Therapeutic Exon Skipping Through a CRISPR-Guided Cytidine Deaminase Rescues Dystrophic Cardiomyopathy in Vivo.

Background: Loss of dystrophin protein causes Duchenne muscular dystrophy (DMD), characterized by progressive degeneration of cardiac and skeletal muscles, and mortality in adolescence or young adulthood. Although cardiac failure has risen as the leading cause of mortality in patients with DMD, effective therapeutic interventions remain underdeveloped, in part, because of the lack of a suitable preclinical model.

Methods: We analyzed a novel murine model of DMD created by introducing a 4-bp deletion into exon 4, one of the exons encoding the actin-binding domain 1 of dystrophin (referred to as mice). Echocardiography, microcomputed tomography, muscle force measurement, and histological analysis were performed to determine cardiac and skeletal muscle defects in these mice. Using this model, we examined the feasibility of using a cytidine base editor to install exon skipping and rescue dystrophic cardiomyopathy in vivo. AAV9-based CRISPR/Cas9-AID (eTAM) together with AAV9-sgRNA was injected into neonatal mice, which were analyzed 2 or 12 months after treatment to evaluate the extent of exon skipping, dystrophin restoration, and phenotypic improvements of cardiac and skeletal muscles.

Results: mice recapitulated many aspects of human DMD, including shortened life span (by ≈50%), progressive cardiomyopathy, kyphosis, profound loss of muscle strength, and myocyte degeneration. A single-dose administration of AAV9-eTAM instituted >50% targeted exon skipping in the transcripts and restored up to 90% dystrophin in the heart. As a result, early ventricular remodeling was prevented and cardiac and skeletal muscle functions were improved, leading to an increased life span of the mice. Despite gradual decline of AAV vector and base editor expression, dystrophin restoration and pathophysiological rescue of muscular dystrophy were long lasted for at least 1 year.

Conclusions: Our study demonstrates the feasibility and efficacy to institute exon skipping through an enhanced TAM (eTAM) for therapeutic application(s).