Re-administration of adeno-associated virus (AAV)-mediated gene therapy remains challenging due to neutralizing antibodies (NAbs) induced by the initial dose. We previously conducted a single-arm trial showing that single-dose administration of AAV-hOTOF gene therapy in individuals with OTOF-related deafness is safe and leads to hearing improvements. Here we initially demonstrate that AAV1-hOTOF re-administration to the contralateral ear in Otof-/- mice with peak serum NAb titers successfully rescued hearing with limited immune activation. After a protocol amendment of our trial, four patients (aged 2.2-3.4 years) with pre-existing NAbs (titers 1:135-1:3,645), who had previously received a single dose of the gene therapy, were enrolled to receive a second dose in the contralateral ear, as part of the ongoing trial, with a follow-up ranging from 26 weeks to 52 weeks. The primary endpoint was the occurrence of dose-limiting toxicities at 6 weeks and secondary endpoints included safety and auditory function. No dose-limiting toxicity occurred within 6 weeks. In the secondary outcomes, the 26-week average auditory brainstem response threshold in the second treated ear improved from >95 dB at baseline to 43 dB, 63 dB, 80 dB and 53 dB in patients 1-4, respectively. Safety assessment showed that all adverse events were grade 1-2, except one grade 3 decreased neutrophil count; no serious adverse events occurred. These data provide preliminary insights on the safety and efficacy of re-administration of AAV1-hOTOF gene therapy in patients with congenital deafness. Longer follow-up and larger cohorts are needed to establish the safety and efficacy of repeated administration of gene therapies. Trial registration no.: ChiCTR2200063181 .

Autosomal recessive deafness 9, caused by OTOF gene mutations, is characterized by severe-to-complete congenital deafness1. Although gene therapy has shown benefits in a small number of patients2-5, its safety and efficacy across broader age ranges and longer follow-up periods, as well as predictors of treatment outcomes, remain unclear. In this single-arm, multicentre trial conducted at eight centres, 42 participants (aged 0.8-32.3 years) received adeno-associated virus (AAV) serotype 1 carrying a human OTOF coding transgene (AAV1-hOTOF) at three vector dose groups, with up to 2.5-year follow-up. The primary end point was dose-limiting toxicity within 6 weeks. The secondary end point assessed efficacy and adverse events. No dose-limiting toxicities were observed. Grade 3 adverse events included decreased neutrophil count. Hearing was recovered in 90% of participants treated with AAV1-hOTOF, with gradual and stable improvement in auditory brainstem response threshold from greater than 97 ± 1 dB normalized hearing level at baseline to 54 ± 3, 51 ± 3, 50 ± 3 and 42 ± 5 dB normalized hearing level at 1, 1.5, 2 and 2.5 years, respectively, and behavioural audiometry improving from greater than 96 ± 3 dB hearing level at baseline to 37 ± 5 dB hearing level at 2.5 years. Participants aged 0.5-18 years showed greater hearing improvement than adults. A higher number of present distortion product otoacoustic emissions at baseline or biallelic non-truncated OTOF variants was associated with better hearing recovery. Participants with hearing recovery demonstrated gradual improvement in speech perception. AAV1-hOTOF is well-tolerated and efficacious across a broader patient population, with sustained therapeutic benefits for up to 2.5 years. Chinese Clinical Trial Registry registration: ChiCTR2200063181 .

Recent breakthroughs in gene therapy for autosomal recessive deafness 9 (DFNB9) caused by OTOF mutations have transformed treatment paradigms for hereditary hearing loss (HHL). To date, eight clinical trials targeting DFNB9 have been registered in 51 centers across eight countries, demonstrating the rapid progress of gene therapy in auditory medicine. These pioneering studies establish the framework for the clinical translation of gene therapy targeting HHL. This review synthesizes progress in OTOF-related clinical trials, highlighting translational foci such as inner ear drug delivery, trial design, safety assessments, and auditory restoration outcomes. Key challenges in optimizing future therapeutic strategies - including addressing anatomical constraints, refining patient selection criteria, and standardizing outcome measures - are critically examined.

