CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation
- Hanxiao Sun
- Qi Teng
- Wenqing Liu
- Rui Guo
- Menghua Li
- Wei Xiong
- Qiang Huang
- Qianru Yu
- Nan Luo
- Yang Li
- Jinghui Song
- Shusheng Gong
- Xi Shi
- Chengqi Yi
- Ke Liu
2025-12-06
The gene therapy achieved by AAV-mediated otoferlin-overexpression is an effective therapeutic strategy for congenital deafness. However, achieving its physiological and endogenous patterns of expression remains challenging. Here, we generate the homologous mutation Otof c.1315 C > T (p.R439*), equivalent to OTOF c.1273 C > T (p.R425*) found in humans with profound deafness, to create a nonsense mutation-induced deaf mouse model. We then deliver the ‘RESTART v3’ system, which is a CRISPR-free RNA base editor for nonsense mutation suppression, into the cochlea of the mice. We achieve physiological otoferlin expression, and the edited premature termination codon is reverse-mutated to the original amino acid. We observe significant hearing restoration and enhancement of the behavioral auditory startle reflex. Thus, our study presents a successful RNA editing strategy to significantly restore hereditary deafness in mice carrying the specific Otof nonsense mutation, which holds great promise for future clinical translation.