Expression was assessed in six-week old c.216GA and AAV2/Anc80L65.CMV.harmonin-b1 (0.8 μl; 1.9 × 10
12 gc/ml) injected and non-injected c.216AA mice (a) Semi-quantitative RT-PCR of correctly spliced (450 bp) and truncated (415 bp) mRNA expression revealed vector-derived expression of correctly spliced Ush1c in injected (“I”) and contralateral ears (“C”) of c.216AA rescued mice #1 and #2 (thresholds ≤ 35 dB response at 11.3 kHz from injected ears). Mouse #3 with poor ABR response (thresholds ≥90 dB at 11.3 kHz) showed modest recovery of correct mRNA expression and mouse #4 (thresholds ≥100 dB at 11.3 kHz) showed none. While the correctly spliced form is not detected in uninjected left (“L”) and right (“R”) ears of c.216AA mice (mice #5, 6), both the correct and truncated splice forms were detected in c.216GA mice (mice #7, 8, 9). Corresponding mouse Gapdh shown in the bottom panel was amplified to confirm a consistent amount of material was used. (b) Semi-quantitative radiolabeled PCR analysis confirmed the presence AAV-mUsh1c DNA in injected and contralateral ears of Ush1c.216AA mice. AAV-Ush1c DNA was present but reduced in mice #3 and #4. (c) The relative amount of viral DNA correlates with ABR thresholds. The 11.3 and 16 kHz responses from injected and contralateral cochleas of the four injected mice (#1-4) are illustrated. Linear regressions show high correlation between the amount of viral DNA and ABR threshold responses. Tissues were collected at 6 weeks of age. (d) Immunostaining of harmonin confirmed restored expression of harmonin protein in stereocilia of c.216AA mice that recovered hearing following Anc80L65.CMV.harmonin-b1 P1 RWM injection. Panels show immunofluorescence of harmonin (green) and F-actin (red) in outer hair cell (OHC) bundles in mid apical regions of wild type (WT, 6 weeks old), homozygous c.216AA (4 weeks), injected c.216AA mice with no rescue (4 weeks, no ABR response at 110 dB at any frequency tested), and injected c.216AA mice with rescue (4 weeks, 30 dB at 11 kHz). Scale bar: 10 μm.