Pathogenic mutations in the OTOF gene cause autosomal recessive hearing loss (DFNB9), one of the most common forms of auditory neuropathy. There is no biological treatment for DFNB9. Here, we designed an OTOF gene therapy agent by dual-adeno-associated virus 1 (AAV1) carrying human OTOF coding sequences with the expression driven by the hair cell-specific promoter Myo15, AAV1-hOTOF. To develop a clinical application of AAV1-hOTOF gene therapy, we evaluated its efficacy and safety in animal models using pharmacodynamics, behavior, and histopathology. AAV1-hOTOF inner ear delivery significantly improved hearing in Otof-/- mice without affecting normal hearing in wild-type mice. AAV1 was predominately distributed to the cochlea, although it was detected in other organs such as the CNS and the liver, and no obvious toxic effects of AAV1-hOTOF were observed in mice. To further evaluate the safety of Myo15 promoter-driven AAV1-transgene, AAV1-GFP was delivered into the inner ear of Macaca fascicularis via the round window membrane. AAV1-GFP transduced 60%-94% of the inner hair cells along the cochlear turns. AAV1-GFP was detected in isolated organs and no significant adverse effects were detected. These results suggest that AAV1-hOTOF is well tolerated and effective in animals, providing critical support for its clinical translation.

BACKGROUND:Gene therapy shows promising potential for patients with autosomal recessive deafness 9 (DFNB9), with ongoing clinical trials (ChiCTR2200063181). A deeper understanding of changes in audiological characteristics is crucial for optimizing the monitoring and evaluation of patients' recovery post-treatment. METHODS:Audiological data were collected from 10 DFNB9 patients who underwent gene therapy, including auditory brain stem response (ABR), auditory steady-state response (ASSR), distortion product otoacoustic emission (DPOAE), and pure-tone audiometry (PTA) tests. FINDINGS:A clear ABR wave V was observed in all participants by 13 weeks. By 52 weeks, distinct ABR waves I and III were visible in some participants. The 1-kHz ABR wave V latency at 85 dB decreased significantly from 9.220 (range 9.015-9.810) ms at 4 weeks to 8.190 (range 7.780-8.530) ms at 52 weeks (p = 0.004), with a trend toward increased wave V amplitude (p = 0.055). Significant correlations were observed between PTA, ABR, and ASSR thresholds at 0.5-4 kHz. The DPOAE signal-to-noise ratio (SNR) at 26 weeks post-treatment showed no significant difference compared with pre-treatment SNR values, nor were there significant correlations between the pre-treatment SNR values and the post-treatment ABR thresholds. CONCLUSIONS:The study demonstrates that ABR and ASSR are reliable objective tools for assessing hearing recovery in DFNB9 patients after gene therapy. ABR reveals positive changes in the auditory pathway over time after gene therapy, enhancing our understanding of the impact of gene therapy on auditory pathway recovery. FUNDING:This work was funded by the National Natural Science Foundation of China.

Individuals with congenital deafness that have received gene therapy represent a unique group who experience hearing recovery and speech development. However, it is unclear how hearing-related cortex changes because of gene therapy. Here we study neural processing in ten patients using functional near-infrared spectroscopy and electroencephalography during a six-month follow-up period. Patients showed an enhancement of activation in the auditory cortex, particularly in parts of the Sylvian parietotemporal area while listening to music. Activation in the right anterior temporal lobe and left Sylvian parietotemporal area was also enhanced when listening to speech. The electroencephalography data showed that the power of the resting-state electroencephalography beta band at time points T2 and T3 was statistically significantly increased after gene therapy, and mismatch negativity amplitudes at T2 and T3 were statistically significantly higher than those at T0. The mental developmental level of the patients also increased after gene therapy. These preliminary findings illuminate the neural and cognitive effects of gene therapy, supporting its potential effectiveness in auditory and mental development.

