Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models

Nat Commun. 2021 Nov 24;12(1):6769. doi: 10.1038/s41467-021-26830-7.


Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. 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.

Publication types

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

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • CRISPR-Cas Systems / immunology*
  • Dependovirus / genetics
  • Disease Models, Animal
  • Dogs
  • Dystrophin / genetics
  • Dystrophin / immunology
  • Gene Editing / methods
  • Genes, Reporter / genetics
  • Genes, Reporter / immunology
  • Genetic Therapy / adverse effects*
  • Genetic Therapy / methods
  • Genetic Vectors / administration & dosage
  • Genetic Vectors / immunology*
  • Humans
  • Muscle, Skeletal / immunology*
  • Muscle, Skeletal / pathology
  • Muscular Dystrophy, Duchenne / genetics
  • Muscular Dystrophy, Duchenne / immunology
  • Muscular Dystrophy, Duchenne / pathology
  • Muscular Dystrophy, Duchenne / therapy*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / genetics
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / immunology


  • DMD protein, human
  • Dystrophin
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • ATP2A2 protein, human