Antisense oligonucleotide-induced (ASO) exon skipping is a promising treatment strategy for Duchenne muscular dystrophy (DMD), an X-linked fatal neuromuscular disease caused by mutations in the dystrophin gene. It affects 1 in 3,500-5,000 male births. By modulating the splicing of pre-mRNA, ASOs can correct the mRNA reading frame by removing the out-of-frame exon to generate a truncated but partially functional protein. Limitations of systemic administration of ASOs in patients with DMD include poor distribution to target tissues and a limited ability to escape the target cell endosome. These insufficiencies result in minimal dystrophin protein restoration in skeletal muscle and almost no expression in cardiac muscle.
We have developed a novel ASO delivery system by conjugation of Entrada’s proprietary Endosomal Escape Vehicle (EEVTM) to a phosphorodiamidate morpholino oligomer (PMO), a stable and effective oligonucleotide for therapeutic use in DMD. The EEV was rationally designed to facilitate intracellular delivery, endosomal escape, and importantly localization to the nucleus. A single dose of EEV-PMO targeting the mutated DMD gene significantly improved the systemic delivery of PMOs. It resulted in enhanced exon skipping and restoration of dystrophin protein in skeletal and cardiac muscle in the D2-mdx mice, a severe model of DMD. Next we performed a sixteen-week repeat dose study to test longitudinal functional efficacy. We measured significant improvement in muscle function using wire hang and grip strength assessments, and creatine kinase levels were normalized to WT levels. Additionally, four weeks after the last dose, we detected robust dystrophin expression at the sarcolemma with marked amelioration of dystrophic pathology in skeletal and cardiac muscle. These results illustrate the significant therapeutic potential of the EEV-PMO delivery system for DMD and provide a path forward for broad utility across other diseases amenable to exon skipping.