Irreversible cisplatin-induced hearing loss (CIHL) is a refractory chemotherapy-related adverse effect with limited clinical treatments. Schisantherin B (STB), a lignan isolated from Schisandra chinensis, is widely recognized for its neuroprotective properties, while its role in auditory injury remains unclear. Herein, we found that STB alleviated cisplatin-induced ototoxicity in House Ear Institute Organ of Corti 1 (HEI-OC1) cells and guinea pig models, protecting cochlear hair cells, synaptic ribbons and spiral ganglion neurons, and partially restoring auditory brainstem response (ABR) thresholds. Furthermore, combined drug affinity responsive target stability (DARTS) assay, the cellular thermal shift assay (CETSA), and the surface plasmon resonance (SPR) assay, we confirmed STB directly binds to the canopy FGF signaling regulator 2 (CNPY2), a key initiator of endoplasmic reticulum (ER) stress. Notably, consistent dual in vitro and in vivo validation confirmed that STB exerts no regulatory effect on CNPY2 protein abundance, yet suppressed the downstream Protein kinase R-like endoplasmic reticulum kinase / C/EBP homologous protein (PERK/CHOP) signaling cascade and ER stress-mediated apoptosis. Moreover, molecular docking and co-immunoprecipitation (co-IP) validated the physical binding of STB to CNPY2 and the endogenous interaction between CNPY2 and PERK. Additionally, CNPY2 overexpression and shRNA knockdown further verified this functional relationship. Integrated proteomic and transcriptomic analyses showed STB partially reversed cisplatin-triggered inflammation and excessive ER stress. Collectively, our results suggest STB may serve as a potential otoprotective agent. The CNPY2-PERK/CHOP axis is closely linked to cisplatin-induced cochlear damage and offers a feasible target for intervention against CIHL. Abbreviations: CIHL, cisplatin-induced hearing loss; STB, Schisantherin B; HEI-OC1, house ear institute organ of corti 1; ABR, auditory brainstem response; DARTS, drug affinity responsive target stability; CETSA, cellular thermal shift assay; SPR, surface plasmon resonance; CNPY2, canopy FGF signaling regulator 2; ER, endoplasmic reticulum; PERK, protein kinase R-like endoplasmic reticulum kinase; CHOP, C/EBP homologous protein; co-IP, co-immunoprecipitation; STA, Schisantherin A; STC, Schisantherin C; dB SPL, decibels sound pressure level; EDTA, ethylenediaminetetraacetic acid; dB SPL, decibels sound pressure level; SGN, spiral ganglion neuron; IHCs, inner hair cells; OHCs, outer hair cells; CCK-8, Cell Counting Kit-8; OD, optical density; ODb, blank sample, ODc, control sample; NC, negative control; PVDF, polyvinylidene difluoride; RT, room temperature; LC-MS/MS, liquid chromatography tandem mass spectrometry; MS, mass spectrometry; DMSO, dimethyl sulfoxide; KDs, equilibrium dissociation constants; SP, standard precision; SEM, standard error of the mean; HSD, honestly significant difference; Ctrl, control group; CV, cell viability; Kd, dissociation rate constant; Ka, association rate constant; STS, sodium thiosulfate; UPR, unfolded protein response; BLB, blood-labyrinth barrier.

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 .

