Gene Therapy for Hereditary Hearing Loss: Comprehensive Overview and Clinical Applications

Abstract

With advances in researching the mechanisms of hereditary hearing loss (HHL) and the development of genetic engineering technology, gene therapy demonstrates enormous potential treating HHL. Adeno-associated virus mediated gene therapy has restored hearing in multiple genetic mouse models of HHL. Notably, gene therapy targeting OTOF mutations has successfully advanced to clinical trials, improve the patient’s auditory and speech functions, marking a paradigm shift in the treatment of hearing loss. In this review, we introduce the typical classifications of HHL, the progress in gene delivery tools and major gene therapy strategies, and summarize the preclinical and clinical progress in different gene types.

Graphical abstract

1 Introduction

According to World Health Organization (WHO), over 700 million people are projected to suffer from disabling hearing loss by 2050^[1]. Congenital hearing loss impacts around 26 million people globally, with genetic factors responsible for as much as 60% of these cases. To date, more than 200 genes associated with hereditary hearing loss (HHL) have been identified, primarily expressed in cells and structures such as auditory hair cells (HCs), supporting cells (SCs), spiral ganglion neurons (SGNs) and the stria vascularis (SV)^[2]. While current interventions−most notably cochlear implantation−provide transformative functional hearing restoration that allows patients to lead largely unhindered lives, they function as neural prostheses that bypass the biological pathology rather than reversing the underlying cellular damage. Furthermore, the efficacy of emerging biological treatments, such as gene therapy, remains contingent upon the preservation of the inner ear’s structural integrity, as these methods cannot yet rescue lost hair cells or neurons in cases of advanced degeneration.

Gene therapy is a strategy that delivers exogenous genetic material into target tissues or cells to treat gene-related diseases. In light of continuous discoveries of deafness-related genes and innovations in genetic manipulation tools and gene delivery vectors, gene therapy for HHL has achieved notable progress. Clinical studies have shown that hearing can be restored in patients with hereditary autosomal recessive deafness 9 (DFNB9) caused by OTOF mutations^[3-^9]. This advancement not only provides an effective treatment option for DFNB9 patients but also lays the groundwork for gene therapy for other types of hereditary hearing loss, thus advancing the field of hearing loss treatment. This review introduces primary delivery tools and gene therapy strategies for HHL, highlights the advancements in the treatment of different HHL types and discusses future prospects for the development of gene therapy in both preclinical research and clinical translation.

2 Common Frameworks of HHL

HHL can be classified into non-syndromic hearing loss (NSHL) and syndromic hearing loss (SHL), depending on whether it is accompanied by systemic symptoms other than hearing impairment.

2.1 Non-syndromic hearing loss (NSHL)

NSHL accounts for about 70% of HHL. According to the inheritance pattern, NSHL can be divided into autosomal dominant deafness (DFNA), autosomal recessive deafness (DFNB), X-linked deafness (DFNX), Y-linked deafness (DFNY) and mitochondrial deafness. When classifying and naming HHL with different gene mutation, sorting is conducted in accordance with the chronological order of locus mapping. Pathogenic mutation in 156 genes have been found to be associated with NSHL^[10].

DFNB is the most common type of hereditary deafness. So far, 88 deafness genes have been found in DFNB^[11], while 64 deafness genes have been reported in DFNA. The genetic variants of DFNX include 6 genes (PRPS1, POU3F4, SMPX, AIFM1, COL4A6, GPRASP2), while DFNY include 1 gene (TBL1Y). There are 9 genes found in mitochondrial hearing loss, in which MT-RNR1, MT-CO1, and MT-TS1 variants are associated with susceptibility to aminoglycoside otoxicity that causes permanent hearing loss.

Primarily caused by single-gene mutations, NSHL has well-defined genetic targets and is more suitable for intervention with gene therapy strategies. Therefore, most research efforts focus on this type of hearing loss, encompassing a range of studies that include basic research to understand the underlying mechanisms, preclinical studies to evaluate the safety and efficacy of potential gene therapies in animal models^[12]. Among these, deafness caused by OTOF mutantions has successfully undergone clinically translation, providing a transformation paradigm for other forms of genetic deafness^[3-⁶,¹³,^14].

2.2 Syndromic hearing loss (SHL)

SHL accounts for about 30% of HHL. In addition to hearing loss, SHL is usually combined with symptoms of other systems such as eyes, heart, kidney, nervous system, skin and bone^[2]. The prevalent symptoms and genetic variants of these diseases are shown in Table 1.

SHL often involves mutations in genes with broad tissue expression and critical physiological roles, leading to multisystem involvement and making gene therapy approaches more challenging. At present, research on gene therapy for SHL primarily focus on single gene mutations, such as Usher syndrome (USH), Jervell and Lange-Nielsen syndrome, and Norrie disease.

3 Design of Gene Therapy Platforms

3.1 Delivery system for inner ear

In the context of gene therapy for HHL, the precise delivery of therapeutic genes is crucial for ensuring both effictive and safe treatment. Therefore, the selection of delivery systems, including vectors and promoters, are critical elements that significantly impact treatment outcomes. With the advancement of genetic engineering, a variety of delivery vectors for efficient inner ear transduction have been developed. Additionally, the integration of cell-specific promoters and other regutary elements enhanced the precision and safety of gene therapy for HHL. Table 2 presents Adeno-associated virus (AAV) vectors and promoters used by the research institutions in the field of ear gene therapy over the past five years.

3.1.1 Vectors for gene delivery

The selection of vectors for HHL gene therapy has undergone extensive experimentation and exploration. In earlier studies, Adenovirus was used for the intracochlear delivery of reporter genes, which demonstrated that Adenovirus could transduce inner hair cells (IHCs), outer hair cells (OHCs), and SCs^[15]. However, Adenovirus exhibited severe ototoxicity, and the first-generation Adenovirus showed an inhibitory effect on mechanical electrical conduction^[16]. Lentivirus was also investigated as a potential gene delivery vector, which could effectively transduce in hair cells while the risk of random insertion may cause safety issues^[17]. AAV is proved to be the most efficient biological delivery tool for gene therapy in the inner ear, due to its high transduction efficiency, low immunogenicity, ability to permanently transduce both mitotic and quiescent cells, and the modifiability of viral capsid^[18].

AAVs are non-pathogenic parvovirus characterized by a single-stranded DNA genome of 4.7 kb, which is enclosed within a nonenveloped icosahedral capsid. For neonatal mice, commonly used AAV capsids such as AAV1, AAV2, AAV5, AAV8 and AAV9 demonstrated efficiently trandution in IHCs, while they showed lower transduction efficiency in OHCs and SCs, especially in adult inner ear^[19]. To obtain more efficient AAV vectors, various strategies have been employed, including random mutagenesis, rational design, directed evolution, and in silicon design^[20]. Multiple new AAV variants have been developed, demonstrating broader targeting, higher transduction efficiency or delivery. For instance, AAV-S, a rational designed variant of AAV9, can simultaneously transduce multiple cell types in the inner ear of mice and primates (IHCs, OHCs, SCs and fibrous cells)^[21]. Another AAV9 variant, AAV-HGHC, exhibits nearly 100% transduction efficiency in IHCs^[22]. Anc80L65, an in silicon designed capsid, exhibit extensive inner and outer hair cells transduction ability in murine cochleas. AAV-ie, a variant engineered by inserting peptide “DGTLAVPFK” into AAV-DJ capsid, showed significant robust transduction efficiency in neonatal cochlear.^[23] The AAV-sia6e vector developed by Ukaji et al.^[24] shows a tropism in Deiters cells, Hensen cells, inner sulcus cells, outer sulcus cells, and fibrous cells. It can effectively deliver adenine base editors and has shown gene repair function in both neonatal and adult GJB2-defective mouse models.

