Duchenne muscular dystrophy (DMD) is a progressive X-linked muscle disease caused by mutations that disrupt the open reading frame (ORF) of the DMD gene, resulting in the absence of dystrophin, a muscle-specific structural protein essential for muscle cell integrity.
U7snRNA gene corrective therapy resulting in a selective exclusion of exon 2 in Dup2 patients is an approach that is already in clinical trial (NCT04240314). Similar strategies to alter splicing to exclude the target exons in DMD transcripts with duplication and deletion mutations, restoring translation of a full-length or internally truncated, yet functionally active protein is a powerful tool for the prevention and cure of diseases and developing personalized therapies for patients with very rare mutations in the DMD gene.
Here, we developed a viral-based exon-skipping platform utilizing rAAV to encapsidate a single copy of a non-coding U7snRNA with additional sequences targeting the splice acceptor (SAS), splice donor (SDS) sites or splice enhancer (ESE) site of different DMD exons. We found that DMD patient-derived cell lines (Immortalized tet-inducible-MyoD fibroblasts, FM cells) treated with various doses of rAAV.U7snRNA vectors showed robust skipping of the targeted dystrophin exons in a dose-dependent manner. Semi-quantitative analysis of RT-PCR gel images confirmed WT or in-frame (IF) dystrophin mRNA transcripts in FM cells after rAAV infection following 8-14 days of differentiation.
Overall, these data demonstrate that AAV-U7snRNA-mediated therapy is a powerful therapeutic tool that could benefit DMD patients with various mutations, including single exon duplications and single- and multiple- exon deletions.