Cardiac Myoediting Attenuates Cardiac Abnormalities in Human and Mouse Models of Duchenne Muscular Dystrophy

Abstract

Supplemental Digital Content is available in the text. Rationale: Absence of dystrophin in Duchenne muscular dystrophy (DMD) results in the degeneration of skeletal and cardiac muscles. Owing to advances in respiratory management of patients with DMD, cardiomyopathy has become a significant aspect of the disease. While CRISPR/Cas9 genome editing technology holds great potential as a novel therapeutic avenue for DMD, little is known about the potential of DMD correction using CRISPR/Cas9 technology to mitigate cardiac abnormalities in DMD. Objective: To define the effects of CRISPR/Cas9 genome editing on structural, functional, and transcriptional abnormalities in DMD-associated cardiac disease. Methods and Results: We generated induced pluripotent stem cells from a patient with a deletion of exon 44 of the DMD gene (ΔEx44) and his healthy brother. We targeted exon 45 of the DMD gene by CRISPR/Cas9 genome editing to generate corrected DMD induced pluripotent stem cell lines, wherein the DMD open reading frame was restored via reframing or exon skipping. While DMD cardiomyocytes demonstrated morphological, structural, and functional deficits compared with control cardiomyocytes, cardiomyocytes from both corrected DMD lines were similar to control cardiomyocytes. Bulk RNA-sequencing of DMD cardiomyocytes showed transcriptional dysregulation consistent with dilated cardiomyopathy, which was mitigated in corrected DMD cardiomyocytes. We then corrected dysfunctional DMD cardiomyocytes by adenoviral delivery of Cas9/gRNA and showed that correction of DMD cardiomyocytes postdifferentiation reduces their arrhythmogenic potential. Single-nucleus RNA-sequencing of hearts of DMD mice showed transcriptional dysregulation in cardiomyocytes and fibroblasts, which in corrected mice was reduced to similar levels as wild-type mice. Conclusions: We show that CRISPR/Cas9-mediated correction of DMD ΔEx44 mitigates structural, functional, and transcriptional abnormalities consistent with dilated cardiomyopathy irrespective of how the protein reading frame is restored. We show that these effects extend to postnatal editing in induced pluripotent stem cell-derived cardiomyocytes and mice. These findings provide key insights into the utility of genome editing as a novel therapeutic for DMD-associated cardiomyopathy.