Variants in the aminoacylase 1 (ACY1) gene are associated with diverse clinical phenotypes, but their roles in hearing loss remain unclear. This study aimed to investigate the potential role of ACY1 in hearing loss. Exome sequencing was performed on 396 patients with sensorineural hearing loss. We identified a homozygous splicing variant, c.1063-1G>A, in ACY1 as a plausible candidate variant in a 5-year-old girl with congenital hearing loss and normal developmental milestones over a 4-year follow-up. Functional assays, including urinary organic acid analysis and aminoacylase 1 enzyme activity testing in Epstein-Barr virus (EBV)-transformed lymphoblasts, confirmed aminoacylase 1 deficiency in the patient. In vitro splicing assays showed that the c.1063-1G>A variant activated a cryptic splice site, causing aberrant splicing, a frameshift, and a premature stop codon. A zebrafish model with morpholino-mediated acy1 knockdown revealed a significant reduction in hair cells and impaired auditory function, which were effectively rescued by wild-type but not mutant human ACY1 mRNA. Furthermore, transcriptomic profiling of zebrafish auditory hair cells, combining fluorescence-activated cell sorting (FACS) with RNA sequencing, revealed downregulation of critical inner ear development genes, including gfi1ab and atoh1a/b, which was validated by RT-qPCR. These results provide clinical and experimental evidence supporting the functional impact of the ACY1 c.1063-1G>A variant and its potential involvement in congenital hearing loss. Our findings support ACY1 as a candidate gene for hereditary hearing impairment, although additional unrelated patients and independent validation are needed to further establish the gene-disease relationship.

Abstract A major challenge in gene therapy for GJB2 -related hearing loss (DFNB1)—the most common form of hereditary deafness—is achieving efficient and precise connexin 26 delivery. Herein, we engineered two cell type-specific promoters (GJB2-1 and WFS1-2274) and developed an AAV capsid, AAV-MAS012, with enhanced transduction efficiency in mature cochlear cells. Our AAV-mediated gene therapy systems restored hearing of low-to-mid-frequencies in newborn Gjb2 cKO mice to wild-type levels and maintained for 45 weeks. Additionally, our therapeutic systems restored low-to-mid-frequencies hearing function to wild-type levels in adult Gjb2 cKO mice. A humanized version of the therapy, AAV-MAS012-WFS1-2274-hGJB2, rescued hearing function in two distinct Gjb2 -deficient mouse models, and demonstrated a favorable safety profile in nonhuman primates. This study represents the first successful hearing restoration in adult Gjb2 -deficient mice. The significant therapeutic efficacy of the humanized gene therapy system shows great potential for clinical translation in DFNB1 patients.

Adeno-associated virus (AAV)-based gene therapy has demonstrated transformative potential in treating hereditary hearing loss (HHL). Investigating factors that influence AAV transduction in target and non-target organs is critical for improving therapeutic precision and safety. Given the postnatal maturation of the murine inner ear and compartmentalized structure of the cochlea, we evaluated whether developmental age or delivery routes affect AAV-PHP.eB tropism in cochlear hair cells (HCs) and adjacent brain regions. Following round window membrane (RWM), posterior semicircular canal (PSCC), or utricle delivery, neonatal mice showed robust OHC transduction (vs. minimal in juveniles), while IHC transduction remained consistently high across age groups. Across all three delivery routes, brain AAV transduction was significantly higher in neonates than in juveniles. Despite this, AAV genome copies were more highly enriched in the injected inner ear than in the brain at both ages. Dye-tracing experiments demonstrated distinct spatial distribution patterns following three inner ear delivery routes, with the cochlear aqueduct (CA) identified as the primary conduit for intracranial spread post-injection. These findings provide guidance for the design of studies in mouse models of deafness, particularly with respect to cochlear hair cell subtype targeting, therapeutic timing, and safety assessment of AAV-PHP.eB-based therapies for hearing loss.

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.

Hearing loss is one of the most prevalent sensory disorders, but no commercial biological treatments are currently available. Here, we identify an East Asia-specific founder mutation, the homozygous c.220 C > T mutation in MPZL2, that contributes to a significant proportion of hereditary deafness cases in our cohort study. We find that the disease-causing mutation can be targetable by adenine base editors (ABEs) that enable A·T-to-G·C base corrections without DNA double-strand breaks. To demonstrate this, we develop a humanized mouse model (hMPZL2Q74X/Q74X) that recapitulates human MPZL2 deafness and leads to progressive hearing loss. A PAM-flexible ABE variant with reduced bystander and off-target effects (ABE8eWQ-SpRY:sgRNA3) is packaged in dual adeno-associated viruses (AAVs) and injected into the inner ear of hMPZL2Q74X/Q74X mice and effectively corrects the mutation. This treatment significantly restores hearing function, improves inner ear structural integrity, and reverses altered gene expression. Base editing may hold therapeutic potential for hereditary deafness, including most cases of MPZL2 deafness.

