Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness
Wei Wei, Wenliang Zhu, Stewart Silver, Ariel M. Armstrong, Fletcher S. Robbins, Arun Prabhu Rameshbabu, Katherina Walz, Yizhou Quan, Wan Du, Yehree Kim, Artur A. Indzhykulian, Yilai Shu, Xue-Zhong Liu, Zheng-Yi Chen
Wei Wei, Wenliang Zhu, Stewart Silver, Ariel M. Armstrong, Fletcher S. Robbins, Arun Prabhu Rameshbabu, Katherina Walz, Yizhou Quan, Wan Du, Yehree Kim, Artur A. Indzhykulian, Yilai Shu, Xue-Zhong Liu, Zheng-Yi Chen
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Research Article Otology

Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness

Abstract

Genome editing has the potential to treat genetic hearing loss. However, current editing therapies for genetic hearing loss have shown efficacy only in hearing rescue. In this study, we evaluated a rescue strategy using adeno-associated virus (AAV) type 2–mediated delivery of Staphylococcus aureus Cas9-sgRNA in the mature inner ear of the P2rx2V61L/+ mouse model of autosomal dominant deafness-41 (DFNA41), a dominant, delayed-onset, and progressive hearing loss in humans. We demonstrate that local injection in adult mice results in efficient and specific editing that abolishes the mutation without notable off-target effects or AAV genome integration. Editing effectively restores long-term auditory and vestibular function. Editing further protects P2rx2V61L/+ mice from hypersensitivity to noise-induced hearing loss, a phenotype also observed in patients with DFNA41. Intervention in mice at a juvenile stage broadens the frequency range rescued, highlighting the importance of early intervention. An effective and specific gRNA for the human P2RX2 V60L mutation has been identified. Our study establishes the feasibility of editing to treat DFNA41 caused by P2RX2 V60L mutation in humans and opens an avenue for using editing to rescue hearing and vestibular function while mitigating noise-induced hearing loss.

Authors

Wei Wei, Wenliang Zhu, Stewart Silver, Ariel M. Armstrong, Fletcher S. Robbins, Arun Prabhu Rameshbabu, Katherina Walz, Yizhou Quan, Wan Du, Yehree Kim, Artur A. Indzhykulian, Yilai Shu, Xue-Zhong Liu, Zheng-Yi Chen

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Figure 7

AAV2–SaCas9–sgRNA-1 adult injection rescues utricle HCs and vestibular function in the P2rx2V61L/+ mouse model of DFNA41.

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AAV2–SaCas9–sgRNA-1 adult injection rescues utricle HCs and vestibular f...
(A) Schematic representation of the experimental design. (B–D) MYO7A-immunolabeled utricle of WT uninjected, P2rx2V61L/+ injected, and P2rx2V61L/+ uninjected ears at 9 months after injection at 4 weeks old. (E) MYO7A+ cell counts from WT utricles (black), P2rx2V61L/+ injected (blue), and P2rx2V61L/+ uninjected (red). n = 5. (F–H) Representative recording of open field test tracking the movements for 3 minutes for WT (F), injected (G), and uninjected (H) P2rx2V61L/+ mice, 9 months after injection at 4 weeks old. (I) The number of full-body rotations per minute in the open field test among P2rx2V61L/+ uninjected, P2rx2V61L/+ injected, and WT groups. (J) Distance covered per minute for mice tested in the open field tests among P2rx2V61L/+ uninjected, injected, and WT groups. (K) The result of rotarod performance in 5 trials at 9 months after injection of WT, uninjected, and injected P2rx2V61L/+ mice. All data are presented as mean ± SEM. A 2-way ANOVA with Tukey’s multiple comparison test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Scale bar: 50 μm. IF, immunofluorescence. NLS, nuclear localization signals.