For genes espressed in multiple cell types, a single AAV capsid may not efficient for delivery, to address the limitations, strategy of utilizes multiple AAV capsids was employed. Isgrig et al.^[25] used AAV2.7m8 to target cochlear HCs and SCs, alongside AAV8BP2 aimed at lateral wall cells, to deliver the ILDR1 gene to the inner ear, resulting in significantly improvement in auditory function in mice. Similary, Sun et al.^[26] employed AAV-ie to deliver the GJB2 gene to SCs, in combination with AAV1 targeting lateral wall cells, demonstrating pronounced therapeutic effects. Iwasa et al.^[27] combined AAV2 and AAV9 which target IHCs and OHCs respectively, carrying miRNAs to inhibit the expression of endogenous mutant TMC1 genes.

For limitation of packaging capacity of traditional AAV vectors, dual- or multiple-AAV approaches were used to overcome this issue. For example, dual vectors are used to separately deliver SpCas9 and sgRNA in the CRISPR/Cas9 system^[22]. For gene replacement strategies, dual-AAV systems have also been employed to deliver large genes such as STRC and OTOF, utilizing recombination strategies at either the nucleic acid or protein level in inner ear gene therapy^[20,^28]. Additionally, some researchers tried shortening gene sequences or using overloaded AAVs. Ivanchenko et al.^[29] designed mini-PCDH15 with a 25%−40% reduction in length by deleting non-critical EC repeat sequences based on the structural analysis of PCDH15, enabling it to fit into AAV vectors. Rankovic et al.^[30] demonstrated the viability of using a single overloaded AAV vector for gene replacement therapy in OTOF mutations using six AAV capsids .

In recent years, non-viral vectors have also been invested in the research of gene therapy. These innovative delivery tools offer new possibilities for gene therapy targeting hereditary hearing loss, particularly in the context of gene editing systems. Le et al.^[31] combined RH-PAMAM G2 dendritic polymers with hexanol chitosan thermogel to enhance gene delivery to the inner ear. Tao et al.^[32] used a liposome-mediated CRISPR-Cas9 ribonucleoprotein (RNP) complex delivery system for dual gene editing of ATP2B2 and TMC1, achieving significant therapeutic effects. Pan et al.^[33] developed a high-throughput microfluidic droplet-based electroporation system (µDES)-based extracellular vesicle (EVs) delivery technology, loading Cas9-sgRNA RNP complexes into EVs to achieve MYO7A gene editing in hair cells.

3.1.2 Promoters and other regulatory elements for precise gene delivery

Although engineered AAV capsid variants have demonstrated broader transduction ranges and improved expression, no vectors have yet been developed that can achieve cell-specific expression in the inner ear. Some deafness genes, such as GJB2 and MPZL2, exhibit severe toxicity when expressed ectopically^[34,³⁵,^36]. Therefore, achieving precise delivery of genes is an important objective in HHL gene therapy.

To achieve hair cell-specific gene expression, Myo15 promoter or minimized Myo15 promoters were used to specifically deliver the OTOF gene to IHCs in the DFNB9 mouse model, aiming to rescue hearing function^[37-^39]. GFAP promoter or engineered GJB2 specific promoters were also administrated in GJB2 overexpression gene therapy to avoid ectopic expression toxicity in recent preclinical researches^[26,³⁴,⁴⁰,^41]. In addition, optimization of expression efficiency and strength, enabled by intronic module screening and enhancer engineering. Zhao et al.^[42] introduced an AAV-reporter-based in vivo transcriptional enhancer reconstruction (ARBITER) workflow, screening conserved noncoding elements E1 and E2 in the intron of the SLC26A5 gene as enhancer sequences. Through recombinant module design, the synthetic enhancer B8 was created, enhancing the specificity and expression intensity of SLC26A5 in OHCs.

3.2 Common gene therapy strategies

In the preclinical research and clinical translation of gene therapy for hearing loss, three core strategies have emerged: gene replacement, gene suppression, and gene editing. These approaches aim to reverse the auditory impairment caused by genetic mutations based on different gene regulation methods (Figure 1).

3.2.1 Gene replacement: loss-of-function

Gene replacement therapy is a treatment method that identifies faulty genes in autosomal recessive or haploinsufficient dominant disorders (loss-of-function) and overlays their expression with exogenous transgenes. Such mutations can cause genes to fail to encode normally functional proteins or result in abnormally structured proteins. In 2012, Akil et al.^[43] delivered AAV1-Vglut3 to the inner ear of Vglut3 knockout mice, restoring hearing function for at least 7 weeks in p10−12 mice, and partially reversed the morphologic change of afferent IHC ribbon synapse. This represents the first study utilizing gene replacement therapy for the treatment of HHL. Currently, gene replacement therapy has been proven effective in various DFNB cases. Among them, the therapy for OTOF has achieved progress in clinical research, both in child and adult^[3-^6].

3.2.2 Gene suppression: gain-of-function/dominant-negative variants

Gene suppression involves two distinct strategies: antisense oligonucleotides (ASOs) and RNA interference (RNAi).

ASOs are single-stranded oligonucleotide molecules typically comprising 15−25 nucleotide residues^[2]. After entering a cell, ASO regulate gene expression in two ways: one is to bind to their complementary target messenger RNA (mRNA) through base pairing under the action of RNase H1, inhibiting the expression of the target gene; the other is to target splice sites, exons, or introns for selective splicing in order to exclude or include target exons through splice site switching. ASOs have been reported as a therapeutic option for USH1C^[44-^48] and has the therapeutic potential in SLC26A4 mutation^[49]. RNAi is a process by which the expression of target genes is knocked down by suppressing or degrading the translation of endogenous mRNA using exogenous small interfering RNAs (siRNAs) and microRNAs (miRNAs). Traditional siRNAs and miRNAs have been proven effective in treating hereditary deafness associated with the GJB2 p.R75W mutation in animal models^[50]. Iwasa et al.^[27] used AAV to deliver artificial miRNA-miTmc1 into the inner ears of neonatal Bth mouse models, selectively suppressing the mutant TMC1 allele and enhancing the survival time of cochlear IHCs.

3.2.3 Precision gene editing

Gene editing involves precise modification of DNA or RNA sequences through the addition, deletion, or substitution of bases at specific locations. CRISPR/Cas system is particularly notable for its capability of precise editing and its wide applicability, and has been used in diseases such as NSHL and USH, proving its therapeutic potential for HHL. This delivery method has been applied in various NSHL studies, including OTOF^[51], KCNQ4^[52], PCDH15^[53], Klhl18^[54], MIR96^[55], and TMC1^[22]. Ukaji et al.^[24] developed SaABE_V106W#1 and sgRNA, which can precisely repaired the dominant negative mutation of GJB2 R75W through base editing. Tao et al.^[32] injected Cas9:Atp2b2-mut1 complexes into Atp2b2^Obl/+ mice, which restored OHCs function and recovered hearing.

Recent studies have demonstrated that the RNA base editor offers higher specificity for treating hereditary deafness. Mini dCas13X.1-based adenosine base editor (mxABE) has been proven to have certain therapeutic effects in mutations of MYO6^[56]. It can directly correct the base pair and only functions at the RNA level, avoiding the risk of permanent DNA alteration.

4 Preclinical Progress in HHL Therapy

HHL displays genetic heterogeneity, with more than 200 genes associated with its development. A number of these genes have been identified as promising candidates for gene replacement therapy due to their clearly defined molecular roles, the absence of structural changes in Corti’s organ due to mutations, or associated with progressive forms of deafness that have a critical therapeutic window, as well as high-prevalence pathogenic genes in hereditary hearing loss. Successful preclinical studies focusing on these genes have established a strong basis for translational approaches. In this review, we will provide a detailed overview of the preclinical progress in gene therapy for OTOF, GJB2, STRC, SLC26A4 and USH1C.

