CRISPR-Cas9 corrects Duchenne muscular dystrophy exon 44 deletion mutations in mice and human cells

Abstract

Mutations in the dystrophin gene cause Duchenne muscular dystrophy (DMD), which is characterized by lethal degeneration of cardiac and skeletal muscles. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. Here, we present a simple and efficient strategy for correction of exon 44 deletion mutations by CRISPR-Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, we also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings represent a significant step toward possible clinical application of gene editing for correction of DMD.

Genome Editing for the Understanding and Treatment of Cardiomyopathies

“…Studies on delivery have been limited, however, owing to the more prevalent use of in vitro hiPSC systems for research. Interestingly, studies from DMD models suggest that the heart is surprisingly well-favored for viral vector-based delivery of genome editing agents [106][107][108][109][110][111]113,163]. It would be interesting to see if this is the case for other in vivo cardiomyopathy models or how non-viral means of delivery may compare.…”

“…In one study, tripling the dose of a single Cas9-gRNA vector increased the number of dystrophin-positive fibers to about 40%, when initially there were only a few, scattered dystrophin-positive fibers present in the heart [112]. Min et al (2019) published an interesting study on dosing, which showed that the ratio of Cas9 to gRNA vector amounts administered in vivo was a critical determinant of therapeutic efficacy [113]. In the study, they treated mice carrying a deletion in Dmd exon 44 through the CRISPR-mediated skipping or reframing of exon 45.…”

“…In that study, the authors similarly demonstrated that increasing the amount of gRNA vector provided to mdx mice, at a 1:3 Cas9:gRNA vector ratio, led to increased dystrophin rescue in the heart post-treatment. It is surmised that the limiting property of gRNAs is related to how the gRNA vector adopts an unstable hairpin/cruciform structure in solution, how gRNAs are critical in helping stabilize the conformation of Cas9 to its active form, how fast gRNA turnover is, or how increasing gRNA vector dose ensures that more nuclei express gRNA and are thus amenable to being acted upon by Cas9 [109,113]. Whichever the reason, further investigating this relationship may lead to substantial improvements in the development of CRISPR therapies.…”

Genetically Modified Humans: The X-Men of Scientific Research

“…In their most recent work, the research group have demonstrated the ability of the CRISPR/Cas9 system to correct the deletion mutation of exon 44 of the DMD gene in mice and human iPSC-derived cardiomyocytes [11]. They found that when targeting this section of the gene, a different region to that targeted in their previous studies, the standard 1-to-1 ratio of Cas9 and guide RNA was not as effective.…”

“…Dystrophin (red) restoration shown in a CRISPR edited DMD-affected heart muscle cell (right) relative to unedited cell (left) [11]. …”