The human retina, a complex and highly specialized structure, includes multiple cell types that work synergistically to generate and transmit visual signals. However, genetic predisposition or age-related degeneration can lead to retinal damage that severely impairs vision or causes blindness. Treatment options for retinal diseases are limited, and there is an urgent need for innovative therapeutic strategies. Cell and gene therapies are promising because of the efficacy of delivery systems that transport therapeutic genes to targeted retinal cells. Gene delivery systems hold great promise for treating retinal diseases by enabling the targeted delivery of therapeutic genes to affected cells or by converting endogenous cells into functional ones to facilitate nerve regeneration, potentially restoring vision. This review focuses on two principal categories of gene delivery vectors used in the treatment of retinal diseases: viral and non-viral systems. Viral vectors, including lentiviruses and adeno-associated viruses, exploit the innate ability of viruses to infiltrate cells, which is followed by the introduction of therapeutic genetic material into target cells for gene correction. Lentiviruses can accommodate exogenous genes up to 8 kb in length, but their mechanism of integration into the host genome presents insertion mutation risks. Conversely, adeno-associated viruses are safer, as they exist as episomes in the nucleus, yet their limited packaging capacity constrains their application to a narrower spectrum of diseases, which necessitates the exploration of alternative delivery methods. In parallel, progress has also occurred in the development of novel non-viral delivery systems, particularly those based on liposomal technology. Manipulation of the ratios of hydrophilic and hydrophobic molecules within liposomes and the development of new lipid formulations have led to the creation of advanced non-viral vectors. These innovative systems include solid lipid nanoparticles, polymer nanoparticles, dendrimers, polymeric micelles, and polymeric nanoparticles. Compared with their viral counterparts, non-viral delivery systems offer markedly enhanced loading capacities that enable the direct delivery of nucleic acids, mRNA, or protein molecules into cells. This bypasses the need for DNA transcription and processing, which significantly enhances therapeutic efficiency. Nevertheless, the immunogenic potential and accumulation toxicity associated with non-viral particulate systems necessitates continued optimization to reduce adverse effects in vivo . This review explores the various delivery systems for retinal therapies and retinal nerve regeneration, and details the characteristics, advantages, limitations, and clinical applications of each vector type. By systematically outlining these factors, our goal is to guide the selection of the optimal delivery tool for a specific retinal disease, which will enhance treatment efficacy and improve patient outcomes while paving the way for more effective and targeted therapeutic interventions.

Hearing loss profoundly affects social engagement, mental health, cognition, and brain development, with sensorineural hearing loss (SNHL) being a major concern. Linked to ototoxic medications, ageing, and noise exposure, SNHL presents significant treatment challenges, highlighting the need for effective prevention and regeneration strategies. Ferroptosis, a distinct form of cell death featuring iron-dependent lipid peroxidation, has garnered interest due to its potential role in cancer, ageing, and neuronal degeneration, especially hearing loss. The emerging role of ferroptosis as a crucial mediator in SNHL suggests that it may offer a novel therapeutic target for otoprotection. This review aims to summarise the intricate connection between ferroptosis and SNHL, offering a fresh perspective for exploring targeted therapeutic strategies that could potentially mitigate cochlear cells damage and enhance the quality of life for individuals with hearing impairments.

Gene therapy is an innovative medical approach that offers new treatment options for congenital and acquired diseases by transferring, correcting, inactivating or regulating genes to supplement, replace or modify a gene function. The approval of voretigene neparvovec (Luxturna), a gene therapy for RPE65-associated retinopathy, has marked a milestone for the field of retinal gene therapy, but has also helped to accelerate the development of gene therapies for genetic diseases affecting other organs. Voretigene neparvovec is a vector based on adeno-associated virus (AAV) that delivers a functional copy of RPE65 to supplement the missing function of this gene. The AAV-based gene delivery has proven to be versatile and safe for the transfer of genetic material to retinal cells. However, challenges remain in treating additional inherited as well as acquired retinopathies with this technology. Despite the high level of activity in this field, no other AAV gene therapy for retinal diseases has been approved since voretigene neparvovec. Ongoing research focuses on overcoming the current restraints through innovative strategies like AAV capsid engineering, dual-AAV vector systems, or CRISPR/Cas-mediated genome editing. Additionally, AAV gene therapy is being explored for the treatment of complex acquired diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) by targeting molecules involved in the pathobiology of the degenerative processes. This review outlines the current state of retinal gene therapy, highlighting ongoing challenges and future directions.

