strategies. The immediate impact of our work will be to provide preclinical data that supports rapid clinical development of therapies to provide a clinically meaningful benefit to boys with DMD and BMD, which will be facilitated by our established collaborations with biopharmaceutical companies and by our own extensive experience in investigator-initiated pre-IND interactions
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