Single-cut genome editing restores dystrophin expression in a new mouse model of muscular dystrophy

Abstract

Duchenne muscular dystrophy (DMD) is a severe, progressive muscle disease caused by mutations in the dystrophin gene. The majority of DMD mutations are deletions that prematurely terminate the dystrophin protein. Deletions of exon 50 of the dystrophin gene are among the most common single exon deletions causing DMD. Such mutations can be corrected by skipping exon 51, thereby restoring the dystrophin reading frame. Using clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9), we generated a DMD mouse model by deleting exon 50. These ΔEx50 mice displayed severe muscle dysfunction, which was corrected by systemic delivery of adeno-associated virus encoding CRISPR/Cas9 genome editing components. We optimized the method for dystrophin reading frame correction using a single guide RNA that created reframing mutations and allowed skipping of exon 51. In conjunction with muscle-specific expression of Cas9, this approach restored up to 90% of dystrophin protein expression throughout skeletal muscles and the heart of ΔEx50 mice. This method of permanently bypassing DMD mutations using a single cut in genomic DNA represents a step toward clinical correction of DMD mutations and potentially those of other neuromuscular disorders.

Therapeutic approaches for cardiac regeneration and repair

“…For example, Duchenne muscular dystrophy, an X-linked recessive monogenic disease caused by mutations in the dystrophin gene (DMD), is a severe progressive muscle disease that causes premature death usually in the mid-twenties owing to cardiac and respiratory failure 175 . In vivo genome editing with the CRISPR- Cas9 system restored muscle function by correcting the Dmd mutation in a mouse model of Duchenne muscular dystrophy in both the germline and the postnatal stage 176–180 . In a proof-of-principle study, genome editing technology also corrected mutations in human iPSC- derived cardiomyocytes from patients with Duchenne muscular dystrophy 181–185 .…”

“…Cells were immunostained with antibodies for dystrophin (green) and troponin I (red). Adapted with permission from REF 180 , AAAS.…”

Multiplex in situ hybridization within a single transcript: RNAscope reveals dystrophin mRNA dynamics

“…If correct, this surprising finding might in fact be of considerable value to therapeutic approaches for DMD such as gene editing: if under healthy conditions many more transcripts are produced than are needed, in dystrophic conditions, beneficial levels of dystrophin protein might be restored with only modest levels of genomic correction (and studies suggest this may be the case (64)(65)(66)(67)(68)).…”

Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy

“…1C). The deletions identified with this method encompassed a highly predicted exonic splicing enhancer (ESE) site for exon 51 (18, 27, 28) (fig. S3A).…”