4.1 DFNB9 (OTOF)

The mutation of the OTOF gene is the main cause of DFNB9, the proportion in the hearing loss population is approximately 2%−8%^[57]. This mutation leads to the absence of otoferlin, disrupting the process of synaptic vesicle release from IHCs to SGNs in the cochlea, causing severe congenital hearing loss. Deafness caused by OTOF mutations is currently the only form of congenital hearing loss for which gene therapy has advanced to the clinical trial phase.

The size of the OTOF gene (about 6 kb) exceeds the length that a single AAV vector can accommodate. Previously, researchers split the cDNA coding sequence of the OTOF gene, loaded the fragments into two separate AAV vectors respectively, and successfully achieved the complete expression of the OTOF gene cDNA sequence. Al-Moyed et al.^[58] developed two strategies: a trans-splicing AAV system constructed with AAV2/6 and the β-actin promoter/CMV promoter, and a hybrid AAV system capable of fully expressing the full-length expression cassette. Murine OTOF was delivered via these two strategies into the cochleae of postnatal day 6−7 (p6−7) mice. This approach completely restored IHCs function and partially improved the mice’s hearing; however, it failed to rescue the loss of 40% of synaptic ribbons in IHCs, a phenomenon that may be associated with the timing of administration. In 2019, Akil et al.^[28] divided the cDNA sequence of murine otoferlin into 5' fragments (nucleotides 1−2448 bp) and 3' fragments (nucleotides 2449−5979 bp) using the DFNB9 mouse model. The 5' fragment was fused with a splicing donor (SD) plus a dual AAV hybrid sequence (AP/AK), and the 3' fragment with the same hybrid sequence plus a splice acceptor (SA). The two constructs were separately packaged into AAVs, delivered to mouse cochleae via microinjection, and their therapeutic efficacy was evaluated in otoferlin-deficient deaf mice. Results showed that treated neonatal mice exhibited reduced the Auditory brainstem response (ABR) thresholds and significant hearing recovery at 4 weeks post-injection; adult mice achieved similar restoration at 3 weeks post-injection, with stable thresholds over an extended period. Another strategy to address the challenge of large gene size is to use overloaded AAV for delivering the complete gene sequence. Rankovic et al.^[30] demonstrated the viability of using a single overloaded AAV vector for gene replacement therapy in OTOF mutations using six AAV capsids. However, it lags behind on conductive efficiency. In humanized mouse models, the therapeutic approach using dual AAV vectors is equally effective. Tang et al.^[59] constructed the Rma-hOTOF dual AAV system based on the principles of protein trans-splicing, restored the hearing of OTOF^-/- mice to near wild-type levels. To strengthen the specificity of the transgene expression, Myo15 promoter (about 1611 bp) was used in OTOF delivery system, which is an HC-specific promotor that can reduce the expression of the exogenous gene in the central nervous system and other types of cochlear cells apart from HCs. Research have shown that the Myo15-mediated delivery of AAV. PHPeB-OTOF results in a higher otoferlin expression level in IHCs than that mediated by the CAG promoter^[37]. The length of Myo15 was further shortened into mini-Myo15 (about 956 bp), which does not affect its specificity and expression intensity^[38]. The OTOF therapeutic system mediated by the mMyo15 promoter has also been validated for safety in multiple animal models, laying a foundation for subsequent clinical trials^[38]. Myo15 promoter-driven AAV1-hOTOF was also proved to be safe in mouse and Macaca fascicularis^[12]. In non-human primate experiments, the safety of the Anc80L65−mMyo15 system in treating OTOF was verified using the cynomolgus monkey model^[57].

Furthermore, DNA and RNA base editing therapy also progress in DFNB9 treatment. Xue et al.^[60] developed an enhanced mini dCas13X RNA base editor (emxABE), using AAV-HGHC, which specifically targets IHCs, as the vector. It can efficiently and specifically correct the OTOF c.2485C > T (p.Q829X) nonsense mutation, achieving an approximately 80% A-to-I (TAG > TGG) conversion efficiency, restoring the expression of Otoferlin in IHCs and auditory function, and maintaining long-term effectiveness. Cui et al.^[51] developed an efficient and low off-target editing NG-ABE7.10max, which was delivered by dual-AAV system. The research results demonstrated that this ABE system could restore hearing to the wild-type level in both OTOF^Q828X/Q828X mice and humanized mouse model. The recovery of auditory function in OTOF^Q828X/Q828X mice lasted for up to 1.5 years. Moreover, it was proved to be safe, providing potential for clinical translation for gene editing therapy for DFNB9 and other recessive hereditary deafness.

4.2 DFNB1A (GJB2)

The GJB2 mutation leads to DFNB1A, which is the most common causative gene for non-syndromic recessive hereditary deafness, account for 50% of all prelingual hearing loss cases^[61]. GJB2 encodes Connexin 26 (Cx26), a gap junction protein expressed in various cochlear cells and tissues, responsible for forming channels between cells. It plays a crucial role in maintaining potassium ion homeostasis and facilitating intercellular signal transmission within the organ of Corti^[62]. Over 467 different variant sites have been identified, including missense mutations, nonsense mutations, frameshift mutations, insertions, and deletions^[61]. Some drugs, such as Narciclasine and Triiodothyronine, have a certain degree of salvage effect on hair cell degeneration and tunnel of Corti deformity caused by Cx26 defect, but the therapeutic effect is limited^[63,^64].

A number of preclinical studies have shown that by delivering overexpressed GJB2 gene through AAV vectors is possible to restore the protein expression of Cx26 and the gap junction function in the cochlea of GJB2 knockout mice, while the auditory restoration is limited. In 2015, Takashi et al.^[65] utilized an AAV5 vector driven by the CMV promoter to mediate GJB2 gene expression. They administered the vector to p0 neonates and p42 adult mice, respectively. Restoration of cochlear cellular structure and hearing function was only observed in the neonates. In 2021, Guo et al.^[36] performed inner ear injections in Sox10iCre^ERT2-mediated GJB2-inducible knockout mice using Anc80L65 and CAG promotor carrying the human GJB2 gene (hGJB2). They found that this system could express connexin 26 in both SCs and HCs. However, IHCs degenerated and the hearing threshold was not improved.

Due to species differences between humans and mice, homozygous GJB2 mutations are lethal in mice, preventing researchers from verifying the efficacy of therapeutic drugs in GJB2-mutant mouse models and limiting the further development of GJB2 gene therapy drugs. In 2023, Li et al.^[66] successfully constructed heterozygous mutant mice (Gjb2^+/35delG and Gjb2^+/235delC) using androgenetic haploid embryonic stem cell-mediated semicloning technology combined with the CRISPR−Cas9 system. Moreover, by employing enhanced tetraploid embryo complementation, they overcame the placental developmental defects and embryonic lethality caused by homozygous Gjb2 mutations, thereby successfully obtaining homozygous mutant mice (Gjb2^{35delG/35delG}). This study is the first to successfully establish heterozygous and homozygous mouse models carrying high-frequency pathogenic mutations in humans, which perfectly mimic the phenotypes of human DFNB1A-related hereditary deafness and provide an ideal tool for investigating pathogenic mechanisms and testing therapeutic regimens.

The ectopic GJB2 expression in hair cells could cause ototoxicity. Xu et al.^[67] found that dexamethasone (DEX) has a protective effect against HCs damage and hearing loss caused by Cx26 deficiency, which may be related to the regulation of immune responses in the inner ear. The subsequent research demonstrated that the combined use of gene therapy and DEX can suppress the inflammatory response caused by Cx26 produced by exogenous GJB2 expression^[68]. It provides a new solution against the IHC injury caused by gene replacement therapy. Use SC-specific promoter to reduce the ectopic expression and enhance therapeutic safety was another strategy. Sun et al.^[26] developed a combined AAV strategy with SCpro, which specifically transduce SCs, restored the expression of Cx26, recovered the hearing in mice model and conducted the transduction efficiency and safety assessments in large animals (bama miniature pigs and cynomolgus monkeys).