Importance:OTOF gene therapy (GT) has been shown to improve hearing and speech. The efficacy of GT remains to be compared against cochlear implantation (CI), the current gold standard for congenital deafness. Objective:To evaluate treatment outcomes in auditory and speech perception between patients with congenital deafness treated with GT, CI, or both. Design, Setting, and Participants:This nonblind cohort study was conducted between December 2022 and November 2024. GT patients received follow-up at 3, 6, and 12 months; CI patients received 1-time evaluation at the corresponding time intervals or longer (3, 6, or 12 months). The study was conducted at a single class A tertiary hospital in China. Participants with congenital severe to complete hearing loss, aged 1 to 18 years, who received GT or CI were enrolled. They were matched on duration of deafness, hearing thresholds, and speech ability at the presurgical baseline. Of 1568 participants screened, 72 participants enrolled. Participants were excluded if they had inner ear malformations or vestibular-cochlear nerve abnormalities. Exposures:GT only vs CI; bimodal (unilateral GT plus contralateral CI) vs bilateral CI; GT (CI turned off [CI-off]) vs unilateral CI. Main Outcomes and Measures:The primary outcomes were auditory and speech perception evaluated by questionnaires, including the Infant-Toddler Meaningful Auditory Integration Scale/Meaningful Auditory Integration Scale (IT-MAIS/MAIS), and tests, including audiometry, speech, and music tests. The main secondary outcome was auditory information processing ability assessed by mismatch negativity (MMN). Results:A total of 11 GT patients (6 male [55%]; mean [SD] age at baseline, 3.7 [2.8] years) and 61 CI patients (34 male [56%]; mean [SD] age at baseline, 1.9 [1.5] years) were enrolled. The mean (SD) auditory brainstem response thresholds were restored from greater than 95.0 (0.0) decibels normalized hearing level (dB nHL) to 54.8 (15.9) dB nHL in 9 GT patients at 12 months. For GT-only vs CI in auditory and speech perception, GT patients performed better in IT-MAIS/MAIS at 6 months (median [IQR] score, 31.0 [30.0-32.0] vs 23.5 [19.0-26.3]; P = .01) and 12 months (median [IQR] score, 32.0 [31.0-32.0] vs 28.0 [24.5-30.5]; P = .007). GT patients showed shorter latencies of MMN at 6 months (median [IQR], 0.20 [0.05-0.21] seconds vs 0.23 [0.22-0.25] seconds; P = .006). For bimodal patients at 12 months, GT (CI-off) patients performed better than unilateral CI patients in speech in a noisy environment (median [IQR] disyllable, -1.0 [-3.0 to 2.4] dB sound pressure level (SPL) vs 5.3 [3.1 to 12.1] dB SPL; P = .03); GT plus CI patients performed better than bilateral CI patients in singing in-tune rates (median [IQR], 66.6% [53.7%-83.9%] vs 37.1% [30.3%-56.3%]; P = .04); GT plus CI patients showed shorter latencies of MMN at 12 months (median [IQR], 0.08 [0.07-0.10] seconds vs 0.21 [0.15-0.23] seconds, P = .01). Conclusions and Relevance:GT patients showed stable hearing recovery and exhibited more rapid improvements in auditory and speech performance than CI patients, while outperforming CI patients in speech in noise performance and music perception. These findings suggest that GT may provide a novel effective treatment alternative for patients with genetically driven congenital deafness.

Gene therapy is a promising approach for hereditary deafness. We recently showed that unilateral AAV1-hOTOF gene therapy with dual adeno-associated virus (AAV) serotype 1 carrying human OTOF transgene is safe and associated with functional improvements in patients with autosomal recessive deafness 9 (DFNB9). The protocol was subsequently amended and approved to allow bilateral gene therapy administration. Here we report an interim analysis of the single-arm trial investigating the safety and efficacy of binaural therapy in five pediatric patients with DFNB9. The primary endpoint was dose-limiting toxicity at 6 weeks, and the secondary endpoint included safety (adverse events) and efficacy (auditory function and speech perception). No dose-limiting toxicity or serious adverse event occurred. A total of 36 adverse events occurred. The most common adverse events were increased lymphocyte counts (6 out of 36) and increased cholesterol levels (6 out of 36). All patients had bilateral hearing restoration. The average auditory brainstem response threshold in the right (left) ear was >95 dB (>95 dB) in all patients at baseline, and the average auditory brainstem response threshold in the right (left) ear was restored to 58 dB (58 dB) in patient 1, 75 dB (85 dB) in patient 2, 55 dB (50 dB) in patient 3 at 26 weeks, and 75 dB (78 dB) in patient 4 and 63 dB (63 dB) in patient 5 at 13 weeks. The speech perception and the capability of sound source localization were restored in all five patients. These results provide preliminary insights on the safety and efficacy of binaural AAV gene therapy for hereditary deafness. The trial is ongoing with longer follow-up to confirm the safety and efficacy findings. Chinese Clinical Trial Registry registration: ChiCTR2200063181.