The autosomal recessive deafness 9 (DFNB9) is caused by mutations in the otoferlin gene that accounts for 2-8% of all inherited deafness cases. In a previous study, we demonstrated that Adeno-associated virus (AAV) gene therapy restored hearing in a preclinical mouse model of profound DFNB9 deafness caused by a frameshift mutation leading to a complete loss of otoferlin expression. However, it remains to be demonstrated that it can address the full spectrum of DFNB9 deafness severity, while also restoring central auditory processing essential for speech understanding.

Using homologous recombination in mouse embryonic stem cells, we created a knock-in mouse model carrying the E1799del otoferlin mutation, which mirrors the human E1804del variant linked to DFNB9 deafness, characterized by moderate-to-profound deafness during febrile episodes in affected individuals. A mixture of male and female mice was used at P2, P8, and P30. Some were followed for up to 4 months for longevity monitoring and behavioral tests.

The mouse model exhibits abnormal otoferlin distribution, failure of synaptic transmission in inner hair cells, and profound hearing loss, all of which is restored to normal through AAV gene therapy. Notably, we conduct objective behavioral testing to provide the first evidence that gene therapy administered to the cochlea, which is part of the peripheral auditory system, can restore frequency discrimination, indicating the recovery of central auditory processing. This is achieved even when treatment is administered late at the end of the critical period.

These findings indicate that gene therapy can address the entire spectrum of DFNB9 hearing loss, and that profound deafness during critical period may not impede the restoration of central auditory processing.

Noise-induced hearing loss (NIHL) poses an emerging global health problem with only ear protection or sound avoidance as preventive strategies. The cochlea receives some protection from medial olivocochlear efferent neurons, providing a potential target for therapeutic enhancement. Cholinergic efferents release acetylcholine (ACh) to hyperpolarize and shunt the outer hair cells (OHCs), reducing sound-evoked activation. The (α9)2(α10)3 nicotinic ACh receptor (nAChR) on the OHCs mediates this effect. Transgenic knockin mice with a gain-of-function nAChR (α9L9'T) suffer less NIHL. α9 knockout mice are more vulnerable to NIHL but can be rescued by viral transduction of the α9L9'T subunit. In this study, an HA-tagged gain-of-function α9 isoform was expressed in wild-type mice to reduce NIHL. Synaptic integration of the virally expressed nAChR subunit was confirmed by HA immunopuncta localized to the postsynaptic membrane of OHCs. After noise exposure, AAV2.7m8-CAG-α9L9'T-HA (α9L9'T-HA)-injected mice had less hearing loss (auditory brainstem response [ABR] thresholds and threshold shifts) than did control mice. ABRs of α9L9'T-HA-injected mice also had larger wave-1 amplitudes and better recovery of wave-1 amplitudes post noise exposure. Thus, virally expressed α9L9'T combines effectively with native α9 and α10 subunits to mitigate NIHL in wild-type cochleas.

Hearing loss is a common disability affecting the world's population. Currently, its treatment options are limited. Adeno-associated virus (AAV)-mediated inner ear gene therapy has shown great promise as a treatment for hereditary hearing loss. However, the host immune responses to AAV-mediated gene therapy in the mammalian inner ear is not well understood. In this study, two serotypes of AAV vectors were injected individually into the mouse inner ear to evaluate the host innate and adaptive immune responses up to 1 month after inner ear gene delivery. Our results suggest that the host innate and adaptive immune responses to AAV-mediated inner ear gene delivery are limited and mild, which is favorable for its clinical translation.

