# CRISPR editing therapy for Duchenne muscular dystrophy

> **NIH NIH R01** · UNIVERSITY OF MISSOURI-COLUMBIA · 2024 · $552,403

## Abstract

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.

## Key facts

- **NIH application ID:** 10881958
- **Project number:** 5R01NS131416-02
- **Recipient organization:** UNIVERSITY OF MISSOURI-COLUMBIA
- **Principal Investigator:** Dongsheng Duan
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $552,403
- **Award type:** 5
- **Project period:** 2023-07-15 → 2028-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10881958

## Citation

> US National Institutes of Health, RePORTER application 10881958, CRISPR editing therapy for Duchenne muscular dystrophy (5R01NS131416-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10881958. Licensed CC0.

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