Adeno-associated viral (AAV) vectors are increasingly used as vehicles for gene delivery to treat hearing loss. However, lack of specificity of the transgene expression may lead to overexpression of the transgene in nontarget tissues. In this study, we evaluated the expression efficiency and specificity of transgene delivered by AAV-PHP.eB under the inner ear sensory cell-specific Myo15 promoter. Compared with the ubiquitous CAG promoter, the Myo15 promoter initiates efficient expression of the GFP fluorescence reporter in hair cells, while minimizing non-specific expression in other cell types of the inner ear and CNS. Furthermore, using the Myo15 promoter, we constructed an AAV-mediated therapeutic system with the coding sequence of OTOF gene. After inner ear injection, we observed apparent hearing recovery in Otof-/- mice, highly efficient expression of exogenous otoferlin, and significant improvement in the exocytosis function of inner hair cells. Overall, our results indicate that gene therapy mediated by the hair cell-specific Myo15 promoter has potential clinical application for the treatment of autosomal recessive deafness and yet for other hereditary hearing loss related to dysfunction of hair cells.

A prevalent recessive mutation (c.2485C>T, p.Q829X) within the OTOF gene leads to profound prelingual hearing loss. Here we show that in Otof mice harbouring a mutation (c.2482C>T, p.Q828X) homozygous to human OTOF that faithfully mimics the hearing-loss phenotype, a base editor (consisting of the deaminase ABE7.10max and the Cas9 variant SpCas9-NG) packaged in adeno-associated viruses and injected into the inner ear of the mice via the round-window membrane effectively corrected the pathogenic mutation, with no apparent off-target effects. The treatment restored the levels of the otoferlin protein in 88

Objective:This study aims to analyze the threshold changes in distortion product otoacoustic emissions（DPOAE） and auditory brainstem response（ABR） in adult Otof-/- mice before and after gene therapy, evaluating its effectiveness and exploring methods for assessing hearing recovery post-treatment. Methods:At the age of 4 weeks, adult Otof-/- mice received an inner ear injection of a therapeutic agent containing intein-mediated recombination of the OTOF gene, delivered via dual AAV vectors through the round window membrane（RWM）. Immunofluorescence staining assessed the proportion of inner ear hair cells with restored otoferlin expression and the number of synapses.Statistical analysis was performed to compare the DPOAE and ABR thresholds before and after the treatment. Results:AAV-PHP. eB demonstrates high transduction efficiency in inner ear hair cells. The therapeutic regimen corrected hearing loss in adult Otof-/- mice without impacting auditory function in wild-type mice. The changes in DPOAE and ABR thresholds after gene therapy are significantly correlated at 16 kHz. Post-treatment,a slight increase in DPOAE was observeds,followed by a recovery trend at 2 months post-treatment. Conclusion:Gene therapy significantly restored hearing in adult Otof-/- mice, though the surgical delivery may cause transient hearing damage. Precise and gentle surgical techniques are essential to maximize gene therapy's efficacy.

Adeno-associated virus (AAV)-mediated gene therapy is widely applied to treat numerous hereditary diseases in animal models and humans. The specific expression of AAV-delivered transgenes driven by cell type-specific promoters should further increase the safety of gene therapy. However, current methods for screening cell type-specific promoters are labor-intensive and time-consuming. Herein, we designed a “multiple vectors in one AAV” strategy for promoter construction in vivo. Through this strategy, we truncated a native promoter for Myo15 expression in hair cells (HCs) in the inner ear, from 1,611 bp down to 1,157 bp, and further down to 956 bp. Under the control of these 2 promoters, green fluorescent protein packaged in AAV-PHP.eB was exclusively expressed in the HCs. The transcription initiation ability of the 2 promoters was further verified by intein-mediated otoferlin recombination in a dual-AAV therapeutic system. Driven by these 2 promoters, human otoferlin was selectively expressed in HCs, resulting in the restoration of hearing in treated Otof −/− mice for at least 52 weeks. In summary, we developed an efficient screening strategy for cell type-specific promoter engineering and created 2 truncated Myo15 promoters that not only restored hereditary deafness in animal models but also show great potential for treating human patients in future.