The clinical need for minimally invasive inner ear diagnostics and therapeutics has grown rapidly in recent years, particularly with the development of gene therapies for treating hearing and balance disorders. These therapies often require delivery of large injectate volumes that can cause hearing damage. In response to this challenge, dual-lumen microneedles, with two separate fluidic pathways controlled independently by micropumps, were designed for simultaneous aspiration and delivery to the inner ear across the round window membrane (RWM) and were fabricated using 2-photon polymerization (2PP). To assess the proof of concept of the dual-lumen microneedle device, simultaneous injection of 5 µL of adeno-associated virus (AAV) expressing green fluorescent protein (GFP) and aspiration of 5 µL of perilymph was performed in guinea pigs in vivo. Hearing thresholds were measured using auditory brainstem response (ABR) at time points before and 1 week after the procedure. Confocal imaging of the cochlea, the utricle, and the contralateral inner ear was employed to quantify and characterize the spatial distribution of hair cells with AAV transduction. Dual-lumen microneedle devices were found to be functional in the surgical setting. There was hearing loss limited to higher frequencies of 24 kHz and 28 kHz with ABR mean threshold shifts of 13 dB sound pressure level (SPL) (p = 0.03) and 23 dB SPL (p < 0.01), respectively. Furthermore, cochlear AAV transduction with a stereotypical basoapical gradient was observed in all animals (n = 5). Thus, dual-lumen microneedles can facilitate delivery of large volumes of therapeutic material into the inner ear, overcoming the limitations of single-lumen microneedles.

To assess outcomes of CI in adolescent patients with ANSD, a population which has not yet been comprehensively reviewed through a scoping review.

A scoping review in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive search of MEDLINE, EMBASE, Cochrane DSR, Cochrane CENTRAL, CINAHL, and Web of Science was performed. Cohort and case studies evaluating outcomes of CI in adolescents with ANSD were selected. A case report of an adolescent ANSD patient who underwent CI from our tertiary care academic hospital setting is also reported.

Our search identified a total of 17 articles after screening 488 articles. Among the 24 patients isolated from the selected articles, the vast majority showed some level of improvement in their hearing ability (n = 21, 87.5%). Additionally, of the 20 individuals whose hearing outcomes had a comparison cohort of subjects under 10 years old, nearly half showed better or similar levels of hearing improvement (n = 9, 45%).

Adolescents with ANSD receiving CI have notable improvements in hearing outcomes, but to a lesser degree than younger cohorts with ANSD. As such, CI should be considered as a valid treatment option for adolescents with ANSD. However, the benefit of such intervention has a wide variability, presumably based on the different pathologies that can cause their hearing loss and not necessarily the age at implantation.

Hearing loss is extremely common, yet limited treatment options are available to restore hearing, and those that are available provide incomplete recovery of hearing detection. For patients who are born with normal hearing, the most common cause of hearing loss is the loss of the sensory hair cells located in the cochlea of the inner ear. Non-mammals, including birds, fish, and amphibians, naturally regenerate new hair cells after damage and this natural process results in functional recovery. While some limited hair cell regeneration also occurs in the immature cochlea of mice, the mature mammalian cochlea does not naturally produce replacement hair cells, and thus hearing loss is permanent. Since the late 1980s, researchers have been investigating mechanisms to convert supporting cells, the cells that remain once hair cells have been killed, into new replacement hair cells. Here we review the current status of hair cell regeneration in the adult cochlea, highlighting recent achievements, as well as challenges that have yet to be resolved.

OTOF-gene therapy for profound deafness in children has entered clinical trials. Given that there is an approved alternative therapy with cochlear implants, it is imperative to scrutinize the risks, while also highlighting the novel benefits, of this experimental gene therapy. Since the untreated inner ear subsequently degenerates in this form of inherited deafness, the OTOF-gene therapy will be most effective in young children. Moreover, the best outcome in terms of hearing and speech comprehension is expected when the gene therapy is applied before the age of 3 years. Given such "earlier the better" considerations, the optimal time for these clinical trials and this particular therapy is at an age when children are too young to give informed consent. Enrolling children, which are a vulnerable category of persons, in clinical trials where the balance of benefits and risks is uncertain, raises a series of ethical considerations. In this article, we outline how this research can be pursued in an ethically responsible manner.

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 50-55dB. 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.

