Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disease caused by mutations in the X-linked DMD gene. These mutations result in a loss of expression of the dystrophin protein, which provides structural support to the muscle cell membrane and protects it from contraction induced damage. Without dystrophin, a cyclical process of skeletal muscle degeneration and regeneration occurs until it is ultimately replaced with adipose cells and fibrotic tissue, rendering it non-functional. The most critical dystrophin domains are the N-terminus, which binds to cytoplasmic filamentous actin and connects it to the extracellular matrix through C-terminus binding to β-dystroglycan, and by extension the associated dystrophin-glycoprotein complex. An allelic disease, Becker muscular dystrophy, is caused by in-frame mutations within the dispensable central domain and manifests as a less severe phenotype.
The most promising DMD therapy is antisense oligonucleotide approaches in which single exons adjacent to the mutation are targeted for pre-mRNA removal by the splicing machinery to restore the mRNA reading frame. This has been successful in cell culture, animal models, and now has shown promise in clinical trials. However, rescued dystrophin protein levels remain variable suggesting the need for improvements in exon skipping efficiencies. We performed a high-throughput screen to identify and repurpose FDA approved drugs that potentiate this antisense exon skipping strategy. We found that one drug, dantrolene, enhanced exon skipping, rescued dystrophin protein levels, and improved overall function in a DMD mouse model. In reprogrammed patient myotubes, dantrolene, and other Ryanodine Receptor (RyR1) antagonists increased DMD exon 51 skipping, suggesting the RyR1 calcium channel as the relevant molecular target. An independent high-throughput screen found a cohort of small molecules with similar 2-D structures that enhanced DMD exon 51 skipping and shared a known protein target, calmodulin. Identified AO potentiating small molecules bind and inhibit either calcium channels (RyR1) or calcium regulatory proteins (Calmodulin) suggesting the importance of Ca2+ regulation and its impact on Ca2+-binding proteins in directing exon skipping activity
Antisense-based exon skipping is highly sequence specific and benefits from the availability of diverse genetic mutations for the evaluation of potential therapies or molecular mechanisms. We have collected and derived 49 DMD patient fibroblasts and have reprogrammed a subset into muscle cells as a resource for the research community. In summary, we have established a research environment facilitating the discovery of novel therapies for the treatment of DMD, and have identified Ca2+ modulation as a key regulator of antisense mediated splicing activity.