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AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice
Chady H. Hakim, … , Charles A. Gersbach, Dongsheng Duan
Chady H. Hakim, … , Charles A. Gersbach, Dongsheng Duan
Published December 6, 2018
Citation Information: JCI Insight. 2018;3(23):e124297. https://doi.org/10.1172/jci.insight.124297.
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Research Article Therapeutics

AAV CRISPR editing rescues cardiac and muscle function for 18 months in dystrophic mice

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Abstract

Adeno-associated virus–mediated (AAV-mediated) CRISPR editing is a revolutionary approach for treating inherited diseases. Sustained, often life-long mutation correction is required for treating these diseases. Unfortunately, this has never been demonstrated with AAV CRISPR therapy. We addressed this question in the mdx model of Duchenne muscular dystrophy (DMD). DMD is caused by dystrophin gene mutation. Dystrophin deficiency leads to ambulation loss and cardiomyopathy. We treated 6-week-old mice intravenously and evaluated disease rescue at 18 months. Surprisingly, nominal dystrophin was restored in skeletal muscle. Cardiac dystrophin was restored, but histology and hemodynamics were not improved. To determine the underlying mechanism, we evaluated components of the CRISPR-editing machinery. Intriguingly, we found disproportional guide RNA (gRNA) vector depletion. To test whether this is responsible for the poor outcome, we increased the gRNA vector dose and repeated the study. This strategy significantly increased dystrophin restoration and reduced fibrosis in all striated muscles at 18 months. Importantly, skeletal muscle function and cardiac hemodynamics were significantly enhanced. Interestingly, we did not see selective depletion of the gRNA vector after intramuscular injection. Our results suggest that gRNA vector loss is a unique barrier for systemic AAV CRISPR therapy. This can be circumvented by vector dose optimization.

Authors

Chady H. Hakim, Nalinda B. Wasala, Christopher E. Nelson, Lakmini P. Wasala, Yongping Yue, Jacqueline A. Louderman, Thais B. Lessa, Aihua Dai, Keqing Zhang, Gregory J. Jenkins, Michael E. Nance, Xiufang Pan, Kasun Kodippili, N. Nora Yang, Shi-jie Chen, Charles A. Gersbach, Dongsheng Duan

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Figure 4

Increasing the gRNA AAV vector dose did not alter dystrophin expression following intramuscular AAV CRISPR therapy.

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Increasing the gRNA AAV vector dose did not alter dystrophin expression ...
The Cas9 and gRNA AAV vectors were coinjected to the tibialis anterior muscle of 6-week-old mdx mice at the Cas9 vector-to-gRNA vector ratio of 1:0 (1 × 1011 vg Cas9 only), 0:1 (1 × 1011 vg gRNA only), 1:1 (1 × 1011 vg Cas9 and 1 × 1011 vg gRNA), or 1:3 (1 × 1011 vg Cas9 and 3 × 1011 vg gRNA) and evaluated at 3 months of age. (A) Representative dystrophin immunostaining photomicrographs. Scale bar: 200 μm. (B) Quantification of dystrophin-positive myofibers. (C) Representative dystrophin Western blot. (D) Quantification of dystrophin Western blot. (E) Quantification of the AAV genome copy number. Statistical analyses were done using following tests: B, 1-way ANOVA; D and E, Mann-Whitney test. *P < 0.05.

Copyright © 2023 American Society for Clinical Investigation
ISSN 2379-3708

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