U7snRNA-mediated skipping of intron-derived pseudoexons restores full-length DMD expression in patient-derived cell...

X-linked Duchenne muscular dystrophy (DMD) is caused by pathogenic variants of the DMD gene that result in absent or significantly altered dystrophin expression. Deep intronic mutations within the DMD locus account for 1%-7% of all DMD patients. These are typically point mutations that create splice acceptor (SA) or splice donor (SD) sites, resulting in the inclusion of an intronic fragment as a pseudoexon within the DMD mRNA. Because such patients possess all of the canonical DMD coding exons, efficient exclusion of the pseudoexon from the mature mRNA restores a full-length transcript. We induced pseudoexon skipping in patient-derived cells by employing a modified U7 small nuclear RNA (U7snRNA) that targets an SA, SD, or exonic splice enhancer (ESE) sequence of the frame-shifting pseudoexons. The U7snRNA vector repurposes an endogenous U7 small nuclear ribonucleoprotein to induce exon skipping at the pre-mRNA processing step of mRNA maturation. U7snRNA treatment resulted in dose-dependent pseudoexon skipping. In all four cell lines treated, we achieved skipping efficiencies over 90%, at times resulting in over 100% of wild-type dystrophin protein levels. This approach addresses an orphan class of DMD mutations and demonstrates the potential of bespoke gene therapies to restore full-length dystrophin in individual patients.