CRISPR editing therapy for Duchenne muscular dystrophy

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Project Description Duchenne muscular dystrophy (DMD) is caused by null mutations in the dystrophin gene. CRISPR/Cas9 editing holds promise to treat DMD at its genetic root. Since DMD affects all muscles in the body, effective therapy for DMD would require bodywide muscle delivery. Adeno-associated virus (AAV) vector is the only delivery system that can efficiently reach all body muscles. For this reason, AAV has been the vector of choice for CRISPR-mediated gene repair therapy for DMD. The AAV vector leads to persistent transgene expression. Continuous Cas9 expression creates two problems. First, it increases the odds of off-target editing. Second, the cytotoxic T lymphocyte (CTL) response to the bacterial-derived Cas9 protein can eliminate the treated cells and abolish the therapy. Many approaches have been developed to monitor off-target editing and improve gene editing fidelity. However, it has been elusive to model the Cas9-specific CTL response. Mouse studies revealed a limited cellular response that failed to eliminate Cas9 transduced cells. In fact, we and others have observed nearly lifelong Cas9 expression, muscle pathology amelioration, and function improvement in mdx mice, the most used mouse DMD model. In contrast to the mouse model, dystrophic canines are considered better models for informing human trials. To determine whether Cas9 immunity is a hurdle for AAV-mediated DMD CRISPR therapy, we performed a comprehensive study in four independent canine models (normal canines and three different canine DMD models) using both Cas9 and non-Cas9 AAV vectors via local and systemic delivery. We found compelling evidence suggesting that the Cas9, but not non-Cas9, AAV vector induced a robust CTL response and eliminated gene-edited dystrophin-positive myofibers. Our studies established a reliable model system to study Cas9 immunity. Importantly, it opens the door to developing and validating strategies that may mitigate the Cas9-specific CTL response in a clinically relevant large animal model. In this proposal, we will leverage our findings to explore novel strategies that may support persistent therapeutic editing without inducing the CTL response in the canine DMD model. Our studies will pave the way for translating AAV CRISPR therapy to DMD patients in the future. Our findings will also inform the translation of AAV CRISPR therapy for other human diseases.