Hereditary hearing loss accounts for about 60% of congenital deafness. Although adeno-associated virus (AAV)-mediated gene therapy shows substantial potential for treating genetic hearing impairments, there remain significant concerns regarding the specificity and safety of AAV vectors. The sophisticated nature of the cochlea further complicates the challenge of precisely targeting gene delivery. Here, we introduced an AAV-reporter-based in vivo transcriptional enhancer reconstruction (ARBITER) workflow, enabling efficient and reliable dissection of enhancers. With ARBITER, we successfully demonstrated that the conserved non-coding elements (CNEs) within the gene locus collaboratively regulate the expression of Slc26a5, which was further validated using knockout mouse models. We also assessed the potential of identified enhancers to treat hereditary hearing loss by conducting gene therapy in Slc26a5 mutant mice. Based on the original Slc26a5 enhancer with limited efficiency, we engineered a highly efficient and outer hair cell (OHC)-specific enhancer, B8, which successfully restored hearing of Slc26a5 knockout mice.

Pathogenic variants in OTOF are a major cause of auditory synaptopathy. However, challenges remain in interpreting OTOF variants, including difficulties in confirming haplotype phasing using traditional short-read sequencing (SRS) due to the large gene size, the potential incomplete penetrance of certain variants, and difficulties in assessing variants at non-canonical splice sites. This study aims to revisit the genetic landscape of OTOF variants in a Taiwanese non-syndromic auditory neuropathy spectrum disorder (ANSD) cohort using a combination of sequencing technologies, predictive tools, and experimental validations.

We performed SRS to analyze OTOF variants in 65 unrelated Taiwanese patients diagnosed with non-syndromic ANSD, complemented by long-read sequencing (LRS) for haplotype phasing. A prediction-to-validation pipeline was implemented to assess the pathogenicity of cryptic variants using SpliceAI software and minigene assays.

Biallelic pathogenic OTOF variants were identified in 33 patients (50.8%), while monoallelic variants were found in five patients. Three novel variants, c.3864G > A (p.Ala1288 =), c.4501G > A (p.Ala1501Thr), and c.5813 + 2T > C, were detected. The pathogenicity of two non-canonical mis-splicing variants, c.3894 + 5G > C and c.3864G > A (p.Ala1288 =), was confirmed by minigene assays. LRS-based haplotype phasing revealed that the common missense variant c.5098G > C (p.Glu1700Gln) and the novel variant c.5975A > G (p.Lys1992Arg) are in cis and form a founder pathogenic allele in the Taiwanese population.

Our study highlights the genetic heterogeneity of DFNB9 and emphasizes the importance of population-specific variant interpretation. The integration of advanced sequencing technologies, predictive algorithms, and functional validation assays will improve the accuracy of molecular diagnosis and inform personalized treatment strategies for individuals with DFNB9.

Gene therapy shows promising potential for patients with autosomal recessive deafness 9 (DFNB9), with ongoing clinical trials (ChiCTR2200063181). A deeper understanding of changes in audiological characteristics is crucial for optimizing the monitoring and evaluation of patients' recovery post-treatment.

Audiological data were collected from 10 DFNB9 patients who underwent gene therapy, including auditory brain stem response (ABR), auditory steady-state response (ASSR), distortion product otoacoustic emission (DPOAE), and pure-tone audiometry (PTA) tests.

A clear ABR wave V was observed in all participants by 13 weeks. By 52 weeks, distinct ABR waves I and III were visible in some participants. The 1-kHz ABR wave V latency at 85 dB decreased significantly from 9.220 (range 9.015-9.810) ms at 4 weeks to 8.190 (range 7.780-8.530) ms at 52 weeks (p = 0.004), with a trend toward increased wave V amplitude (p = 0.055). Significant correlations were observed between PTA, ABR, and ASSR thresholds at 0.5-4 kHz. The DPOAE signal-to-noise ratio (SNR) at 26 weeks post-treatment showed no significant difference compared with pre-treatment SNR values, nor were there significant correlations between the pre-treatment SNR values and the post-treatment ABR thresholds.

The study demonstrates that ABR and ASSR are reliable objective tools for assessing hearing recovery in DFNB9 patients after gene therapy. ABR reveals positive changes in the auditory pathway over time after gene therapy, enhancing our understanding of the impact of gene therapy on auditory pathway recovery.

This work was funded by the National Natural Science Foundation of China.

The number of people with hearing loss disorders is enormous, causing great physical and mental stress to patients, as well as a huge social burden. Among these patients, hearing loss caused by inner ear lesions accounts for a large proportion. Therefore, treatment of the inner ear is important. Inner ear drug delivery systems, which can reduce the side effects of systemic drug administration by delivering drugs directly to the inner ear, are important in sensorineural hearing loss. Here, the development of inner ear drug delivery systems is focused, including the complex physiological structure that they face, types of drugs delivered, routes of administration, and forms of drug delivery carrier platforms. Recent studies in this process are presented and it is concluded with a summary and outlook on the problems faced and possible solutions.