Pathological mutations in the OTOF gene cause autosomal recessive deafness 9 (DFNB9). Although dual-adeno-associated virus (AAV) gene replacement therapy has been shown to partially rescue the hearing of patients with DFNB9, the therapeutic effects still need further exploration. To investigate the impact of different recombination strategies on the efficacy of OTOF gene therapy, we constructed five dual-AAV1 therapeutic agents using RNA or protein splicing principles. Based on the recombination strategy using RNA splicing, the AAV1-AK system rescued the hearing of Otof-/- mice to 55-70 dB, which outperformed the AAV1-AP and AAV1-TS systems. Based on protein splicing, the AAV1-intein system rescued the hearing of Otof-/- mice to 35-70 dB, which outperformed that of AAV1-AK, and the effects lasted up to 12 months after injection in both newborn and adult mice. The efficacy of otoferlin re-expression and the number and functional restoration of ribbon synapses in the AAV1-intein system were also better than the AAV1-AK system. These results show that protein recombination is more efficient than nucleic acid recombination for gene therapy in DFNB9. Therefore, this work provides not only data for optimization of DFNB9 gene therapy but also a reference for the delivery of other large genes.

Cisplatin has a high efficacy for treating solid tumors, but it is generally accompanied by ototoxic side effects. Leonurine (LEO) has anti-oxidative and anti-apoptotic effects, although its role in the treatment of cisplatin-induced hearing impairment (CIHI) remains unclear. Here, we explored in vitro and in vivo models of cisplatin injury and analyzed the efficacy of LEO on cisplatin-induced ototoxicity by immunofluorescence, otoacoustic assessment, qRT-PCR, and Western blot. At the cellular level, LEO reduced oxidative stress and apoptosis, while at the organism level LEO protected guinea pigs against CIHI and maintained the hearing thresholds of cisplatin-treated guinea pigs at 50-55 dB. LEO effectively prevented cisplatin-induced decreases in hair cells, supporting cells, spiral ganglion neurons and ribbon synapses; reduced Cleaved Caspase 3 expression through activation of Bcl-2 and reducing reactive oxygen species (ROS) accumulation and improving mitochondrial membrane potential and reduced cisplatin-induced apoptosis by increasing the expression of Nrf2/Nqo1. In conclusion, the present study expands the application range of LEO and suggests that LEO is a potential therapeutic agent for preventing cisplatin ototoxicity.

The sense of balance,mediated by the vestibular system,is crucial in our three-dimensional world.In a recent study published in Cell Research,Yang et al.revealed an essential role of a mechanosensitive G protein-coupled receptor as a tip link-independent equilibrioceptor actively participating in the mechano-electrical transduction process.

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.

Aminoglycosides are commonly used for the treatment of life-threatening bacterial infections, however, aminoglycosides may cause irreversible hearing loss with a long-term clinical therapy. The mechanism and prevention of the ototoxicity of aminoglycosides are still limited although amounts of studies explored widely. Specifically, advancements in programmed cell death (PCD) provide more new perspectives. This review summarizes the general signal pathways in programmed cell death, including apoptosis, autophagy, and ferroptosis, as well as the mechanisms of aminoglycoside-induced ototoxicity. Additionally, novel interventions, especially gene therapy strategies, are also investigated for the prevention or treatment of aminoglycoside-induced hearing loss with prospective clinical applications.

Gene therapy has made significant progress in the treatment of hereditary hearing loss. However, most research has focused on deafness-related genes that are primarily expressed in hair cells with less attention given to multisite-expressed deafness genes. MPZL2, the second leading cause of mild-to-moderate hereditary deafness, is widely expressed in different inner ear cells. We generated a mouse model with a deletion in the Mpzl2 gene, which displayed moderate and slowly progressive hearing loss, mimicking the phenotype of individuals with DFNB111. We developed a gene replacement therapy system mediated by AAV-ie for efficient transduction in various types of cochlear cells. AAV-ie-Mpzl2 administration significantly lowered the auditory brainstem response and distortion product otoacoustic emission thresholds of Mpzl2-/- mice for at least seven months. AAV-ie-Mpzl2 delivery restored the structural integrity in both outer hair cells and Deiters cells. This study suggests the potential of gene therapy for MPZL2-related deafness and provides a proof of concept for gene therapy targeting other deafness-related genes that are expressed in different cell populations in the cochlea.