Gene editing could also be one of the treatment strategy for GJB2. Ukaji et al.^[24] developed an all-in-one AAV vector, which encoded all the component of base editing (SaABE_V106W#1). This editor can mediate an A-to-G base conversion at the GJB2 R75W mutation. The result shows that the system can repair abnormal gap junction plaques caused by mutations and restore their physiological function. The improvement of hearing has not been provided.

4.3 DFNB16 (STRC)

The STRC gene mutation (DFNB16) is the second most common cause of ARNSHL, the proportion in the population is approximately 16%^[69]. STRC mutations in humans are usually copy number variations (CNVs), and manifest as moderate progressive hearing loss. STRC encodes the protein stereocilin, and the absence of stereocillin induces the loss of the cross connection between OHCs^[70].

Shubina-Okeinik et al.^[69] delivered STRC to the inner ear of newborn STRC^Δ/Δ mice using a dual-vector system. The results showed that 59% of OHC bundles re-expressed STRC protein, and 61%−64% of HC bundles developed apical connectors. Meanwhile, distortion product otoacoustic emission (DPOAE) was restored in 50% of the mice, and the ABR thresholds of the mice with restored DPOAEs improved by 50−60 dB, with the effect lasting up to 12 weeks. This study verify the long-term effectiveness of gene therapy for DFNB16, and it was the first time that the intein-mediated protein recombination technology was used in hearing loss.

However, pathogenic STRC mutations in mice result in profound deafness immediately after birth, which is inconsistent with the slowly progressive mild-to-moderate hearing loss phenotype observed in humans. Therefore, in the research and development of STRC gene therapy, it is necessary to take into account the genotypic characteristics of human patients and avoid directly extrapolating the results of mouse experiments to humans.

4.4 DFNB4 (SLC26A4)

SLC26A4 gene mutation can lead to DFNB4. The SLC26 gene family encodes transmembrane anion exchangers and anion channels. SLC26A4 is a common pathogenic gene associated with enlarged vestibular aqueduct-related deafness, and the encoded protein Pendrin is necessary for normal reabsorption of endolymph^[71].

Among all the SLC26A4 mutations, the splice site mutation c.919-2A > G (A−2G) is the most common in Asian populations. It impairs the 3' splice site of intron7, leading to the skipping of exon8 during pre-mRNA splicing, which in turn causes a frameshift and the generation of a premature termination codon in subsequent exons^[49]. Feng et al.^[49] used ASOs to correct the erroneous splicing caused by the SLC26A4 A−2G mutation. The researchers designed ASO1029, whose mechanism involves targeting the heterogeneous nuclear ribonucleoprotein (hnRNP) A1/A2 intronic splicing silencer in intron8, promoting the inclusion of exon8. ASO1029 significantly increased the full-length transcription in peripheral blood mononuclear cells cultured from homozygous patients, and the same effect was observed in tissues of treated humanized SLC26A4 A−2G mice. ASO1029 holds potential for treating hereditary hearing loss caused by the A−2G mutation. However, there is currently a lack of verification regarding its therapeutic effect on hearing loss.

4.5 Usher syndrome type 1

USH is an autosomal recessive syndromic ciliopathy characterized by sensorineural hearing loss, retinitis pigmentosa, and sometimes vestibular dysfunction. USH is classified under three distinct subtypes based on the onset age, the severity of symptoms, and the presence of vestibular symptoms: type I (USH1), type II (USH2), type III (USH3)^[72]. USH1 is the most severe subtype and is characterized by a severe to profound prelingual SNHL, early RP onset and vestibular alterations. Six genes have been identified to be associated with USH1 (MYO7A, USH1C, CDH23, PCDH15, USH1G and CIB2), and several studies have achieved preliminary results targeting different mutations.

Ivanchenko et al. constructed a mini-PCDH15 with a shorter coding sequence by deleting 3−5 non-essential extracellular cadherin (EC) repeats based on the PCDH15 gene (about 5.3 kb) that causes USH1F mutations. This mini-PCDH15, along with the CMV promoter and BGH poly(A) without the WPRE sequence, was packaged into AAV9-PHP.B. It was injected into newborn USH1F mice via the round window membrane (RWM), which partially restored the morphology of hair cell bundles in the inner ear of the mice, recovered the stereocilia tip links and mechanotransduction in PCDH15^R245X/R245X mice, and improved ABR and DPOAE^[29]. Peters et al.^[73] constructed a dual-AAV8(Y733F)−MYO7A system to deliver the gene into USH1B model mice (shaker-1 mice, MYO7A^{4626SB/4626SB}). This system restored the expression of MYO7A in IHCs and OHCs and improved vestibular function. However, the stereocilia structure and hearing were not improved, and ototoxicity was observed in wild-type mice. Schott et al.^[74] delivered the full-length MYO7A cDNA by third-generation lentivirus, driven by the SFFV promoter, via injection into the posterior semicircular canal (PSC) of Shaker-1 mice. LV-MYO7A efficiently transduced the cochlea and vestibular organs, with a transduction efficiency of 89%−95%. In heterozygous mice, ABR was fully restored, and late-onset hearing loss was successfully prevented. In homozygous mice, the ABR showed an average improvement of 25 dB at 8 kHz, which did not reach the level of wild-type mice. HC degeneration was delayed but gradually decreased at 6 months of age. Du et al.^[75] Injected Anc80L65 carried harmonin-b1 cDNA into the RWM/PSCC of p2 and p4 USH1C knockout newborn mice. They found that one month after treatment, both the ABR and vestibular function of the mice were improved. The hearing recovery in the round window membrane injection group was more long-lasting, and the recovery effect in the p2 group was better than that in the p4 group.

Researchers have also attempted to treat USH1F through gene editing approaches. Peters et al.^[73] injected dual AAV9-PHP.B encapsulated split-intein ABE8e and gRNA1 into newborn PCDH15^R245X/R245X mice to repair the C-to-T mutation. In the conditional knockout mouse model with well-preserved HC structure, the mechanotransduction function of hair cells was partially restored after administration, and the ABR threshold was improved by an average of 20 dB, lasting for more than two months. However, the mRNA editing efficiency of ABE8e in HCs was only 3%, and it failed to restore the hair cell bundle morphology and hearing in constitutive PCDH15^R245X/R245X mice.

5 Clinical Progress

In recent years, gene therapy on HHL has been gradually put into clinical research. Since 2022, 7 clinical trials have been registered successively, all targeting the OTOF gene for treatment. They are respectively conducted by Eye & ENT Hospital of Fudan University, Eli Lilly, Regeneron Pharmaceuticals, Otovia Therapeutics, Sensorion, Shanghai Ninth People’s Hospital (Table 3).