Immune responses to adeno-associated virus (AAV) have long been perplexing, from its first discovery to the latest clinical trials of recombinant AAV (rAAV) therapy. Wild-type AAV (wtAAV) does not cause any known disease, making it an ideal vector for gene therapy, as viral vectors retain virus-like properties. Although AAV stimulates only a mild immune response compared with other viruses, it is still recognized by the innate immune system and induces adaptive immune responses. B cell responses against both wtAAV and rAAV are robust and can hinder gene therapy applications and prevent redosing. T cell responses can clear transduced cells or establish tolerance against gene therapy. Immune responses to AAV gene therapy are influenced by many factors. Most clinical immunotoxicities that develop in response to gene therapies have emerged as higher doses of AAV vectors have been utilized and were not properly modeled in preclinical animal studies. Thus, several strategies have been undertaken to reduce or mitigate immune responses to AAV. While we have learned a considerable amount about how the immune system responds to AAV gene therapy since the discovery of AAV virus, it still remains a curious case that requires more investigation to fully understand.

Congenital hearing loss is a major type of sensorineural deafness. Recently, Dmxl2 has been identified as a new gene associated with familial deafness. However, its role in auditory development remains unclear. This study investigated the expression and localization of DmX-like protein 2 (DMXL2), encoded by Dmxl2, in the mouse cochlea at various postnatal stages. DMXL2 was predominantly expressed in inner and outer hair cells, with the highest levels at postnatal day 7, followed by a rapid decline, nearly disappearing by day 14. To elucidate Dmxl2's function, we administered short hairpin RNA (shRNA) targeting Dmxl2 to the cochlea within 24 h post-birth, effectively knocking down its expression in the mouse inner ear. This resulted in profound hearing loss in treated mice, accompanied by disruption of development of cochlear ribbon synapses and spiral ganglion cells (SGCs). In conclusion, our study demonstrates the critical role of Dmxl2 in hearing development, suggesting it as a potential molecular target for future gene therapy in hearing loss treatment.

Recent clinical trials employing AAV (adeno-associated virus)-mediated gene therapy for hereditary deafness have demonstrated significant therapeutic potential. However, immune responses triggered by AAV delivery in the inner ear remain poorly characterized, despite their critical implications for treatment safety and efficacy.

This study systematically evaluates serotype-specific (AAV1 vs. AAV9) and promoter-dependent (CMV vs CBA) immune responses in murine cochleae following local AAV delivery.

Recombinant AAV1/AAV9 vectors expressing tdTomato under CMV or CBA promoters were injected via the posterior semicircular canal. Macrophage infiltration (F4/80+/CD68+ cells) was tracked for five weeks via immunohistochemistry.

Temporal analysis revealed inflammation onset at one week, peaking at two weeks, and resolving to baseline by five weeks. CMV-driven vectors provoked significantly stronger immune activation than CBA. Serotype comparisons showed AAV9 induced greater immunogenicity, with elevated F4/80+ cells at two weeks (p < .001) and prolonged CD68+ cell elevation through four weeks (p < .001) versus AAV1. AAV9 triggered diffuse inflammation, while AAV1 responses were modiolus-restricted.

These findings highlight serotype- and promoter-dependent immunogenicity as key determinants of cochlear inflammation. Strategic selection of AAV components is essential to balance transduction efficiency and immune tolerance in inner ear gene therapy.

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.

