Duchenne muscular dystrophy (DMD) is a neuromuscular disorder that leads to progressive muscle deterioration, loss of ambulation, and respiratory complications. It is caused by genetic mutations that result in the absence of dystrophin protein expression needed for muscle function. Despite significant advances in our understanding of the pathogenesis of DMD, no curative treatment has been identified to date and the disorder has a life-limiting disease trajectory. Recently, we have pioneered an approach to successfully remove large duplications in patient cells. We first tested this approach in vitro by removing a multi-exon (18-30) duplication of 139 kb in the DMD gene using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated Nuclease (Cas9) with a single guide. To test our treatment approach in vivo, I first generated a mouse model harboring a multiexon duplication of 136.8 kb in Dmd using CRISPR/Cas9. This first multiexon duplication model of DMD specifically mimics a patient duplication of Exons 18-30. Molecular and functional characterization of this model reveals dystrophin deficiency with characteristic markers of dystrophic muscle. Furthermore, using our previously described CRISPR/Cas9 single guide strategy, we have for the first time treated a large genomic duplication in vivo and shown successful removal of the duplication fragment leading to restoration of full-length dystrophin in skeletal and cardiac muscles. Additionally, histopathological analysis shows that treated mice have less indications of dystrophy including fewer centrally localized nuclei as well as significantly improved muscle function. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders caused by duplications.Ph.D.2021-06-22 00:00:0