In December 2022, the world’s first patient with congenital deafness caused by OTOF gene mutations received treatment with RRG-003, a drug developed by Professor Yilai Shu, at Eye & ENT Hospital of Fudan University. This represented the first gene therapy administered for congenital deafness, as well as the first dual-AAV gene replacement therapy applied in humans, with the relevant research findings being reported for the first time^[8]. Lv et al.^[3] from Shu’s team conducted a single-center, open-label, individual patient, non-randomized controlled interventional clinical study on 6 children with severe deafness caused by OTOF mutations who received AAV1-hOTOF (RRG-003) unilateral injection, partially restored the hearing and enabled language recognition. This was the first gene therapy used for congenital deafness, and the first dual-AAV gene replacement therapy applied in human, which was published on Lancet. Furthermore, the team demonstrated the efficacy and safety of AAV1-hOTOF bilateral injection for DFNB9 deafness on four children and one adolescent, provided important data for gene therapy for other genetic hearing losses, expanded the treatment window to include adolescents^[4]. Valid conclusions have also been drawn from the assessments of auditory cortex development, speech perception and music perception in patients undergoing AAV1-hOTOF therapy^[13,¹⁴,^76]. In the subsequent multicenter trial, Qi et al.^[5,^6] reported the theraputic effect in 5-year-old and 8-year-old child patient, and also reported that a 23-year-old adolescent exhibited a significant decrease in hearing threshold 9 months after Anc80L65-OTOF therapy, demonstrating that adult patients can also benefit from gene therapy. In the DB-OTO study, a total of 12 patients (aged 10 months to 16 years) were included. Among them, 9 patients had reached the primary endpoint (average threshold of behavioral pure tone audiometry (PTA) ≤ 70 dB HL at 24 weeks of treatment) and the secondary endpoint (ABR threshold ≤ 90 dB nHL at 24 weeks). Both of the two 16-year-old patients reached the primary endpoint and no special adverse events occurred^[7]. In the AK-OTOF study, improvements in hearing were observed in two children, confirmed through behavioral audiometry^[77-^79]. Meanwhile, in the SENS-501 trial, three children received treatment, with one showing a response to sound within a month^[80,^81].

Except for OTOF, no other genotypes of deafness have been successfully invested in clinical trials so far. The clinical translation of gene therapies for different HHL genes is associated with multiple factors, including the mutation characteristics and clarity of pathogenic mechanisms of the target genes, as well as the therapeutic time window. For instance, genes such as OTOF and GJB2—featuring well-defined pathogenic mechanisms and explicit cellular targets—exhibit greater potential for gene therapy development. In contrast, clinical translation proves more challenging for complex cases characterized by ambiguous pathogenic mechanisms, numerous interfering factors or resultant structural alterations. As for the control of therapeutic time window, gene therapy can effectively reconstruct auditory signal pathways only when the target cells have not undergone irreversible degeneration and the central auditory system retains strong plasticity.

Current animal studies have proved the possibility of gene replacement therapy for GJB2-related deafness, and some studies have shown that the combined use of gene therapy and dexamethasone can suppress the inflammatory response caused by Cx26 produced by exogenous GJB2 expression^[34]. Specific promotor has also been developed to limit transduction in SCs. In the future, it is necessary to establish a long-term safety evaluation system for large animal models, verify the transduction efficiency and immune response of vectors in adult individuals, so as to promote the clinical transformation of GJB2.

6 Conclusion

Gene therapy for deafness offers novel therapeutic options beyond traditional cochlear implant surgeries, and holds the potential for curing HHL. Recent years, gene therapy for HHL has seen various enhancements and progress in delivery system, treatment methods and therapeutic effects.

There are several limitations to the use of AAV for HHL. For instance, the size of many known deafness genes and gene editing tools exceeds the carrying capacity of AAV, which significantly restricts its application in gene therapy for HHL. However, dual-vector systems that employ nucleic acid or protein recombination technologies,along with minigene construction, provide viable solutions for delivering these larger genes.

Another issue is the current lack of AAV capsid types that can accurately target specific types of cochlear cells. The strategy of combining efficient AAV capsids with specific promoters offers a novel approach for precise delivery. This strategy has already been successfully applied in gene therapy for OTOF and GJB2.

The inherent safety is a significant issue in AAV-mediated gene replacement therapy for hereditary deafness. Current clinical trials of OTOF gene therapy have demonstrated a favorable safety profile. To date, only two severe adverse events (SAEs) have been reported in the DB‑OTO clinical trial, neither of which was directly attributable to the gene therapy intervention, and both resolved without sequelae^[7]. Regarding AAV capsid selection, AAV1, Anc80L65, and AAV8 have been employed in these trials. Although these vectors may mediate off-target transgene expression in OHCs, SCs, and other cell types, safety assessments indicate that off-target expression of OTOF rarely leads to toxic phenotypes. However, for genes associated with epi-toxicity, such as GJB2 and MPZL2, the selection of AAV capsids and promoters is critically important^[82]. For GJB2 gene replacement therapy, a commonly adopted strategy involves the use of an AAV capsid with broad inner ear tropism combined with a cell‑specific promoter .

Neutralizing antibodies (NAbs) represent a critical factor influencing the efficacy of gene therapy. In studies involving systemic administration of the AAV8-hFIX vector in non-human primates, elevated NAb titers were shown to significantly impair transduction efficiency during subsequent AAV administrations^[83]. Therefore, in future clinical investigations of gene therapy, it is essential to monitor the dynamics of NAb titers following vector administration and evaluate their potential impact on therapeutic efficacy. Additionally, attention should be paid to the distribution of NAbs both locally within the inner ear and systemically after unilateral treatment, to assess the feasibility of repeated dosing in either the ipsilateral or contralateral ear. In the ongoing development of gene therapy products, efforts should also be directed toward minimizing the potential of AAV vectors to induce neutralizing antibody responses.

For gene therapy of HHL, different strategies have their own specific applicable scenarios. For loss-of-function genetic hearing loss, gene replacement therapy is the most suitable approach, especially in cases where the underlying mechanism is well understood, and the loss of gene function does not affect the development of inner ear structures or lead to hair cell death. OTOF serves as an excellent example, as gene replacement therapy for OTOF mutations has successfully reached the clinical stage. Other genes, such as GJB2 and STRC, have also achieved preclinical success through gene replacement therapy. For gain-of-function/dominant-negative variants of HLL, novel gene suppression stratagies using ASO, siRNA/shRNA or miRNA was performed to inhibit pathogenic gene expression, and epigenic tools were used to inhibit or activate therupetic genes. In addition to gene replacement and gene suppression, precise gene editing is also an effective strategy for the treatment of genetic hearing loss. Among them, DNA single-base editing and RNA single-base editing are commonly used tools that achieve more precise site-specific therapy based on conventional gene editing tools. This greatly enhances safety and addresses types of hearing loss that gene replacement or gene suppression cannot manage.

Currently, multiple clinical trials targeting mutations in the OTOF gene are underway^[2]. Studies has shown that gene replacement therapy is effective in children’s auditory thresholds and speech perception, and no dose-limiting toxicity was found. Further, one adult patient in the study demonstrated significant therapeutic efficacy. Recently, the first international expert consensus on gene therapy for HHL has been published^[76]. It covers multiple aspects including ethical review, patient selection criteria, diagnosis and preoperative evaluation, drug delivery methods, follow-up, and post-treatment auditory-verbal rehabilitation, establishing for the first time a standardized framework for gene therapy in hereditary hearing loss. The clinical treatment paradigm of the OTOF deafness gene provides an important reference template for other genes, such as GJB2, which have achieved preliminary therapeutic effects in gene therapy for animals.

