584. Myoblast Fusion Mediates Morpholino Entry and Exon Skipping In Dystrophic Muscle

Duchenne muscular dystrophy (DMD) is a severe, progressive, X-linked myopathy involving cycles of muscle cell degeneration, regeneration, and inflammation. DMD results from mutations in the dystrophin gene that result in a consequent failure to translate dystrophin protein. Exon skipping is a promising therapeutic strategy employing antisense oligonucleotides (AO) to exclude exons that disrupt the open reading frame, so as to produce a truncated, partially functional dystrophin protein. Several different AOs, including the phosphorodiamidate morpholino (PMO) and the 2’O-methyl phosphorothioate (2’OMe), have been shown to induce dystrophin expression and are currently being investigated in clinical trials. However, drug uptake and efficacy is inconsistent and highly-variable between and within individual muscles. Our objective was to identify factors within dystrophic muscle responsible for the observed variability in myofiber penetration, exon skipping, and dystrophin expression following systemic AO administration. Specifically, we investigated the role of myofiber regeneration on exon skipping. By treating dystrophin-null mdx mice with a single high-dose of the PMO together with staggered pulses of bromodeoxyuridine (BrdU) we can precisely identify timeframes of myocyte proliferation and relate this to the efficiency of PMO delivery. Initially, we utilized a tagged-PMO to track its efficiency of entry into muscle fibers and determine how long after administration it persists. Intriguingly, we observed that the efficiency of entry of PMO into myofibers is strongly associated with the late stages of differentiation and fusion of satellite cells into regenerating dystrophic mdx myofibers. We also observed a specific co-localization of BrdU-positive myonuclei with dystrophin-positive myofibers when the pulse of BrdU immediately preceded PMO delivery, implying that satellite cell proliferation and fusion into regenerating myofibers play a crucial role in the effectiveness of PMO-mediated exon skipping. Our investigation of the mechanisms involved in PMO uptake and efficacy will provide valuable insights for optimizing protocols for this promising approach to therapy for DMD.