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.

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.

Cisplatin is a highly effective anti-tumor drug that is often used to treat a variety of cancers, such as ovarian, cervical, and brain cancers. However, cisplatin can cause serious side effects, such as ototoxicity. Clinically, chemotherapy with cisplatin often causes permanent inner ear damage, thereby leading to progressive, bilateral, and irreversible hearing loss in patients. One mechanism underlying cisplatin-induced ototoxicity is the excessive production and accumulation of reactive oxygen species (ROS) and the ensuing apoptosis of hair cells (HCs) and spiral ganglion neurons (SGNs). Several molecules that can prevent excessive ROS or apoptosis have been identified for the treatment of cisplatin-induced ototoxicity.1Brock P.R. Maibach R. Childs M. Rajput K. Roebuck D. Sullivan M.J. Laithier V. Ronghe M. Dall'Igna P. Hiyama E. et al.Sodium Thiosulfate for Protection from Cisplatin-Induced Hearing Loss.N. Engl. J. Med. 2018; 378: 2376-2385Crossref PubMed Scopus (201) Google Scholar,2Thakur N.S. Rus I. Sparks E. Agrahari V. Dual stimuli-responsive and sustained drug delivery NanoSensoGel formulation for prevention of cisplatin-induced ototoxicity.J. Control. Release. 2024; 368: 66-83Crossref Scopus (0) Google Scholar However, since chemotherapy often involves several cycles of treatment, the short half-life of anti-ototoxic drugs limits their therapeutic effects within the cochlea.3Araya C.E. Fennell R.S. Neiberger R.E. Dharnidharka V.R. Sodium thiosulfate treatment for calcific uremic arteriolopathy in children and young adults.Clin. J. Am. Soc. Nephrol. 2006; 1: 1161-1166Crossref PubMed Scopus (87) Google Scholar Moreover, repeated injection of anti-ototoxic drugs is inconvenient and could cause adverse local reactions. Thus, more permanent anti-ototoxic treatment strategies are necessary. Gene therapy is a promising strategy that offers durable efficacy with one-time administration and may revolutionize the treatment of many diseases, such as ear diseases, eye diseases, and blood disorders. Currently, adeno-associated viruses (AAVs), adenoviruses, lentiviruses (LVs), and retroviruses have been developed as delivery vehicles in gene therapy. Viral vectors are highly efficient at transfecting cells, and in general, viral vector-mediated gene therapy provides relatively strong and long-term expression of therapeutic transgenes. Moreover, many gene therapy clinical trials have used viral vectors as delivery tools.4Wei Hu S. Ding T. Tang H. Guo H. Cui W. Shu Y. Nanobiomaterial vectors for improving gene editing and gene therapy.Mater. Today. 2023; 66: 114-136Crossref Scopus (9) Google Scholar,5Lv J. Wang H. Cheng X. Chen Y. Wang D. Zhang L. Cao Q. Tang H. Hu S. Gao K. et al.AAV1-hOTOF gene therapy for autosomal recessive deafness 9: a single-arm trial.Lancet. 2024; https://doi.org/10.1016/S0140-6736(23)02874-XAbstract Full Text Full Text PDF PubMed Scopus (8) Google Scholar In a recent study published in Molecular Therapy Nucleic Acids, Nassauer et al. developed a gene therapy approach to protect cochlear cells from cisplatin-induced toxicity by using third-generation self-inactivating (SIN) LV vectors in vitro and in vivo.6Nassauer L. Staecker H. Huang P. Renslo B. Goblet M. Harre J. Warnecke A. Schott J.W. Morgan M. Galla M. Schambach A. Protection from cisplatin-induced hearing loss with lentiviral vector-mediated ectopic expression of the anti-apoptotic protein BCL-XL.Mol. Ther. Nucleic Acids. 