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Updated: Oct 12, 2025
CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors
Published on: September 14, 2019
Cas9-specific immune responses compromise local and systemic AAV CRISPR therapy in multiple dystrophic canine models
Chady H Hakim1,2, Sandeep R P Kumar3,4, Dennis O Pérez-López1
1Department of Molecular Microbiology and Immunology, The University of Missouri, Columbia, MO, USA.
Abstract:
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.
Insights
Immune responses to Cas9 in dogs treated with adeno-associated virus (AAV)-CRISPR gene therapy can cause inflammation. This Cas9 immunity may hinder AAV-CRISPR
Area of Science:
- Gene therapy
- Immunology
- Molecular biology
Background:
- Adeno-associated virus (AAV)-CRISPR gene editing shows therapeutic potential for various diseases.
- The immune response to bacterial-derived Cas9 is a potential obstacle for AAV-CRISPR therapies.
- Immunological effects of AAV-mediated Cas9 expression in large mammals remain under-investigated.
Purpose of the Study:
- To investigate Cas9-specific immune responses in canine models following AAV-CRISPR therapy for Duchenne muscular dystrophy (DMD).
- To assess the impact of intramuscular and intravenous delivery of AAV-CRISPR on immune reactions.
- To evaluate the efficacy of immune suppression and muscle-specific promoters in mitigating these responses.
Main Methods:
- Canine models of Duchenne muscular dystrophy (DMD) received intramuscular or intravenous AAV-CRISPR therapy.
- Immune responses, including humoral and cytotoxic T-lymphocyte (CTL) activity, were monitored.
- Muscle inflammation and dystrophin expression were assessed.
- Comparisons were made with other therapeutic (micro-dystrophin, SERCA2a) and reporter (alkaline phosphatase, AP) AAV vectors.
Main Results:
- AAV-CRISPR treatment led to initial dystrophin restoration in DMD dogs but also induced muscle inflammation.
- Cas9-specific humoral and CTL responses were observed.
- Prednisolone and muscle-specific promoters did not prevent these immune responses.
- Normal dogs showed similar but milder immune responses to AAV-mediated Cas9 expression.
- Other AAV vectors (micro-dystrophin, SERCA2a, AP) achieved persistent expression without inducing inflammation.
Conclusions:
- Cas9-specific immunity, including inflammation and T-cell responses, is induced by AAV-mediated Cas9 expression in large mammals.
- These immune responses were not mitigated by standard immune suppression or muscle-specific promoters.
- Cas9 immunity poses a significant challenge for the clinical application of AAV-CRISPR gene therapy in large animals and potentially humans.
