@article{90b55a9033d2424abbf34e7fefd3693f,
title = "Long-term maintenance of dystrophin expression and resistance to injury of skeletal muscle in gene edited DMD mice",
abstract = "Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by mutations in the dystrophin gene. CRISPR/Cas9 genome editing has been used to correct DMD mutations in animal models at young ages. However, the longevity and durability of CRISPR/Cas9 editing remained to be determined. To address these issues, we subjected ΔEx44 DMD mice to systemic delivery of AAV9-expressing CRISPR/Cas9 gene editing components to reframe exon 45 of the dystrophin gene, allowing robust dystrophin expression and maintenance of muscle structure and function. We found that genome correction by CRISPR/Cas9 confers lifelong expression of dystrophin in mice and that corrected skeletal muscle is highly durable and resistant to myofiber necrosis and fibrosis, even in response to chronic injury. In contrast, when muscle fibers were ablated by barium chloride injection, we observed a loss of gene edited dystrophin expression. Analysis of on- and off-target editing in aged mice confirmed the stability of gene correction and the lack of significant off-target editing at 18 months of age. These findings demonstrate the long-term durability of CRISPR/Cas9 genome editing as a therapy for maintaining the integrity and function of DMD muscle, even under conditions of stress.",
keywords = "AAV, CRISPR/Cas9, Duchenne muscular dystrophy, exon reframing, gene editing",
author = "Karri, {Dileep R.} and Yu Zhang and Francesco Chemello and Min, {Yi Li} and Jian Huang and Jiwoong Kim and Mammen, {Pradeep P.A.} and Lin Xu and Ning Liu and Rhonda Bassel-Duby and Olson, {Eric N.}",
note = "Funding Information: We thank A. Mireault for tissue harvesting and performing the grip strength assay; E. Sanchez-Ortiz for performing IHC; H. Li for TIDE analysis and protocols for amplicon-based deep sequencing; X. Men?dez Caravia for help with the BaCl2 study; J. Cabrera for graphics; the Boston Children's Hospital Viral Core for AAV production; the Metabolic Phenotyping Core for serum CK analysis; the Sanger Sequencing Core for sequencing services; and the Histology Core for H&E staining. We are grateful to S. Hauschka (University of Washington) for providing the muscle-specific CK8e promoter, to D. Grimm (Heidelberg University Hospital, Germany) for providing TRISPR-sgRNA expression plasmid, and S. Gray (University of Texas Southwestern Medical Center) for providing the self-complementary AAV plasmid. This work was supported by the National Institutes of Health (NIH) (grants HL130253), Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center (grant P50 HD 087351), and Robert A. Welch Foundation (grant 1-0025 to E.N.O.). D.R.K. N.L. R.B.-D. and E.N.O. wrote and edited the manuscript. D.R.K. Y.Z, R.B.-D. and E.N.O. designed the experiments. D.R.K. performed downhill running studies and BaCl2 injury and analyzed data. D.R.K. and F.C. performed the older mouse study and analyzed data. Y.-L.M. generated the mouse model. Y.-L.M. and Y.Z. designed the genome editing strategy. J.H. performed the electrophysiology studies. P.P.A.M. provided oversight of the electrophysiology analysis. J.K. performed bioinformatic analysis. L.X. provided oversight of the bioinformatic analysis. E.N.O. is a consultant for Vertex Therapeutics. Y.-L.M. is an employee at Vertex Pharmaceuticals. The other authors declare that they have no competing interests. Funding Information: We thank A. Mireault for tissue harvesting and performing the grip strength assay; E. Sanchez-Ortiz for performing IHC; H. Li for TIDE analysis and protocols for amplicon-based deep sequencing; X. Men{\'e}dez Caravia for help with the BaCl 2 study; J. Cabrera for graphics; the Boston Children{\textquoteright}s Hospital Viral Core for AAV production; the Metabolic Phenotyping Core for serum CK analysis; the Sanger Sequencing Core for sequencing services; and the Histology Core for H&E staining. We are grateful to S. Hauschka (University of Washington) for providing the muscle-specific CK8e promoter, to D. Grimm (Heidelberg University Hospital, Germany) for providing TRISPR-sgRNA expression plasmid, and S. Gray (University of Texas Southwestern Medical Center) for providing the self-complementary AAV plasmid. This work was supported by the National Institutes of Health ( NIH ) (grants HL130253 ), Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center (grant P50 HD 087351 ), and Robert A. Welch Foundation (grant 1-0025 to E.N.O.). Publisher Copyright: {\textcopyright} 2022 The Author(s)",
year = "2022",
month = jun,
day = "14",
doi = "10.1016/j.omtn.2022.03.004",
language = "English (US)",
volume = "28",
pages = "154--167",
journal = "Molecular Therapy - Nucleic Acids",
issn = "2162-2531",
publisher = "Nature Publishing Group",
}