Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin (Dmd), a protein required for muscle fiber integrity. DMD affects approximately 1 in 3,500 boys and is characterized by progressive severe muscle weakness and a shortened lifespan. Despite intense efforts to find cures for DMD through a variety of approaches, including myoblast transfer, viral delivery of dystrophin, and oligonucleotide-mediated exon skipping, there remains no cure for this disease. We have recently used clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9)-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germ line of mdx mice, a model for DMD. Genome editing produced mosaic animals containing a wide range of correction (2 to 100%) of the Dmd gene. Interestingly, the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle. The major advantage of this genome editing approach is that it removes the genetic mutation responsible for the disease, allowing for permanent correction of muscle structure and function. The long-term goal of this project is to optimize and adapt CRISPR/Cas9-mediated genome editing to postnatal muscle and ultimately to leverage this approach to correct DMD mutations in humans. This project represents a close collaboration between clinicians and basic scientists sharing the common goal of advancing an entirely new therapeutic strategy to permanently cure DMD. We refer to this new strategy as ?Myoediting?. To achieve our goals we plan to optimize CRISPR/Cas9-mediated permanent Dmd exon skipping (Myoediting) on genomic ?hot spots? in human muscle cells derived from induced pluripotent stem cells (iPSCs) in culture. Based on the knowledge gained from these studies, we will establish a publicly available resource for selecting the optimal sequences for editing individual human DMD mutations. In addition, we will generate a ?humanized? mouse model of DMD as a means of assessing the consequences of specific exon skipping strategies in adult mice. We will also generate dystrophin reporter mice to allow assessment of the phenotypic consequences of Myoediting in living animals. Finally and most importantly, we will optimize the conditions for delivery of CRISPR/Cas9 gene editing components to skeletal muscle and the heart of mice. These studies will involve detailed phenotypic analysis of gene-edited mice and determination of safety of this gene editing approach. Ultimately, the optimized Myoediting method will be tested in pre-clinical studies of a canine model of Dmd as a prelude to eventual therapeutic translation of this approach.
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| Amoasii, Leonela; Hildyard, John C W; Li, Hui et al. (2018) Gene editing restores dystrophin expression in a canine model of Duchenne muscular dystrophy. Science 362:86-91 |
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| Hashimoto, Hisayuki; Olson, Eric N; Bassel-Duby, Rhonda (2018) Therapeutic approaches for cardiac regeneration and repair. Nat Rev Cardiol 15:585-600 |
| Makarewich, Catherine A; Baskin, Kedryn K; Munir, Amir Z et al. (2018) MOXI Is a Mitochondrial Micropeptide That Enhances Fatty Acid ?-Oxidation. Cell Rep 23:3701-3709 |
| Bi, Pengpeng; McAnally, John R; Shelton, John M et al. (2018) Fusogenic micropeptide Myomixer is essential for satellite cell fusion and muscle regeneration. Proc Natl Acad Sci U S A 115:3864-3869 |
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| Makarewich, Catherine A; Olson, Eric N (2017) Mining for Micropeptides. Trends Cell Biol 27:685-696 |
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