Prevention of early-onset cardiomyopathy in Dmd exon 52-54 deletion mice by CRISPR-Cas9-mediated exon skipping

Matthew Rok; Tatianna Wai Ying Wong; Eleonora Maino; Abdalla Ahmed; Grace Yang; Elzbieta Hyatt; Kyle Lindsay; Sina Fatehi; Ryan Marks; Paul Delgado-Olguín; Evgueni A Ivakine; Ronald D Cohn

doi:10.1016/j.omtm.2023.07.004

Prevention of early-onset cardiomyopathy in Dmd exon 52-54 deletion mice by CRISPR-Cas9-mediated exon skipping

Mol Ther Methods Clin Dev. 2023 Jul 17:30:246-258. doi: 10.1016/j.omtm.2023.07.004. eCollection 2023 Sep 14.

Authors

Matthew Rok^{1

2}, Tatianna Wai Ying Wong^{1

2}, Eleonora Maino^{1

2}, Abdalla Ahmed³, Grace Yang⁴, Elzbieta Hyatt¹, Kyle Lindsay¹, Sina Fatehi^{1

2}, Ryan Marks^{1

2}, Paul Delgado-Olguín^{2

3

5}, Evgueni A Ivakine^{1

6}, Ronald D Cohn^{1

2

7

8}

Affiliations

¹ Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada.
² Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
³ Department of Translational Medicine, The Hospital for Sick Children, Toronto, ON, Canada.
⁴ Department of Biochemistry & Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
⁵ Heart & Stroke Richard Lewar Centre of Excellence, Toronto, ON, Canada.
⁶ Department of Physiology, University of Toronto, Toronto, ON, Canada.
⁷ Institute of Medical Science, University of Toronto, Toronto, ON, Canada.
⁸ Department of Pediatrics, The Hospital for Sick Children, Toronto, ON, Canada.

Abstract

Duchenne muscular dystrophy (DMD) is a disease with a life-threatening trajectory resulting from mutations in the dystrophin gene, leading to degeneration of skeletal muscle and fibrosis of cardiac muscle. The overwhelming majority of mutations are multiexonic deletions. We previously established a dystrophic mouse model with deletion of exons 52-54 in Dmd that develops an early-onset cardiac phenotype similar to DMD patients. Here we employed CRISPR-Cas9 delivered intravenously by adeno-associated virus (AAV) vectors to restore functional dystrophin expression via excision or skipping of exon 55. Exon skipping with a solitary guide significantly improved editing outcomes and dystrophin recovery over dual guide excision. Some improvements to genomic and transcript editing levels were observed when the guide dose was enhanced, but dystrophin restoration did not improve considerably. Editing and dystrophin recovery were restricted primarily to cardiac tissue. Remarkably, our exon skipping approach completely prevented onset of the cardiac phenotype in treated mice up to 12 weeks. Thus, our results demonstrate that intravenous delivery of a single-cut CRISPR-Cas9-mediated exon skipping therapy can prevent heart dysfunction in DMD in vivo.

Keywords: AAV; CRISPR; Duchenne muscular dystrophy; cardiomyopathy; dystrophin; exon skipping; genome editing; heart; in vivo; systemic.