Background Autosomal recessive deafness 9, caused by mutations of the OTOF gene, is characterised by congenital or prelingual, severe-to-complete, bilateral hearing loss. However, no pharmacological treatment is currently available for congenital deafness. In this Article, we report the safety and efficacy of gene therapy with an adeno-associated virus (AAV) serotype 1 carrying a human OTOF transgene (AAV1-hOTOF) as a treatment for children with autosomal recessive deafness 9. Methods This single -arm, single -centre trial enrolled children (aged 1-18 years) with severe-to-complete hearing loss and confirmed mutations in both alleles of OTOF , and without bilateral cochlear implants. A single injection of AAV1-hOTOF was administered into the cochlea through the round window. The primary endpoint was dose-limiting toxicity at 6 weeks after injection. Auditory function and speech were assessed by appropriate auditory perception evaluation tools. All analyses were done according to the intention-to-treat principle. This trial is registered with Chinese Clinical Trial Registry, ChiCTR2200063181, and is ongoing. Findings Between Oct 19, 2022, and June 9, 2023, we screened 425 participants for eligibility and enrolled six children for AAV1-hOTOF gene therapy (one received a dose of 9 x 10 11 vector genomes [vg] and five received 15 x 10 12 vg). All participants completed follow-up visits up to week 26. No dose-limiting toxicity or serious adverse events occurred. In total, 48 adverse events were observed; 46 (96%) were grade 1-2 and two (4%) were grade 3 (decreased neutrophil count in one participant). Five children had hearing recovery, shown by a 40-57 dB reduction in the average auditory brainstem response (ABR) thresholds at 05-40 kHz. In the participant who received the 9 x 10 11 vg dose, the average ABR threshold was improved from greater than 95 dB at baseline to 68 dB at 4 weeks, 53 dB at 13 weeks, and 45 dB at 26 weeks. In those who received 15 x 10 12 AAV1-hOTOF, the average ABR thresholds changed from greater than 95 dB at baseline to 48 dB, 38 dB, 40 dB, and 55 dB in four children with hearing recovery at 26 weeks. Speech perception was improved in participants who had hearing recovery. Interpretation AAV1-hOTOF gene therapy is safe and efficacious as a novel treatment for children with autosomal recessive deafness 9.

Background To create and develop a delivery approach for clinical inner ear gene therapy, we conducted a study of trans-round window membrane (RWM) microinjection using a pipetting microneedle via transcanal endoscopic ear surgery (TEES). Methods The implementation of the trans-RWM microinjection surgery involved seven cadaveric specimens, and the surgical procedures and the pipetting microneedle were developed and optimized. The TEES procedures included tympanic cavity visualization, RWM exposure, stapes footplate perforation, and trans-RWM microinjection. The feasibility of different pipetting microneedles was evaluated during microinjection. Results Exposure of the RWM microinjection site could be easily achieved in TEES, and the soft-connected pipetting microneedle was most suitable for the trans-RWM microinjection. The fluid outflow from stapes perforation could be visibly observed during the microinjection, which indicated inner ear drug delivery was successful. This inner ear drug delivery approach was successfully applied in the clinical trial. Conclusion The trans-RWM microinjection via the soft-connected pipetting microneedle in TEES was proved to be a feasible delivery approach of the inner ear gene therapy.

Mutations to the OTOF gene are among the most common reasons for auditory neuropathy. Although cochlear implants are often effective in restoring sound transduction, there are currently no biological treatments for individuals with variants of OTOF . Previous studies have reported the rescue of hearing in DFNB9 mice using OTOF gene replacement although the efficacy needs improvement. Here, we developed a novel dual-AAV-mediated gene therapy system based on the principles of protein trans-splicing, and we show that this system can reverse bilateral deafness in Otof –/– mice after a single unilateral injection. The system effectively expressed exogenous mouse or human otoferlin after injection on postnatal day 0–2. Human otoferlin restored hearing to near wild-type levels for at least 6 months and restored the release of synaptic vesicles in inner hair cells. Our study not only provides a preferential clinical strategy for the treatment of OTOF -related auditory neuropathies, but also describes a route of development for other large-gene therapies and protein engineering techniques.

OTOF基因突变是导致听神经病的主要因素之一,也是隐性遗传性耳聋的常见致病基因.OTOF基因编码的耳畸蛋白(otoferlin)参与Ca2+相关内毛细胞突触囊泡的融合及神经递质的释放.随着对OTOF基因致病机制的研究和生物治疗递送工具的发展,近年成功采用双腺相关病毒(AAV)介导的基因治疗策略恢复了Otof-/-小鼠耳畸蛋白的表达并挽救了听力.国内外陆续已批准OTOF遗传性耳聋的基因治疗临床试验,这将是当前最有希望实现临床转化的耳聋基因治疗.OTOF基因突变所致耳聋的成功治疗将不仅为耳聋药物治疗开辟新方向,开启耳聋基因治疗新时代,也为其他耳聋患者的药物治疗带来希望.

