Fig 2: Anc80L65–SaCas9–Htra2 gRNA injections protected hair cells and improved hearing function in neomycin-exposure mice. a Representative confocal microscopy images at 2 weeks after the last dose of neomycin from an injected cochlea and the contralateral (non-injected) cochlea. IHC inner hair cell, OHC outer hair cell. Arrows indicate the missing IHCs. Scale bar represents 20 μm long and applies to all images. b Quantification of the hair cells 2 weeks after the last dose of neomycin. Individual values are shown. Statistical tests in b are unpaired two-tailed Student’s t-tests. c ABR thresholds in injected and non-injected ears 2 weeks after the last dose of neomycin. Paired t-tests or Wilcoxon matched-pairs signed rank tests were used as applicable. d, e Peak amplitudes (d) and latencies (e) of ABR wave 1 evoked by 90 dB SPL at 4 and 8 kHz in the Anc80L65–SaCas9–Htra2 gRNA-injected ears compared with the non-injected ears 2 weeks after the last dose of neomycin. Unpaired two-tailed Student’s t-tests were used. f 8-kHz ABR waveforms recorded at 2 weeks after the last dose of neomycin from the representative non-injected ear and the single best injected ear. The hearing thresholds are shown in blue and red for the non-injected ear and injected ear, respectively. The scale bar applies to all traces. g ABR thresholds in injected and non-injected ears 4 weeks after the last dose of neomycin. Paired t-tests or Wilcoxon matched-pairs signed rank tests were used as applicable. The lighter red lines in c and g represent the ABR thresholds of individual injected ears. **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 3: HTRA2 plays a critical role in DELE1-VS generation.a WT HEK293T cells were treated with DMSO, CCCP (20 μM), o-Phenanthroline (500 μM) or a combination of CCCP and o-Phenanthroline for the indicated time. Whole-cell lysates were analyzed by WB. b WT HEK293T cells were treated with DMSO, oligomycin (10 μM) or increasing doses of AEBSF (125, 250, 500 μM) for 4 h, or pre-treated with the same increasing doses of AEBSF in (A) for 4 h, and then co-treated with 4 h of 20 μM CCCP or 10 μM oligomycin. Whole-cell lysates were analyzed by WB. c Mitochondrial fractionation of WT HEK293T cells lysates following 4 h of DMSO or CCCP (20 μM) and analyzed by WB. d SC, shHtrA2 A, B and C HEK293T cells were treated with DMSO, CCCP (20 μM) or oligomycin (10 μM) for 4 h. Whole-cell lysates were analyzed by WB. e SC and shHtrA2 C HEK293T cells were transfected with 0.4 μg of empty plasmid, WT HtrA2 plasmid or S306A HtrA2 plasmid overnight, and then treated with 4 h of DMSO or oligomycin (10 μM). Whole cell lysates were analyzed by WB. f SC and shHtrA2 B HEK293T cells were transfected with 0.4 μg of empty plasmid, WT HtrA2 plasmid, S141A HtrA2 plasmid or G399S HtrA2 plasmid overnight, and then treated with 4 h of DMSO or oligomycin (10 μM). Whole cell lysates were analyzed by WB.

Fig 4: Effects of Anc80L65–SpCas9 therapeutic system on hair cell survival and hearing function in neomycin-exposure mice. a Experimental overview. b The individual (dot) and mean ± SD (bar) indel frequency of the three gRNAs. n = 4. c Representative confocal images showing immunofluorescence staining with Myo7a (red) 2 weeks after the last dose of neomycin from an injected cochlea and the contralateral (non-injected) cochlea. IHC inner hair cell, OHC outer hair cell. Arrows indicate the missing IHCs. Scale bar represents 20 μm long and applies to all images. d Quantification of cochlear hair cells 2 weeks after the last dose of neomycin. Individual values are shown; bars represent the mean ± SD. Unpaired two-tailed Student’s t-tests or Mann–Whitney U-tests were used as applicable. e Comparison of the ABR thresholds between the non-injected and injected ears 2 weeks after the last dose of neomycin. f Families of ABR waveforms recorded at 2 weeks after the last dose of neomycin from the representative non-injected ear and the single best ear injected with Anc80L65–SpCas9 system. The blue trace indicates the threshold of the non-injected ear while the red trace indicates the threshold of the injected ear. The scale bar applies to all traces. g Comparison of the ABR thresholds between the non-injected and injected ears 4 weeks after the last dose of neomycin. The lighter red lines in e and g represent the ABR threshold of individual injected ears. For statistical analysis in e and g, two-tailed paired t-tests or Wilcoxon matched-pairs signed rank tests were used as applicable. h, i Peak amplitudes (h) and latencies (i) of ABR wave 1 evoked by 90 dB SPL in the Anc80L65–SpCas9–Htra2 gRNA-injected ears compared with the non-injected ears 4 weeks after the last dose of neomycin. Unpaired two-tailed Student’s t-tests or unpaired t-tests with Welch’s correction were used as applicable. Data are presented as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig 5: The Anc80L65–SpCas9–Htra2 gRNA therapeutic system protected hair cells from neomycin-induced injury in vitro. a Experimental procedures for the in vitro studies with application of the Anc80L65–SpCas9 therapeutic system. b The indel frequency of the gRNAs was measured in the cultured basilar membrane at 8 days post-transduction with the Anc80L65–SpCas9 therapeutic system using high-throughput sequencing. Dots represent individual values, and bars represent the mean ± SD, n = 3. c Indels causing in-frame versus frameshift mutations in the coding sequence after co-transduction of Anc80L65–SpCas9 and Anc80L65–gRNA. d Representative confocal images of neomycin-treated cochlear explants without Anc80L65–SpCas9–Htra2 gRNA treatment. e Representative confocal images of Myo7a and caspase-3 immunofluorescence in neomycin-treated cochlear explants treated with Anc80L65–SpCas9–Htra2 gRNA for 8 days prior to neomycin exposure. IHC inner hair cell, OHC outer hair cell. Both scale bars (in d and e) represent 20 μm long and apply to all images. f–h Comparison of the numbers of Myo7a+ hair cells (f), caspase-3+ cells (g), and Myo7a+/caspase-3+ hair cells (h) per 100-μm sensory epithelium region between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment in the apical, middle, and basal turns of the cochlea. i Comparison of the ratio of Myo7a+/caspase-3+ cells to Myo7a+ cells between the neomycin-treated groups with or without Anc80L65–SpCas9–Htra2 gRNA treatment. Dots represent individual values, and bars represent the mean ± SD in f–i. For statistical analysis in f–i, Mann–Whitney U-tests, unpaired two-tailed Student’s t-tests, or unpaired t-tests with Welch’s correction were used as applicable. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 5