Prevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editing
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Skeletal muscle is one of the largest tissues in the human body and hence muscle diseases caused by genetic mutations have a profound and systemic impact on human health. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder, caused by mutations in the DMD gene on the X chromosome, which consists of 79 exons encoding dystrophin protein. Patients with DMD develop progressive muscle weakness and cardiomyopathy, and ultimately succumb to respiratory and cardiac failure in their mid-20s. The dystrophin gene was identified three decades ago and mutations in the DMD gene are well-characterized. However, there is no effective treatment for this debilitating disease. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) was first discovered as an adaptive immune system in bacteria and archaea for defending against phage infection. Recently, the CRISPR/Cas system has been applied for mammalian genome editing because it provides site-specific DNA double-stranded breaks with simplicity and precision. In this study, I demonstrate the feasibility of using CRISPR/Cpf1 to correct a DMD exon 48-50 out-of-frame deletion mutation in cardiomyocytes derived from patient induced pluripotent stem cells by exon skipping and exon reframing strategies. Next, I precisely correct a Dmd exon 23 nonsense mutation in mdx mouse by CRISPR/Cpf1-mediated germline editing. Furthermore, I apply CRISPR/Cas9-mediated post-natal genome editing to correct a Dmd exon 44 out-of-frame deletion mutation in a DMD mouse model. Finally, I develop an effective strategy to improve CRISPR/Cas9-mediated in vivo genome editing by packaging Cas9 nuclease in conventional single-stranded AAV and CRISPR single guide RNAs in double-stranded self-complementary AAV. This strategy significantly reduces the amount of AAV vector needed for therapeutic genome editing and enhances dystrophin restoration after delivery into a mouse model of DMD harboring an exon 44 deletion. These findings represent an important advancement toward therapeutic translation of genome editing technology for permeant correction of Duchenne muscular dystrophy.
Muscular Dystrophy, Duchenne