2024; 35102157Abstract Full Text Full Text PDF Scopus (0) Google Scholar Their preclinical data suggest that gene therapy has the potential to be translated to clinical use in cancer patients. Nassauer et al. first confirmed that cisplatin can significantly increase ROS generation in HC-like House Ear Institute-organ of Corti 1 (HEI-OC1) cells and lead to cumulative cytotoxicity. To obtain the candidate molecules that could combat cisplatin-induced ototoxicity, they performed a small-scale screening that included the antioxidant enzymes superoxide dismutase 1 (SOD1), catalase (CAT), and glutathione peroxidase (GPX1) as well as the anti-apoptotic proteins B cell lymphoma 2 (BCL-2) and B cell lymphoma-extra large (Bcl-xl). Then they packaged the transgenes of the aforementioned molecules into LV vectors and tested their anti-ototoxic potential in HEI-OC1 cells (Figure 1). The first set of experiments assessed the effects of SIN LV vectors overexpressing human codon-optimized SOD1, CAT, or GPX1 on cisplatin-induced ROS and cell death. The results revealed that none of the SIN LV vectors overexpressing antioxidant enzymes affected ROS production or cell death in HEI-OC1 cells. Next, the authors examined the impact of BCL-2 and Bcl-xl on cisplatin-induced cell death. Compared to the vector control, SIN LV vectors expressing a codon-optimized version of human BCL-2 (LV.BCL-2) or murine BCL-XL (LV.BCL-XL) significantly decreased apoptotic cell death in cisplatin-treated HEI-OC1 cells. However, LV.BCL-XL was more effective at reducing cisplatin-induced cell death than LV.BCL-2; thus, only LV.BCL-XL was assessed in further experiments. Utilizing similar methods, the authors demonstrated that cisplatin treatment can induce cell death in SGN-like phoenix auditory neurons and neuroprogenitors in vitro. Cisplatin treatment also induced morphological changes in phoenix auditory neurons, reduced neuroprogenitor sphere formation, and altered neuroprogenitor sphere morphology. Compared with the vector control, LV.BCL-XL prevented these toxic effects in both phoenix auditory neurons and phoenix auditory neuroprogenitors. Further experiments in primary SGN cells and rat cochlear explants revealed that cisplatin induced cell death in primary SGNs and destroyed the normal HC organization of rat cochlear explants; the administration of LV.BCL-XL also prevented these negative effects. Altogether, these results indicate that the antiapoptotic effects mediated by LV.BCL-XL ultimately decreased the cytotoxicity of cisplatin treatment in HC-like cells, SGN-like cells, primary SGNs, and primary HCs. More importantly, the authors verified the protective effects of BCL-XL protein in a cisplatin challenge experiment in vivo. LV.BCL-XL was administered at post-natal day 30 via canalostomy into one ear of each wild-type C57BL/6 mouse. Auditory brainstem response (ABR) measurements revealed this manipulation did not affect the hearing capacity of the mice. Five days later, 4 mg/kg cisplatin was administered intraperitoneally for five consecutive days. Cisplatin treatment induced damage to outer hair cells (OHCs) and hearing loss in mice that did not receive LV.BCL-XL gene therapy, whereas OHCs in the cochlear base and high-frequency hearing were protected in mice that received LV.BCL-XL gene therapy (Figure 1). It is worth noting that, similar to other studies, Nassauer et al. used local administration of LV.BCL-XL to the inner ear instead of systemic administration. Local administration to the inner ear is typically utilized because the cochlea is relatively anatomically isolated from neighboring organs and is separated from systemic blood circulation through the blood-labyrinth barrier. This unique structure maintains immune privilege within the cochlea and minimizes off-target transfection. Additionally, the blood-labyrinth barrier could prevent systemically administered gene therapy drugs from reaching target cells in the inner ear. Overall, this very well-designed study demonstrates the ability of a novel preclinical gene therapy strategy to protect cochlear cells from cisplatin-induced ototoxicity in vitro, ex vivo, and in vivo. Nevertheless, several limitations need to be further addressed before clinical translation of LV.BCL-XL gene therapy. The first limitation is the choice of viral vector. Although SIN LV has several advantageous characteristics, like high packaging capacity and low immunogenicity, LVs are integrating vectors that could potentially induce insertional mutagenesis.4Wei Hu S. Ding T. Tang H. Guo H. Cui W. Shu Y. Nanobiomaterial vectors for improving gene editing and gene therapy.Mater. Today. 