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

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

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 1

Long-term systemic AAV CRISPR therapy in mdx mice is associated with disproportional depletion of the gRNA vector genome.

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Long-term systemic AAV CRISPR therapy in mdx mice is associated with dis...
Systemic AAV-9 CRISPR therapy was performed in 6-week-old male mdx mice at the dose of 7.2 × 1012 vg/mouse and 3.6 × 1012 vg/mouse for the Cas9 and gRNA vectors, respectively. Mice were evaluated at 18 months of age. (A) Representative dystrophin immunostaining photomicrographs from WT, mdx, and CRISPR-treated mdx mice. Scale bar: 100 μm (top); 200 μm (bottom). (B) Quantification of dystrophin-positive myofibers. (C) Representative dystrophin Western blot from WT and CRISPR-treated mice. (D) Quantification of dystrophin Western blot. (E) Quantification of edited and unedited dystrophin transcripts. (F) Representative H&E and Masson trichrome (MTC) staining of the heart. Scale bar: 100 μm. (G) Deep sequencing quantification of indels in untreated and CRISPR-treated mdx mice. (H) Quantification of Cas9 protein expression. (I) Quantification of the AAV genome copy number. For B, E, and H, statistical analyses were performed using Mann-Whitney test. For D, unpaired t test was used. For G and I, multiple t tests was used for statistical analysis. *P < 0.05. #Loading in this lane was at one-sixth the volume of that in other lanes. The quadriceps or gastrocnemius muscle was used to generate the skeletal muscle data shown in the figure.