Mutations in GJB2 (Gap junction protein beta 2) are the most common genetic cause of non-syndromic hereditary deafness in humans, especially the 35delG and 235delC mutations. Owing to the homozygous lethality of Gjb2 mutations in mice, there are currently no perfect mouse models carrying Gjb2 mutations derived from patients for mimicking human hereditary deafness and for unveiling the pathogenesis of the disease. Here, we successfully constructed heterozygous Gjb2+/35delG and Gjb2+/235delC mutant mice through advanced androgenic haploid embryonic stem cell (AG-haESC)-mediated semi-cloning technology, and these mice showed normal hearing at postnatal day (P) 28. A homozygous mutant mouse model, Gjb235delG/35delG, was then generated using enhanced tetraploid embryo complementation, demonstrating that GJB2 plays an indispensable role in mouse placenta development. These mice exhibited profound hearing loss similar to human patients at P14, i.e., soon after the onset of hearing. Mechanistic analyses showed that Gjb2 35delG disrupts the function and formation of intercellular gap junction channels of the cochlea rather than affecting the survival and function of hair cells. Collectively, our study provides ideal mouse models for understanding the pathogenic mechanism of DFNB1A-related hereditary deafness and opens up a new avenue for investigating the treatment of this disease.

Gene therapy would benefit from the effective editing of targeted cells with CRISPR-Cas9 tools. However, it is difficult to precisely assess the editing performance in vivo because the tissues contain many non-targeted cells, which is one of the major barriers to clinical translation. Here, in the Atoh1-GFP;Kcnq4(+/G229D) mice, recapitulating a novel mutation we identified in a hereditary hearing loss pedigree, the high -effi- ciency editing of CRISPR-Cas9 in hair cells (34.10% on average) was precisely detected by sorting out labeled cells compared with only 1.45% efficiency in the whole cochlear tissue. After injection of the developed AAV_SaCas9-KKH_sgRNA agents, the Kcnq4(+/G229D) mice showed signifi- cantly lower auditory brainstem response (ABR) and distor-tion product otoacoustic emission (DPOAE) thresholds, shorter ABR wave I latencies, higher ABR wave I amplitudes, increased number of surviving outer hair cells (OHCs), and more hyperpolarized resting membrane potentials of OHCs. These findings provide an innovative approach to accurately assess the underestimated editing efficiency of CRISPR-Cas9 in vivo and offer a promising strategy for the treatment of KCNQ4-related deafness.

The pathogenic variants in KCNQ4 cause DFNA2 nonsyndromic hearing loss. However, the understanding of genotype-phenotype correlations between KCNQ4 and hearing is limited. Here, we identified a novel KCNQ4 mutation p.G228D from a Chinese family, including heterozygotes characterized by high-frequency hearing loss that is progressive across all frequencies and homozygotes with more severe hearing loss. We constructed a novel murine model with humanized homologous Kcnq4 mutation. The heterozygotes had mid-frequency and high-frequency hearing loss at 4 weeks, and moved toward all frequencies hearing loss at 12 weeks, while the homozygotes had severe-to-profound hearing loss at 8 weeks. The degeneration of outer hair cells (OHCs) was observed from basal to apical turn of cochlea. The reduced K+ currents and depolarized resting potentials were revealed in OHCs. Remarkably, we observed the loss of inner hair cells (IHCs) in the region corresponding to the frequency above 32 kHz at 8-12 weeks. The results suggest the degeneration of OHCs and IHCs may contribute to high-frequency hearing loss in DFNA2 over time. Our findings broaden the variants of KCNQ4 and provide a novel mouse model of progressive hearing loss, which contributes to an understanding of pathogenic mechanism and eventually treatment of DFNA2 progressive hearing loss.

Endolymphatic potential (EP) is the main driving force behind the sensory transduction of hearing, and K+ is the main charge carrier. Kir5.1 is a K+ transporter that plays a significant role in maintaining EP homeostasis, but the expression pattern and role of Kir5.1 (which is encoded by the Kcnj16 gene) in the mouse auditory system has remained unclear. In this study, we found that Kir5.1 was expressed in the mouse cochlea. We checked the inner ear morphology and measured auditory function in Kcnj16–/– mice and found that loss of Kcnj16 did not appear to affect the development of hair cells. There was no significant difference in auditory function between Kcnj16–/– mice and wild-type littermates, although the expression of Kcnma1, Kcnq4, and Kcne1 were significantly decreased in the Kcnj16–/– mice. Additionally, no significant differences were found in the number or distribution of ribbon synapses between the Kcnj16–/– and wild-type mice. In summary, our results suggest that the Kcnj16 gene is not essential for auditory function in mice.