# CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation

## TL;DR

A new gene therapy using RNA base editing successfully restores hearing in mice with a specific genetic mutation causing deafness.

## Contribution

The study introduces a CRISPR-free RNA base editing system that effectively restores hearing in a mouse model of hereditary deafness.

## Key findings

- RNA base editing with RESTART v3 reverses a nonsense mutation in Otof, restoring functional otoferlin.
- Mice treated with the system showed significant hearing restoration and improved auditory reflexes.
- The approach offers a promising strategy for treating hereditary deafness caused by specific Otof mutations.

## Abstract

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.

Nonsense mutations in OTOF cause hereditary deafness. RESTART v3 achieves efficient, precise readthrough at Otof c.1315 C > T (p.R439*) sites, reinstating arginine to yield full-length, functional otoferlin, markedly restoring hearing in mice and showing strong promise for clinical translation.