Development of a Minimized Exons 45-55 Skipping Cocktail for the Treatment of Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disorder characterized by progressive, body-wide muscle weakness and degeneration. It is the most common inherited neuromuscular disorder worldwide, with 1:3,500-1:5,000 males affected, and there is currently no cure for it. DMD is mostly caused by large out-of-frame deletions of the DMD gene that lead to dystrophin loss in muscle. Exon skipping uses antisense oligonucleotides (AOs) to exclude out-of-frame exons from the DMDtranscript to restore the reading frame and allow for the production of truncated, partially functional dystrophin. While promising, current exon skipping approaches have limited patient applicability since they only target single exons. We aim to develop an AO formulation capable of skipping DMD exons 45-55, which due to the region being in a mutation hotspot, could treat 47% of all patients. We designed phosphorodiamidate morpholino oligomer (PMO) AOs targeting the human DMD exons 45-55 region, one PMO per exon except for exon 48 which required two. We have shown that this 12-PMO cocktail restored up to ~15.9% dystrophin of normal levels in immortalized DMD patient-derived muscle cells with various mutations. Presently, we found that we can reduce the number of PMOs in this cocktail and yet retain appreciable levels of exons 45-55 skipping. Immortalized patient-derived myotubes carrying an exon 52 deletion were transfected with a 5-PMO cocktail at 3 days post-differentiation. At 2 days post-treatment, 34% exons 45-55 skipping on average was observed by RT-PCR in the treated cells, which was not significantly different from the 25% skipping by the full cocktail. On average, Western blot showed 21% dystrophin of normal levels upon treatment with the 5-PMO cocktail; 13% dystrophin was observed for the full cocktail. As we plan to deliver this minimized cocktail in vivo, we have tested peptides that can be conjugated to the PMOs to increase their muscle uptake. A promising peptide is DG9, which we previously identified through a zebrafish-based screen of cell-penetrating peptides. When conjugated to an exon 51-skipping PMO, a single retro-orbital injection of DG9-PMO in hDMDdel52;mdx mice enhanced exon 51 skipping and dystrophin rescue by up to 7-fold and up to 4-fold, respectively, across skeletal muscles and the heart compared to the naked PMO1 week post-treatment. Widespread dystrophin-positive fibers were observed by immunostaining in these tissues as well. Overall, we have developed a minimized exons 45-55 skipping cocktail that effectively restored dystrophin production in vitro. Future work will test the efficacy of the DG9-conjugated version of this cocktail in vivo.