AAV.Cas9 and AAV.SERCA2a strategies to mitigate heart failure in DMD pigs

Abstract

Abstract Aims Duchenne muscular dystrophy is a devastating disease affecting skeletal muscle and heart, which is caused by loss-of-function mutations in the dystrophin (DMD) gene. Since pharmacologic treatment is ineffective over time, gene therapy including gene editing is rapidly evolving. In DMDdelta52 pigs, we have previously demonstrated efficacy of an AAV9-Cas9-gRNA approach, which by excision of exon 51 provided a shortened, but stable dystrophin D51-52. Here, we set out to compare this approach to germline excision of exon 51 in the DMDdelta52 pig, resulting in ubiquitous dystrophin D51-52 expression, a porcine model of Becker muscle dystrophy (BMD). Alternately, we provided locoregional AAV.SERCA2a gene therapy to the DMDdelta52 pigs. Methods and results We generated pigs lacking exon 52 of the DMD gene. Male offspring are characterized by heart failure (EF33±3%), arrhythmogenesis due to distinct regions of low or no-amplitude action potentials and by sudden cardiac death before 4 months of age. Germline correction of the loss of function mutation by additional excision of neighbouring exon 51 induced a Becker phenotype (BMD), with a mildly reduced ejection cardiac function (EF 45±3%) a reduction of vulnerable areas in electrophysiology mapping and a prolonged lifespan. Postnatal AAV9.Cas9-gE51 also improved EF (40±2%) and reduced electrophysiological vulnerability, increasing survival significantly. Using intracoronary delivery of AAV1.SERCA2a at a dose of 3x1013 virus genomes (vg) per pig by slow antegrade infusion of LAD & LCx improved left ventricular ejection fraction (EF 46±5% +AAV1.SERCA2a), but did not alter the amount of no-amplitude areas per LV. Conclusions Compared to ubiquitous excision of exon 51 (DMDdelta51-52) or postnatal AAV.Cas9-gE51 treatment of a DMDdelta52 pig model, which both improved ejection fraction EF and almost normalized action potential amplitudes, a moderate dose of AAV1.SERCA2a sufficed to normalize left ventricular function, but did not affect electrical vulnerability of the heart. Hence, AAV.SERCA2a combined with antitachycardic pacemaker protection may serve as a safe treatment option for DMD cardiomyopathy, whereas Cas9-mediated gene editing may provide additional therapy of electrical vulnerability. Further translational studies will establish novel gene therapeutic options for DMD patients.