There are still challenges in the development of HHL gene therapy. Firstly, the delivery efficiency and precision of the vector in adult mouse inner ear cells still need to be further improved. It is also necessary to take into account the different effects of gene delivery in different species. The efficiency and safety of gene delivery may vary among mice, primates and human being. The possible immune response induced by vectors and genetic tools suggests that further clinical trials with larger sample sizes and longer follow-ups are needed. Secondly, the phenotypes of gene mutations in some hereditary deafness are inconsistent between mice and humans. For example, STRC mutations lead to total deafness in mice, while in humans, they only result in progressive moderate deafness. Therefore, the research results of gene therapy in mouse models should not be directly extrapolated to humans, and there is an urgent need for animal models that are more similar to human phenotypes to conduct more convincing research. Third, it should be noted that the core advantage of OTOF treatment lies in the fact that this mutation has relatively clear mechanism and does not accompany structural damage to the inner ear. HHL genes such as the SLC26A4 gene, which causes enlarged vestibular aqueduct-related deafness, still lack effective treatment methods that can reverse the structural changes at present. However, the clinical genotypes and phenotypes of pathogenic genes associated with hereditary hearing loss are highly complex. For the GJB2 gene, mutations at different sites alter distinct protein domains, thereby affecting specific cell types and resulting in varying degrees and progression rates of hearing loss. The phenotypic heterogeneity associated with different GJB2 mutation sites represents a significant challenge in the development of gene therapy drugs^[84]. Addressing this requires the development of more precise gene delivery systems tailored to specific cell types, as well as the careful selection of appropriate patient populations for treatment. Moreover, the efficacy of gene therapy largely depends on the functional integrity of target cells, such as HCs and SGNs. Given that different genetic mutations affect distinct cell populations, the therapeutic time window should be tailored to specific genotypes individually. Advanced molecular imaging probes capable of quantifying the survival of specific cell types offer a promising strategy for precisely defining these critical treatment windows^[85]. Due to the relatively short lifespan of mice, cellular degeneration proceeds more rapidly in mouse models of genetic hearing loss compared to humans. Therefore, it is essential to align the temporal progression of hearing loss between murine models and human patients to better define the therapeutic window for gene therapy interventions. As research progresses, gene therapy is expected to provide safer, more effective, and more durable treatment options for patients with HHL.

References

[1] Chadha S , Kamenov K , Cieza A . The world report on hearing, 2021. Bull World Health Organ, 2021, 99(4): 242–242A

[2] Jiang L , Wang D , He Y , Shu Y . Advances in gene therapy hold promise for treating hereditary hearing loss. Mol Ther, 2023, 31(4): 934–950

[3] Lv J , Wang H , Cheng X . et al. AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial. Lancet, 2024, 403(10441): 2317–2325

[4] Wang H , Chen Y , Lv J . et al. Bilateral gene therapy in children with autosomal recessive deafness 9: single-arm trial results. Nat Med, 2024, 30(7): 1898–1904

[5] Qi J , Tan F , Zhang L . et al. AAV-Mediated Gene Therapy Restores Hearing in Patients with DFNB9 Deafness. Adv Sci (Weinh), 2024, 11(11): e2306788

[6] Qi J , Zhang L , Lu L . et al. AAV gene therapy for autosomal recessive deafness 9: a single-arm trial. Nat Med, 2025, 31(9): 2917–2926

[7] Valayannopoulos VBance MCarvalho DS, et al. DB-OTO Gene Therapy for Inherited Deafness. N Engl J Med. 2025; Epub ahead of print.

[8] Lustig L . Cochlear gene therapy for otoferlin-related hearing loss. Curr Opin Otolaryngol Head Neck Surg, 2025, 33(5): 312–317

[9] European Society of Gene and Cell Therapy. Abstract book: ESGCT 30th Annual Congress. October 24–27, 2023; Brussels, Belgium. ESGCTcongress.com. Accessed March 26, 2026.

[10] Walls WDAzaiez HSmith RJH. Hereditary Hearing Loss Homepage. Accessed February 19, 2025.

[11] Imtiaz A . ARNSHL gene identification: past, present and future. Mol Genet Genomics, 2022, 297(5): 1185–1193

[12] Zhang L , Wang H , Xun M . et al. Preclinical evaluation of the efficacy and safety of AAV1-hOTOF in mice and nonhuman primates. Mol Ther Methods Clin Dev, 2023, 31: 101154

[13] Cheng X , Zhong J , Zhang J . et al. Gene Therapy vs Cochlear Implantation in Restoring Hearing Function and Speech Perception for Individuals With Congenital Deafness. JAMA Neurol, 2025, 82(9): 941–951

[14] Zhang J , Guo Z , Pan C . et al. Preliminary evidence for enhanced auditory cortex activation and mental development after gene therapy in children with autosomal recessive deafness 9. Nat Hum Behav, 2025, 9(7): 1457–1469

[15] Shu Y , Tao Y , Li W , Shen J , Wang Z , Chen ZY . Adenovirus Vectors Target Several Cell Subtypes of Mammalian Inner Ear In Vivo. Neural Plast, 2016, 2016: 9409846

[16] Wenzel GI , Xia A , Funk E . et al. Helper-dependent adenovirus-mediated gene transfer into the adult mouse cochlea. Otol Neurotol, 2007, 28(8): 1100–1108

[17] Pan S , Wan J , Liu S . et al. Lentivirus carrying the Atoh1 gene infects normal rat cochlea. Neural Regen Res, 2013, 8(17): 1551–1559

[18] Gadenstaetter AJ , Krumpoeck PE , Landegger LD . Inner Ear Gene Therapy: An Overview from Bench to Bedside. Mol Diagn Ther, 2025, 29(2): 161–181

[19] Zhang L , Tan F , Qi J . et al. AAV-mediated Gene Therapy for Hereditary Deafness: Progress and Perspectives. Adv Sci (Weinh), 2024, 11(47): e2402166

[20] Li X , Ma B , Dai J . et al. Metalized polyamide heterostructure as a moisture-responsive actuator for multimodal adaptive personal heat management. Sci Adv, 2021, 7(51): eabj7906

[21] Ivanchenko MV , Hanlon KS , Hathaway DM . et al. AAV-S: A versatile capsid variant for transduction of mouse and primate inner ear. Mol Ther Methods Clin Dev, 2021, 21: 382–398

[22] Wu J , Solanes P , Nist-Lund C . et al. Single and Dual Vector Gene Therapy with AAV9-PHP. B Rescues Hearing in Tmc1 Mutant Mice. Mol Ther, 2021, 29(3): 973–988

[23] Landegger LD , Pan B , Askew C . et al. A synthetic AAV vector enables safe and efficient gene transfer to the mammalian inner ear. Nat Biotechnol, 2017, 35(3): 280–284

[24] Ukaji T , Arai D , Tsutsumi H . et al. AAV-mediated base editing restores cochlear gap junction in GJB2 dominant-negative mutation-associated syndromic hearing loss model. JCI Insight, 2025, 10(5): e185193

[25] Isgrig K , Cartagena-Rivera AX , Wang HJ . et al. Combined AAV-mediated gene replacement therapy improves auditory function in a mouse model of human DFNB42 deafness. Mol Ther, 2023, 31(9): 2783–2795

[26] Sun QTan FZhang L, et al. Combined AAV-mediated specific Gjb2 expression restores hearing in DFNB1 mouse models. Mol Ther. 2025; Epub ahead of print.

[27] Iwasa Y , Klimara MJ , Yoshimura H . et al. Mutation-agnostic RNA interference with engineered replacement rescues Tmc1-related hearing loss. Life Sci Alliance, 2023, 6(3): e202201592

[28] Akil O , Dyka F , Calvet C . et al. Dual AAV-mediated gene therapy restores hearing in a DFNB9 mouse model. Proc Natl Acad Sci U S A, 2019, 116(10): 4496–4501

[29] Ivanchenko MV , Hathaway DM , Klein AJ . et al. Mini-PCDH15 gene therapy rescues hearing in a mouse model of Usher syndrome type 1F. Nat Commun, 2023, 14(1): 2400

[30] Rankovic V , Vogl C , Dörje NM . et al. Overloaded Adeno-Associated Virus as a Novel Gene Therapeutic Tool for Otoferlin-Related Deafness. Front Mol Neurosci, 2020, 13: 600051

[31] Le TP , Le TT , Jin S . et al. Injectable polyplex-loaded glycol chitosan thermogel for efficient and safe inner ear gene delivery. J Control Release, 2025, 380: 1095–1108

[32] Tao Y , Lamas V , Du W . et al. Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo. Nat Commun, 2023, 14(1): 4928

[33] Pan XHuang PAli SS, et al. CRISPR-Cas9 Engineered Extracellular Vesicles for the Treatment of Dominant Progressive Hearing Loss. bioRxiv. Preprint posted December 15, 2023.

