Correction of Hot Spot Mutations in Duchenne Muscular Dystrophy by CRISPR/Cas9 Gene Editing

The ability to efficiently modify the genome using CRISPR technology has rapidly revolutionized biology and genetics and will soon transform medicine. Duchenne muscular dystrophy (DMD) represents one of the first monogenic disorders that has been investigated with respect to CRISPR-mediated correction of causal genetic mutations. DMD results from mutations in the gene encoding dystrophin, a scaffolding protein that maintains the integrity of striated muscles. Thousands of different dystrophin mutations have been identified in DMD patients, who suffer from a loss of ambulation followed by respiratory insufficiency, heart failure, and death by the third decade of life. Most DMD patients have an inherited or spontaneous deletion in the dystrophin gene that disrupts the reading frame resulting in an unstable truncated product. The major DMD mutational hotspots are found between exons 6 to 8, and exons 45 to 53. 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. In this study, a new DMD mouse model was generated by deleting exon 44, thereby creating a human DMD hotspot mutation in a mouse animal model. Using CRISPR/Cas9-mediated genomic editing, the reading frame of the exon 44 DMD mouse model was restored and dystrophin expression was rescued. Furthermore, I present an 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, I also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings provide therapeutic insight to develop possible CRISPR therapies for DMD.