2023; 66: 114-136Crossref Scopus (9) Google Scholar Alternatively, AAVs are non-integrating vectors with high transfection efficiency in a large number of cell types and have the potential to provide long-term expression of therapeutic transgenes such as BCL-XL. Presently, multiple AAV serotypes enable efficient transfection of various types of inner ear cells. The second limitation is the expression region of the BCL-XL protein, as the ectopic expression of protein driven by ubiquitous promoters (e.g., cytomegalovirus immediate‑early enhancer/chicken β‑actin (CAG) and cytomegalovirus (CMV) promoters) may induce cytotoxicity. Administration of LV.BCL-XL into the ear via canalostomy will express the BCL-XL protein in almost all kinds of inner ear cells, whereas combining a cell type-specific viral vector (e.g., AAV.PHP.eB) with a cell type-specific promoter (e.g., the Myo15 promoter7Wang H. Xun M. Tang H. Zhao J. Hu S. Zhang L. Lv J. Wang D. Chen Y. Liu J. et al.Hair cell-specific Myo15 promoter-mediated gene therapy rescues hearing in DFNB9 mouse model.Mol. Ther. Nucleic Acids. 2024; 35102135Abstract Full Text Full Text PDF Scopus (1) Google Scholar) will restrict the expression range of transgenes and further increase the safety of gene therapy. In conclusion, reducing apoptosis by overexpressing the anti-apoptotic protein BCL-XL ameliorated cisplatin-induced cytotoxicity in HEI-OC1 cells, phoenix auditory neurons, primary SGN cultures, and cochlear explants. By contrast, reducing oxidative stress by overexpressing antioxidant enzymes (i.e., SOD1, CAT, or GPX1) failed to protect HEI-OC1 cells from cisplatin-induced toxicity. Moreover, in vivo application of the LV.BCL-XL vector increased the HC survival rate and protected cisplatin-treated mice from high-frequency hearing loss. These results revealed that preventing apoptosis is more effective than reducing oxidative stress in alleviating cisplatin-induced ototoxicity. Research in our laboratory is supported by the National Science Fund for Distinguished Young Scholars (82225014) and the China Postdoctoral Science Foundation (2022M720780). S.W.H., L.H., and Y.S. conceived and wrote this commentary. The authors declare no competing interests.

The adeno-associated virus (AAV) gene therapy has been widely applied to mouse models for deafness. But, AAVs could transduce non-targeted organs after inner ear delivery due to their low cell-type specificity. This study compares transgene expression and biodistribution of AAV1, AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB after round window membrane (RWM) injection in neonatal mice. The highest virus concentration was detected in the injected cochlea. AAV2, Anc80L65, AAV9, AAV-PHP.B, and AAV-PHP.eB transduced both inner hair cells (IHCs) and outer hair cells (OHCs) with high efficiency, while AAV1 transduced IHCs with high efficiency but OHCs with low efficiency. All AAV subtypes finitely transduced contralateral inner ear, brain, heart, and liver compared with the injected cochlea. In most brain regions, the enhanced green fluorescent protein (eGFP) expression of AAV1 and AAV2 was lower than that of other four subtypes. We suggested the cochlear aqueduct might be one of routes for vectors instantaneously infiltrating into the brain from the cochlea through a dye tracking test. In summary, our results provide available data for further investigating the biodistribution of vectors through local inner ear injection and afford a reference for selecting AAV serotypes for gene therapy toward deafness.

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.