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AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice.
Mol Ther. 2019 09 04; 27(9):1568-1585.MT

Abstract

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy.

Authors+Show Affiliations

Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.Department of Biomedical, Biological and Chemical Engineering, College of Engineering, University of Missouri, Columbia, MO 65212, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA; National Center for Advancing Translational Sciences, NIH, Rockville, MD 20850, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.The Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD 21205, USA; Department of Neurology and Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA.Department of Biomedical, Biological and Chemical Engineering, College of Engineering, University of Missouri, Columbia, MO 65212, USA.National Center for Advancing Translational Sciences, NIH, Rockville, MD 20850, USA.Department of Physics, University of Missouri, Columbia, MO 65212, USA.The Hugo W. Moser Research Institute, Kennedy Krieger Institute, Baltimore, MD 21205, USA; Department of Neurology and Neuroscience, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA.Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO 65212, USA; Department of Biomedical, Biological and Chemical Engineering, College of Engineering, University of Missouri, Columbia, MO 65212, USA; Department of Biomedical Sciences, College of Veterinary Medicine, University of Missouri, Columbia, MO 65211, USA; Department of Neurology, School of Medicine, University of Missouri, Columbia, MO 65212, USA. Electronic address: duand@missouri.edu.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, U.S. Gov't, Non-P.H.S.

Language

eng

PubMed ID

31327755

Citation

Nance, Michael E., et al. "AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice." Molecular Therapy : the Journal of the American Society of Gene Therapy, vol. 27, no. 9, 2019, pp. 1568-1585.
Nance ME, Shi R, Hakim CH, et al. AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice. Mol Ther. 2019;27(9):1568-1585.
Nance, M. E., Shi, R., Hakim, C. H., Wasala, N. B., Yue, Y., Pan, X., Zhang, T., Robinson, C. A., Duan, S. X., Yao, G., Yang, N. N., Chen, S. J., Wagner, K. R., Gersbach, C. A., & Duan, D. (2019). AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice. Molecular Therapy : the Journal of the American Society of Gene Therapy, 27(9), 1568-1585. https://doi.org/10.1016/j.ymthe.2019.06.012
Nance ME, et al. AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice. Mol Ther. 2019 09 4;27(9):1568-1585. PubMed PMID: 31327755.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice. AU - Nance,Michael E, AU - Shi,Ruicheng, AU - Hakim,Chady H, AU - Wasala,Nalinda B, AU - Yue,Yongping, AU - Pan,Xiufang, AU - Zhang,Tracy, AU - Robinson,Carolyn A, AU - Duan,Sean X, AU - Yao,Gang, AU - Yang,N Nora, AU - Chen,Shi-Jie, AU - Wagner,Kathryn R, AU - Gersbach,Charles A, AU - Duan,Dongsheng, Y1 - 2019/07/03/ PY - 2019/01/27/received PY - 2019/06/07/revised PY - 2019/06/19/accepted PY - 2019/7/23/pubmed PY - 2020/6/17/medline PY - 2019/7/23/entrez KW - AAV KW - Ai14 KW - CRISPR KW - Cas9 KW - Cre KW - DMD KW - MuSC KW - Pax7 KW - Pax7-ZsGreen KW - dystrophin KW - gRNA KW - gene editing KW - mdx KW - muscle KW - muscle graft KW - muscle stem cell KW - regeneration KW - satellite cell KW - stem cell KW - stem cell renewal SP - 1568 EP - 1585 JF - Molecular therapy : the journal of the American Society of Gene Therapy JO - Mol. Ther. VL - 27 IS - 9 N2 - CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%-47% and 24%-89% of Pax7+ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7+ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin+ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy. SN - 1525-0024 UR - https://www.unboundmedicine.com/medline/citation/31327755/AAV9_Edits_Muscle_Stem_Cells_in_Normal_and_Dystrophic_Adult_Mice_ L2 - https://linkinghub.elsevier.com/retrieve/pii/S1525-0016(19)30307-7 DB - PRIME DP - Unbound Medicine ER -