Poster abstracts
Poster number 7 submitted by Joe Beljan
Restoration of full-length dystrophin using AAV-delivered U7snRNA in patient cells with a DMD pseudoexon: a model for bespoke gene therapies in an orphan mutational class
Joseph Beljan (Molecular, Cellular, and Developmental Biology), Kevin Flanigan, MD (Center for Gene Therapy - Nationwide Childrens Hospital), Liubov Gushchina, PhD (Center for Gene Therapy - Nationwide Childrens Hospital), Stefan Nicolau, MD (Center for Gene Therapy - Nationwide Childrens Hospital), Anthony Stephenson, PhD (Formerly at Center for Gene Therapy - Nationwide Childrens Hospital)
Abstract:
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. The objective of this project was to induce expression of full-length dystrophin in cells carrying an intron 18 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 mutation were treated with increasing doses of AAV1 containing a single U7snRNA antisense sequence targeting the SA, SD, or ESE. Upon treatment we achieved successful skipping of the pseudoexon, restoring up to 93% of full-length dystrophin mRNA transcript, and 34% of dystrophin protein, as compared to wild-type cells, with the SD and ESE antisense sequences resulting in the most robust skipping response. Our preliminary data confirms the pseudoexon class of DMD mutations is amenable to U7snRNA treatment, and we plan to further validate this in other 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.
References:
1. Waldrop, M. A. et al. Intron mutations and early transcription termination in Duchenne and Becker muscular dystrophy. Hum Mutat 43, 511-528, doi:10.1002/humu.24343 (2022).
2. Chaouch, S. et al. Immortalized skin fibroblasts expressing conditional MyoD as a renewable and reliable source of converted human muscle cells to assess therapeutic strategies for muscular dystrophies: validation of an exon-skipping approach to restore dystrophin in Duchenne muscular dystrophy cells. Hum Gene Ther 20, 784-790, doi:10.1089/hum.2008.163 (2009).
3. Wein, N. et al. Absence of Significant Off-Target Splicing Variation with a U7snRNA Vector Targeting DMD Exon 2 Duplications. Hum Gene Ther 32, 1346-1359, doi:10.1089/hum.2020.315 (2021).
Keywords: Duchenne Muscular Dystrophy, U7snRNA, Gene Therapy