Full-length dystrophin restoration in multiple patient cell lines with DMD pseudoexons using AAV-delivered U7snRNA

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by a null allele of the DMD gene. Thousands of pathogenic DMD variants have been described, with deep intronic mutations in the DMD gene causing loss of dystrophin expression in 1-7% of all DMD patients. These mutations create cryptic splice sites that mimic canonical splice acceptor and donor sites, resulting in the creation of a pseudoexon, where an intronic fragment is spliced into the coding sequence of the mRNA transcript. Patients with deep intronic mutations possess all of the correct DMD coding exons, therefore skipping the pseudoexon should restore a full-length transcript. Our objective is to induce expression of full-length dystrophin in patient cells, each patient carrying a unique intronic point mutation, using U7snRNAs targeting either the splice acceptor (SA), splice donor (SD), or exon splice enhancer (ESE) sequences of the resultant frame-shifting pseudoexon. The AAV-delivered U7snRNA vector repurposes an endogenous U7 small nuclear ribonucleoprotein to induce exon skipping at the pre-mRNA processing step of mRNA maturation. To test for pseudoexon skipping efficacy, human MyoD-transformed cells with a c.2292+1024 G>T, c.2949+909 C>T, or c.9163+2510 G>A mutation were treated with increasing doses of AAV1 containing a single U7snRNA antisense sequence targeting the SA, SD, or ESE of each unique pseudoexon. We achieved successful skipping of the pseudoexon induced by c.2292+1024 G>T, restoring up to 93% of full-length dystrophin mRNA transcript, and 34% of dystrophin protein, as compared to wild-type cells. Studies in the additional cells lines are underway, and the results will be presented. Our preliminary data confirm that the pseudoexon class of DMD mutations is amenable to U7snRNA treatment, and we plan to further validate this in additional DMD patient cell lines. This approach addresses an orphaned class of DMD mutations, and demonstrates the potential of bespoke gene therapies to restore full-length dystrophin in individual patients. Duchenne muscular dystrophy (DMD) is an X-linked disease caused by a null allele of the DMD gene. Thousands of pathogenic DMD variants have been described, with deep intronic mutations in the DMD gene causing loss of dystrophin expression in 1-7% of all DMD patients. These mutations create cryptic splice sites that mimic canonical splice acceptor and donor sites, resulting in the creation of a pseudoexon, where an intronic fragment is spliced into the coding sequence of the mRNA transcript. Patients with deep intronic mutations possess all of the correct DMD coding exons, therefore skipping the pseudoexon should restore a full-length transcript. Our objective is to induce expression of full-length dystrophin in patient cells, each patient carrying a unique intronic point mutation, using U7snRNAs targeting either the splice acceptor (SA), splice donor (SD), or exon splice enhancer (ESE) sequences of the resultant frame-shifting pseudoexon. The AAV-delivered U7snRNA vector repurposes an endogenous U7 small nuclear ribonucleoprotein to induce exon skipping at the pre-mRNA processing step of mRNA maturation. To test for pseudoexon skipping efficacy, human MyoD-transformed cells with a c.2292+1024 G>T, c.2949+909 C>T, or c.9163+2510 G>A mutation were treated with increasing doses of AAV1 containing a single U7snRNA antisense sequence targeting the SA, SD, or ESE of each unique pseudoexon. We achieved successful skipping of the pseudoexon induced by c.2292+1024 G>T, restoring up to 93% of full-length dystrophin mRNA transcript, and 34% of dystrophin protein, as compared to wild-type cells. Studies in the additional cells lines are underway, and the results will be presented. Our preliminary data confirm that the pseudoexon class of DMD mutations is amenable to U7snRNA treatment, and we plan to further validate this in additional DMD patient cell lines. This approach addresses an orphaned class of DMD mutations, and demonstrates the potential of bespoke gene therapies to restore full-length dystrophin in individual patients.