Fig. 4: Precision in vivo base editing effectively restores hearing in Pou4f3^WT/Q113* mice.

A Study design of the intein-mediated dual AAV-SchABE8e-sgRNA3 therapy. B G/(A + G) ratios in cochleae of uninjected (gray, n = 14), injected with dual AAV-SchABE8e-sgRNA3 (brown, n = 12), and contralateral ears (orange, n = 12) at 1 week post-injection in DNA level (left). Injected vs uninjected: p = 0.2154, injected vs contralateral: p = 0.0011. In the cDNA level (right), uninjected (gray, n = 9), injected with dual AAV-SchABE8e-sgRNA3 (red, n = 12), and contralateral ears (blue, n = 12) are shown. Injected vs uninjected: p = 0.0001, injected vs contralateral: p = 0.0018. C Off-target activity analysis of the nine candidate sites identified by Benchling in Pou4f3^WT/Q113* treated mice after one week. The on-target sites and the top nine off-target sites (OTs) were analyzed by NGS. Data were collected from five independent experiments. D–F Click ABR (D) and TB-ABR (E, F) thresholds in WT (black) and Pou4f3^WT/Q113* mice injected with AAV-SchABE8e-sgRNA3 (red) and contralateral ears (blue) at 2-, 3-, and 4-month post-injection. D Thresholds in injected ears (red) didn’t differ significantly from age-matched WT controls (black) across time points. WT vs contralateral: p = 0.039710 (2 months), p = 0.001418 (3 months), p = 0.000919 (4 months); contralateral vs injected: p = 0.008767 (2 months), p = 0.003647 (3 months), p = 0.037131 (4 months); WT vs injected: p = 0.862433 (2 months), p = 0.61264 (3 months), p = 0.195394 (4 months). E ABR traces of 12 kHz. F TB-ABR thresholds. 2 months, contralateral vs injected: p = 0.332195 (4k), p < 0.000001 (8k), p < 0.000001 (12k), p = 0.000002 (16k), p = 0.002180 (24k), p = 0.091392 (32k); 3 months, contralateral vs injected: p = 0.000731 (4k), p = 0.000171 (8k), p = 0.000010 (12k), p < 0.000001 (16k), p = 0.031688 (24k), p = 0.229491 (32k); 4 months, contralateral vs injected: p = 0.016965 (4k), p = 0.145704 (8k), p = 0.002659 (12k), p = 0.000270 (16k), p = 0.355918 (24k). At 2 months post-injection, Pou4f3^WT/Q113* mice injected with AAV-SchABE8e-sgRNA3 exhibited threshold improvements of 40 (8 kHz), 30 (12 kHz), and 40 dB SPL (16 kHz) compared to contralateral ears. Hearing recovery remained stable at 3–4 months in the middle- and low-frequency range (4–16 kHz), while hearing recovery in the high-frequency thresholds showed persistent elevation, with a residual deficit of ~25 dB SPL vs WT controls. Data in (B, C, D, F) are represented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-tests, except for comparisons between injected and contralateral groups in (B), which were analyzed by paired two-tailed Student’s t-tests (^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ns no significant difference). Source data are provided as a Source data file.