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The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.<\/jats:p>","DOI":"10.1038\/s41467-021-26830-7","type":"journal-article","created":{"date-parts":[[2021,11,24]],"date-time":"2021-11-24T06:03:25Z","timestamp":1637733805000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":177,"title":["Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models"],"prefix":"10.1038","volume":"12","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-6865-050X","authenticated-orcid":false,"given":"Chady H.","family":"Hakim","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sandeep R. 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The Duan lab has received research supports unrelated to this project from Solid Biosciences and Edgewise in the last three years. RWH has received research support from Luye R&D, royalty payments from Takeda Pharmaceuticals, and consulting fees from AGTC. None of these are related to this project. C.A.G., C.E.N., C.H.H., D.D., N.B.W., Y.Y. have filed patent applications related to genome editing for Duchenne muscular dystrophy. C.A.G. is an advisor to and receives research support from Sarepta Therapeutics, Inc., is a founder of M2X2 Therapeutics, Locus Biosciences, and Element Genomics, and is an advisor to Iveric Bio and Levo Therapeutics. D.G. is a co-founder and shareholder of AaviGen GmbH. The remaining authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"6769"}}