Effects of systemic multiexon skipping with peptide-conjugated morpholinos in the heart of a dog model of Duchenne muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a nextgeneration morpholino: arginine-rich, cell-penetrating peptideconjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMD J ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMD J dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMD J dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart. Duchenne muscular dystrophy | exon skipping | peptide-conjugated morpholinos | cardiac Purkinje fibers | dystrophic dog model

Clinical trials of exon skipping in Duchenne muscular dystrophy

“…One of the potential limitations is that use of Exondys51 with its currently formulated backbone appears to provide no cardiac expression and little to protect the heart in DMD. New formulations will be necessary that are being considered for clinical translation [27,28].…”

Functional correction of dystrophin actin binding domain mutations by genome editing

“…In fact, exons 2-7, which encode part of the ABD-1, are the most frequently mutated portion of the 5′-proximal hot spot (8). Two reports have described genomic editing of a DMD mutation comprising a duplication of exon 2 (30,31), and other reports showed editing of an exon 7 splice site mutation in the golden retriever dog model of DMD (32,33). In-frame deletions and missense mutations of the 5′ region of the DMD gene that affect the ABD-1 structure have been associated with a decrease in dystrophin protein stability, reduced actin binding affinity, and protein mis-folding and degradation (21,27,(34)(35)(36).…”

Canine Models of Inherited Musculoskeletal and Neurodegenerative Diseases

“…Systemic delivery did not improve skeletal muscle histology and function. However, cardiac conduction abnormalities were significantly ameliorated (217,218).…”