Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness
Wei Wei, … , Xue-Zhong Liu, Zheng-Yi Chen
Wei Wei, … , Xue-Zhong Liu, Zheng-Yi Chen
Published August 14, 2025
Citation Information: J Clin Invest. 2025;135(20):e187872. https://doi.org/10.1172/JCI187872.
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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 1

Allele-specific genome editing using SaCas9–sgRNA-1 in mouse P2rx2V61L/+ primary cells.

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Allele-specific genome editing using SaCas9–sgRNA-1 in mouse P2rx2V61L/+...
(A) The DNA sequence of the P2rx2V61L mutation locus and the sgRNAs designs for SpCas9 and SaCas9, respectively. The mutation nucleotide (C) in the P2rx2V61L allele is in red. The protospacer adjacent motifs (PAMs) nucleotides are in blue. (B) Schematic overview of plasmid constructions for different CRISPR systems used for the in vitro study. (C) The experiment design for studying genome editing in the primary fibroblasts from P2rx2V61L/+ mice. (D) Quantification of the indel frequency in P2rx2V61L/+ and P2rx2+/+ primary fibroblasts after genome editing using different Cas9/sgRNA combinations. n = 3. Error bar represents SD. (E and F) Representative NGS results from SaCas9–sgRNA-1–edited P2rx2V61L/+ (E) and P2rx2+/+ primary fibroblasts (F). The red arrowhead indicates the double-stranded DNA cutting site. (G) Indel profiles from the SaCas9–sgRNA-1–edited P2rx2V61L allele in primary fibroblasts. Negative numbers represent deletions, positive numbers represent insertions. (H) Pie chart showing the out-of-frame (3n ± 1) ratio in the indel profile. (I) CIRCLE-Seq analysis of SaCas9–sgRNA-1 in P2rx2V61L/+ primary fibroblasts’ genomic DNA. (J) Quantification of indel frequency of potential off-target sites from the mouse genome. NLS, nuclear localization signal. Puro, puromycin.