Long-Term Rescue of Dystrophin Expression and Improvement in Muscle Pathology and Function in Dystrophic mdx Mice by Peptide-Conjugated Morpholino

Exon skipping is capable of correcting frameshift and nonsense mutations in Duchenne muscular dystrophy (DMD). Phase 2 clinical trials in the United Kingdom and the Netherlands have reported induction of dystrophin expression in muscle of DMD patients by systemic administration of both phosphorodiamidate morpholino oligomers (PMO) and 2′-O-methyl phosphorothioate. Peptide-conjugated PMO (PPMO) offers significantly higher efficiency than PMO, with the ability to induce near-normal levels of dystrophin, and restores function in both skeletal and cardiac muscle. We examined 1-year systemic efficacy of PPMO targeting exon 23 in dystrophic mdx mice. The LD50 of PPMO was determined to be approximately 85 mg/kg. The half-life of dystrophin expression was approximately 2 months in skeletal muscle, but shorter in cardiac muscle. Biweekly injection of 6 mg/kg PPMO produced >20% dystrophin expression in all skeletal muscles and ≤5% in cardiac muscle, with improvement in muscle function and pathology and reduction in levels of serum creatine kinase. Monthly injections of 30 mg/kg PPMO restored dystrophin to >50% normal levels in skeletal muscle (but 15% in cardiac muscle), which was associated with greatly reduced serum creatine kinase levels, near-normal histology, and functional improvement of skeletal muscle. Our results demonstrate for the first time that regular 1-year administration of PPMO can be safely applied to achieve significant therapeutic effects in an animal model.