[34] Wang XZhang LChen S, et al. Viral-Mediated Connexin 26 Expression Combined with Dexamethasone Rescues Hearing in a Conditional Gjb2 Null Mice Model. Adv Sci (Weinh). 2024;e2406510.

[35] Hu SW , Jeong S , Jiang L . et al. PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation. Nat Commun, 2025, 16(1): 7186

[36] Guo J , Ma X , Skidmore JM . et al. GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function. Mol Ther Methods Clin Dev, 2021, 23: 319–333

[37] Wang H , Xun M , Tang H . et al. Hair cell-specific Myo15 promoter-mediated gene therapy rescues hearing in DFNB9 mouse model. Mol Ther Nucleic Acids, 2024, 35(1): 102135

[38] Hu SW , Lv J , Wang Z . et al. Engineering of the AAV-Compatible Hair Cell-Specific Small-Size Myo15 Promoter for Gene Therapy in the Inner Ear. Research (Wash D C), 2024, 7: 0341

[39] Chung Y , Koehler SD , Cancelarich S . et al. Functional, sustained recovery of hearing in Otoferlin-deficient mice using DB-OTO, a hair-cell-specific AAV-based gene therapy. Mol Ther Methods Clin Dev, 2025, 33(4): 101577

[40] Ivanchenko MVBooth KTAKaravitaki KD, et al. Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea. bioRxiv. Preprint posted December 24, 2024.

[41] Seist R , Copeland JS , Tao L , Groves AK . Rational design of a Lfng-enhancer AAV construct drives specific and efficient gene expression in inner ear supporting cells. Hear Res, 2025, 458: 109203

[42] Zhao S , Yang Q , Yu Z . et al. Deciphering enhancers of hearing loss genes for efficient and targeted gene therapy of hereditary deafness. Neuron, 2025, 113(10): 1579–1596.e5

[43] Akil O , Seal RP , Burke K . et al. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron, 2012, 75(2): 283–293

[44] Lentz JJ , Pan B , Ponnath A . et al. Direct Delivery of Antisense Oligonucleotides to the Middle and Inner Ear Improves Hearing and Balance in Usher Mice. Mol Ther, 2020, 28(12): 2662–2676

[45] Hastings ML , Brigande JV . Fetal gene therapy and pharmacotherapy to treat congenital hearing loss and vestibular dysfunction. Hear Res, 2020, 394: 107931

[46] Lentz JJ , Jodelka FM , Hinrich AJ . et al. Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness. Nat Med, 2013, 19(3): 345–350

[47] Ponnath A , Depreux FF , Jodelka FM . et al. Rescue of Outer Hair Cells with Antisense Oligonucleotides in Usher Mice Is Dependent on Age of Treatment. J Assoc Res Otolaryngol, 2018, 19(1): 1–16

[48] Wang L , Kempton JB , Jiang H . et al. Fetal antisense oligonucleotide therapy for congenital deafness and vestibular dysfunction. Nucleic Acids Res, 2020, 48(9): 5065–5080

[49] Feng P , Xu Z , Chen J . et al. Rescue of mis-splicing of a common SLC26A4 mutant associated with sensorineural hearing loss by antisense oligonucleotides. Mol Ther Nucleic Acids, 2022, 28: 280–292

[50] Maeda Y , Fukushima K , Nishizaki K , Smith RJ . In vitro and in vivo suppression of GJB2 expression by RNA interference. Hum Mol Genet, 2005, 14(12): 1641–1650

[51] Cui C , Wang S , Wang D . et al. A base editor for the long-term restoration of auditory function in mice with recessive profound deafness. Nat Biomed Eng, 2025, 9(1): 40–56

[52] Noh B , Rim JH , Gopalappa R . et al. In vivo outer hair cell gene editing ameliorates progressive hearing loss in dominant-negative Kcnq4 murine model. Theranostics, 2022, 12(5): 2465–2482

[53] Liu L , Zou L , Li K . et al. Template-independent genome editing in the Pcdh15(av-3j) mouse, a model of human DFNB23 nonsyndromic deafness. Cell Rep, 2022, 40(2): 111061

[54] Gu X , Hu X , Wang D . et al. Treatment of autosomal recessive hearing loss via in vivo CRISPR/Cas9-mediated optimized homology-directed repair in mice. Cell Res, 2022, 32(7): 699–702

[55] Zhu W , Du W , Rameshbabu AP . et al. Targeted genome editing restores auditory function in adult mice with progressive hearing loss caused by a human microRNA mutation. Sci Transl Med, 2024, 16(755): eadn0689

[56] Xiao Q , Xu Z , Xue Y . et al. Rescue of autosomal dominant hearing loss by in vivo delivery of mini dCas13X-derived RNA base editor. Sci Transl Med, 2022, 14(654): eabn0449

[57] Qi J , Zhang L , Tan F . et al. Preclinical Efficacy And Safety Evaluation of AAV-OTOF in DFNB9 Mouse Model And Nonhuman Primate. Adv Sci (Weinh), 2024, 11(3): e2306201

[58] Al-Moyed H , Cepeda AP , Jung S , Moser T , Kügler S , Reisinger E . A dual-AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock-out mice. EMBO Mol Med, 2019, 11(1): e809396

[59] Tang H , Wang H , Wang S . et al. Hearing of Otof-deficient mice restored by trans-splicing of N- and C-terminal otoferlin. Hum Genet, 2023, 142(2): 289–304

[60] Xue Y , Tao Y , Wang X . et al. RNA base editing therapy cures hearing loss induced by OTOF gene mutation. Mol Ther, 2023, 31(12): 3520–3530

[61] Ma S , Chen X , Wang Y , Guo Y . Mechanisms of congenital hearing loss caused by GJB2 gene mutations and current progress in gene therapy. Gene, 2025, 946: 149326

[62] Zong YJ , Liu XZ , Tu L , Sun Y . Cytomembrane Trafficking Pathways of Connexin 26, 30, and 43. Int J Mol Sci, 2023, 24(12): 10349

[63] Zong YJ , Liu XZ , Shi XY , Zhao ZD , Sun Y . Promotion of Cx26 mutants located in TM4 region for membrane translocation successfully rescued hearing loss. Theranostics, 2025, 15(12): 5801–5825

[64] Liu XXie LJin Y, et al. Connexin 26 Functions as a Direct Transcriptional Regulator During the Cochlea Development. Adv Sci (Weinh). 2026;e14136.

[65] Iizuka T , Kamiya K , Gotoh S . et al. Perinatal Gjb2 gene transfer rescues hearing in a mouse model of hereditary deafness. Hum Mol Genet, 2015, 24(13): 3651–3661

[66] Li Q , Cui C , Liao R . et al. The pathogenesis of common Gjb2 mutations associated with human hereditary deafness in mice. Cell Mol Life Sci, 2023, 80(6): 148

[67] Xu K , Chen S , Xie L . et al. The protective effects of systemic dexamethasone on sensory epithelial damage and hearing loss in targeted Cx26-null mice. Cell Death Dis, 2022, 13(6): 545

[68] Wang X , Zhang L , Chen S . et al. Viral-Mediated Connexin 26 Expression Combined with Dexamethasone Rescues Hearing in a Conditional Gjb2 Null Mice Model. Adv Sci (Weinh), 2025, 12(29): e2406510

[69] Shubina-Oleinik O , Nist-Lund C , French C , Rockowitz S , Shearer AE , Holt JR . Dual-vector gene therapy restores cochlear amplification and auditory sensitivity in a mouse model of DFNB16 hearing loss. Sci Adv, 2021, 7(51): eabi7629

[70] Benoit C , Carlson RJ , King MC , Horn DL , Rubinstein JT . Behavioral characterization of the cochlear amplifier lesion due to loss of function of stereocilin (STRC) in human subjects. Hear Res, 2023, 439: 108898

[71] Lee HJ , Fenollar-Ferrer C , Isgrig K . et al. SLC26A4-AP-2 mu2 interaction regulates SLC26A4 plasma membrane abundance in the endolymphatic sac. Sci Adv, 2024, 10(41): eadm8663

[72] Fuster-García C , García-Bohórquez B , Rodríguez-Muñoz A . et al. Usher Syndrome: Genetics of a Human Ciliopathy. Int J Mol Sci, 2021, 22(13): 6723

[73] Peters CW , Hanlon KS , Ivanchenko MV . et al. Rescue of hearing by adenine base editing in a humanized mouse model of Usher syndrome type 1F. Mol Ther, 2023, 31(8): 2439–2453

[74] Schott JW , Huang P , Morgan M . et al. Third-generation lentiviral gene therapy rescues function in a mouse model of Usher 1B. Mol Ther, 2023, 31(12): 3502–3519

[75] Du WHuang JZhang A, et al. Long-Lasting Auditory and Vestibular Recovery Following Gene Replacement Therapy in a Novel Usher Syndrome Type 1c Mouse Model. Adv Sci (Weinh). 2025;e2410063.

