Prevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editing

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.