@article{a6bcebe4f26c40a2957db2f121d8300e,
title = "A consolidated AAV system for single-cut CRISPR correction of a common Duchenne muscular dystrophy mutation",
abstract = "Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene, is a lethal neuromuscular disease. Correction of DMD mutations in animal models has been achieved by CRISPR/Cas9 genome editing using Streptococcus pyogenes Cas9 (SpCas9) delivered by adeno-associated virus (AAV). However, due to the limited viral packaging capacity of AAV, two AAV vectors are required to deliver the SpCas9 nuclease and its single guide RNA (sgRNA), impeding its therapeutic application. We devised an efficient single-cut gene-editing method using a compact Staphylococcus aureus Cas9 (SaCas9) to restore the open reading frame of exon 51, the most commonly affected out-of-frame exon in DMD. Editing of exon 51 in cardiomyocytes derived from human induced pluripotent stem cells revealed a strong preference for exon reframing via a two-nucleotide deletion. We adapted this system to express SaCas9 and sgRNA from a single AAV9 vector. Systemic delivery of this All-In-One AAV9 system restored dystrophin expression and improved muscle contractility in a mouse model of DMD with exon 50 deletion. These findings demonstrate the effectiveness of CRISPR/SaCas9 delivered by a consolidated AAV delivery system in the correction of DMD in vivo, representing a promising therapeutic approach to correct the genetic causes of DMD.",
keywords = "AAV, CRISPR/Cas, Duchenne muscular dystrophy, SaCas9, exon reframing, exon skipping, gene editing, induced pluripotent stem cells, sgRNA",
author = "Yu Zhang and Takahiko Nishiyama and Hui Li and Jian Huang and Ayhan Atmanli and Efrain Sanchez-Ortiz and Zhaoning Wang and Mireault, {Alex A.} and Mammen, {Pradeep P.A.} and Rhonda Bassel-Duby and Olson, {Eric N.}",
note = "Funding Information: We thank J. Cabrera for graphics, Y. Zhang and the Boston Children{\textquoteright}s Hospital Viral Core for AAV production, the Metabolic Phenotyping Core for serum CK analysis, the Sanger Sequencing Core and the Next Generation Sequencing Core for sequencing services, the Flow Cytometry Core for cell sorting, and the Histology Core for H&E staining. We are grateful to S. Hauschka (University of Washington) for providing the muscle-specific CK8e promoter. This work was supported by the NIH (grant HL130253 ), the Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center (grant P50 HD 087351 ), and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). Funding Information: We thank J. Cabrera for graphics, Y. Zhang and the Boston Children's Hospital Viral Core for AAV production, the Metabolic Phenotyping Core for serum CK analysis, the Sanger Sequencing Core and the Next Generation Sequencing Core for sequencing services, the Flow Cytometry Core for cell sorting, and the Histology Core for H&E staining. We are grateful to S. Hauschka (University of Washington) for providing the muscle-specific CK8e promoter. This work was supported by the NIH (grant HL130253), the Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center (grant P50 HD 087351), and the Robert A. Welch Foundation (grant 1-0025 to E.N.O.). Y.Z. R.B.-D. and E.N.O. wrote and edited the manuscript. Y.Z. designed the experiments, cloned AAV constructs, and performed iPSC culture, animal studies, tissue cryosectioning, imaging, and data analysis. T.N. performed iPSC culture, western blot, and immunocytochemistry. H.L. performed genomic PCR, RT-PCR, and data analysis. J.H. performed the muscle electrophysiology analysis. A.A. performed cardiomyocyte calcium analysis. E.S.-O. performed immunohistochemistry, western blot, and imaging. Z.W. performed deep-sequencing analysis. A.A.M. performed grip-strength analysis, animal dissection, and tissue-processing experiments. P.P.A.M. provided oversight of the electrophysiology analysis. R.B.-D and E.N.O. are consultants for Vertex Therapeutics. The other authors declare that they have no competing interests. Publisher Copyright: {\textcopyright} 2021 The Author(s)",
year = "2021",
month = sep,
day = "10",
doi = "10.1016/j.omtm.2021.05.014",
language = "English (US)",
volume = "22",
pages = "122--132",
journal = "Molecular Therapy - Methods and Clinical Development",
issn = "2329-0501",
publisher = "Nature Publishing Group",
}