[76] Fan XGao ZZhong J, et al. International expert consensus on gene therapy for hereditary hearing loss: Based on clinical trials. Med. 2025;100886.

[77] Eli Lilly and Company. Positive phase 1/2 clinical trial data for an investigational gene therapy for genetic hearing loss to be presented at the Association for Research in Otolaryngology 2024 MidWinter Meeting [press release]. Lilly.com. Published January 23, 2024. Accessed March 26, 2026.

[78] Association for Research in Otolaryngology. ARO 47th Annual MidWinter Meeting program book. February 3–7, 2024, Anaheim, CA. ARO.org. Accessed March 26, 2026.

[79] Zeng F-G . Gene Therapy Restored Partial Hearing in a Dozen Deaf Children. Hear J, 2024, 77(4): 1,2

[80] Sensorion. Sensorion reports new positive clinical results presented at the World Congress of Audiology [press release]. Sensorion.com. Published September 23, 2024. Accessed March 26, 2026.

[81] Sensorion. Sensorion reports full-year 2024 results, provides corporate update and announces availability of full-year report [press release]. Sensorion.com. Published March 14, 2025. Accessed March 26, 2026

[82] Jang SH , Song HG , Joo SY . et al. Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant. Mol Ther, 2026, 34(1): 216–231

[83] Jiang H , Couto LB , Patarroyo-White S . et al. Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood, 2006, 108(10): 3321–3328

[84] Chen LWen CLi W, et al. Natural history and phenotype-genotype correlations in GJB2-related hearing loss: a systematic and comprehensive review. J Genet Genomics. 2026; Epub ahead of print.

[85] Kong C , Wang X , Yu J . et al. Visualization of the Spiral Ganglion Neuron in Vivo Using a Novel (177)Lu Nuclear Molecule Label. Adv Sci (Weinh), 2025, 12(29): e04464

[86] Wu X , Zhang L , Li Y . et al. Gene therapy via canalostomy approach preserves auditory and vestibular functions in a mouse model of Jervell and Lange-Nielsen syndrome type 2. Nat Commun, 2021, 12(1): 697

[87] Taiber S , Cohen R , Yizhar-Barnea O , Sprinzak D , Holt JR , Avraham KB . Neonatal AAV gene therapy rescues hearing in a mouse model of SYNE4 deafness. EMBO Mol Med, 2021, 13(2): e13259

[88] Zhang L , Wang W , Kim SM . et al. Virally Mediated Connexin 26 Expression in Postnatal Scala Media Significantly and Transiently Preserves Hearing in Connexin 30 Null Mice. Front Cell Dev Biol, 2022, 10: 900416

[89] Zhao X , Liu H , Liu H , Cai R , Wu H . Gene Therapy Restores Auditory Functions in an Adult Vglut3 Knockout Mouse Model. Hum Gene Ther, 2022, 33(13-14): 729–739

[90] Lu YC , Tsai YH , Chan YH . et al. Gene therapy with a synthetic adeno-associated viral vector improves audiovestibular phenotypes in Pjvk-mutant mice. JCI Insight, 2022, 7(20): e152941

[91] Jeng JY , Carlton AJ , Goodyear RJ . et al. AAV-mediated rescue of Eps8 expression in vivo restores hair-cell function in a mouse model of recessive deafness. Mol Ther Methods Clin Dev, 2022, 26: 355–370

[92] Marcovich I , Baer NK , Shubina-Oleinik O , Eclov R , Beard CW , Holt JR . Optimized AAV Vectors for TMC1 Gene Therapy in a Humanized Mouse Model of DFNB7/11. Biomolecules, 2022, 12(7): 914

[93] Zheng Z , Li G , Cui C . et al. Preventing autosomal-dominant hearing loss in Bth mice with CRISPR/CasRx-based RNA editing. Signal Transduct Target Ther, 2022, 7(1): 79

[94] Tao Y , Liu X , Yang L . et al. AAV-ie-K558R mediated cochlear gene therapy and hair cell regeneration. Signal Transduct Target Ther, 2022, 7(1): 109

[95] Du W , Ergin V , Loeb C . et al. Rescue of auditory function by a single administration of AAV-TMPRSS3 gene therapy in aged mice of human recessive deafness DFNB8. Mol Ther, 2023, 31(9): 2796–2810

[96] Pauzuolyte V , Patel A , Wawrzynski JR . et al. Systemic gene therapy rescues retinal dysfunction and hearing loss in a model of Norrie disease. EMBO Mol Med, 2023, 15(10): e17393

[97] Ratzan EM , Lee J , Madison MA . et al. TMC function, dysfunction, and restoration in mouse vestibular organs. Front Neurol, 2024, 15: 1356614

[98] Lahlou G , Calvet C , Simon F . et al. Extended time frame for restoring inner ear function through gene therapy in Usher1G preclinical model. JCI Insight, 2024, 9(3): e169504

[99] Jiang L , Hu SW , Wang Z . et al. Hearing restoration by gene replacement therapy for a multisite-expressed gene in a mouse model of human DFNB111 deafness. Am J Hum Genet, 2024, 111(10): 2253–2264

[100] Benamer N , Le Ribeuz H , Felgerolle C . et al. Cochlear gene therapy restores hearing and auditory processing in an atypical DFNB9 mouse model. Commun Med (Lond), 2025, 5(1): 229

[101] Hahn R , Taiber S , Shubina-Oleinik O , Géléoc GSG , Holt JR , Avraham KB . AAV gene therapy rescues hearing and balance in a model of CLIC5 deafness. EMBO Mol Med, 2025, 17(9): 2233–2257

[102] Jang SHSong HGJoo SY, et al. Gene supplementation with precise transgene expression rescues hearing loss in a mouse model with an Mpzl2 East Asian founder variant. Mol Ther. 2025; Epub ahead of print.

[103] Ivanchenko MV , Booth KTA , Karavitaki KD . et al. Cell-specific delivery of GJB2 restores auditory function in mouse models of DFNB1 deafness and mediates appropriate expression in NHP cochlea. Nat Commun, 2025, 16(1): 11157

[104] Wei W , Zhu W , Silver S . et al. Single-dose genome editing therapy rescues auditory and vestibular functions in adult mice with DFNA41 deafness. J Clin Invest, 2025, 135(20): e187872

[105] Wang M , Zhang Z , Wang X . et al. Optimized in vivo base editing restores auditory function in a DFNA15 mouse model. Nat Commun, 2025, 16(1): 8322

[106] Sun HTeng QLiu W, et al. CRISPR-free RNA base editing mediated PTC-readthrough restores hearing in mice with Otof nonsense mutation. Nat Commun. 2025; Epub ahead of print.

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