Neonatal cochlear Lgr5+ progenitors retain limited hair cells (HCs) regenerative capacity, but the regulatory network remains incompletely defined. Serpin family E member 2 (Serpine2) is shown to participate in regulating proliferation and differentiation of cochlear Lgr5+ progenitors in the previous in vitro study. Here, the expression pattern and in vivo roles of Serpine2 in HC regeneration are explored by transgenic mice. It is found that Serpine2 is expressed in the mouse cochlea after birth with a downward trend as the mice age. In addition, Serpine2 conditional overexpression in vivo in Lgr5+ progenitors of neonatal mice cochlea results in an increased number of ectopic HCs in a dose-dependent manner. Serpine2 knockdown ex vivo and in vivo can inhibit HC regeneration. EdU assay and lineage tracing assay demonstrate these ectopic HCs likely originate from Lgr5+ progenitors through direct transdifferentiation rather than through mitotic regeneration. Moreover, single-nucleus RNA sequencing analysis and mRNA level validation reveal that conditionally overexpressed Serpine2 likely induces HC regeneration via inhibiting sonic hedgehog (SHH) signal pathway and inducing Atoh1 and Pou4f3 transcription factor. In brief, these data indicate that Serpine2 plays a pivotal role in HC regeneration from Lgr5+ progenitors in the neonatal mouse cochlea, and this suggests a new avenue for future research into HC regeneration.

Gene therapy offers significant promise for treating inner ear disorders, but its clinical translation requires robust preclinical validation, often reliant on animal models. This review examines the role of these models in advancing gene therapeutics for inherited inner ear disorders, focusing on successes, challenges, and treatment solutions. By providing a precise understanding of disease mechanisms, these models offer a versatile preclinical platform that is essential for assessing and validating therapies. Successful gene supplementation and editing have shown potential in restoring hearing and balance functions and preventing their decline. However, challenges such as limitations in gene delivery methods, surgical access, immune responses, and discrepancies in disease manifestation between animal models and humans hinder clinical translation. Current efforts are dedicated to developing innovative strategies aimed at enhancing the efficiency of gene delivery, overcoming physical barriers such as the blood-labyrinth barrier, improving target specificity, and maximizing therapeutic efficacy while minimizing adverse immune responses. Diverse gene supplementation and editing strategies, along with evolving technologies, hold promise for maximizing therapeutic outcomes using disease relevant models. The future of inner ear gene therapeutics will hinge on personalized therapies and team science fueling interdisciplinary collaborations among researchers, clinicians, companies, and regulatory agencies to expedite the translation from bench to bedside and unlock the immense potential of precision medicine in the inner ear.

GJB2 gene is a common pathogenic gene for non-syndromic hearing loss, located on chromosome 13q12.11, and primarily encodes connexin 26 (Cx26). Cx26, a member of the gap-junction protein family, is mainly expressed in the supporting cells of the cochlea, where it is responsible for intercellular material transfer and signal exchange. Gene therapy, a treatment method that repairs or reconstructs genetic material, has emerged as the most effective approach for hereditary hearing loss. During the initial stages of exploration, researchers need to conduct animal experiments first. By elucidating the mechanisms of GJB2 gene-induced congenital hearing loss, we summarize the commonly used experimental animals (zebrafish, mice) for current research on the Gjb2 gene, and further promote the advancement of gene therapy strategies.

Adeno-associated virus (AAV)-based gene therapy is emerging as a promising treatment for deafness and vestibular deficits, due to the variety of available serotypes that offer a large range of cell targeting capabilities. Nevertheless, the tropism of these AAV serotypes for sensory inner ear cells varies greatly as the cochlea matures, presenting a significant burden for successful preclinical trials. Therefore, identifying serotypes with strong tropism for cochlear and vestibular hair cells during key stages of development in mouse inner ear, the most widely used preclinical model, is essential for advancing clinical applications. We conducted a comparative analysis of the cellular tropism and hair-cell transduction rates of four AAV serotypes in the cochlea and vestibular organs during maturation. We used AAV2, AAV8, AAV9-PHP.eB, and Anc80L65 at the embryonic, neonatal, and adult stages. Our results indicate that the cell transduction rate of these four serotypes varies with age. Notably outer hair cells were mostly targeted during the embryonic stage, inner hair cells were primarily transduced principally at the mature stage, and vestibular hair cells were the most permissive at the neonatal stage. These results provide new insights for preclinical gene therapy studies for the inner ear with potential implications for therapeutic outcomes.

The Universal Neonatal Hearing Screening (UNHS) program is crucial for the early detection and treatment of hearing impairment in newborns. Poland has successfully implemented a nationwide UNHS program, adhering to international standards. Research indicates that hearing loss affects approximately 2-4 per 1000 infants, with sensorineural hearing loss being the most prevalent. Major risk factors include genetic alterations, craniofacial anomalies, prematurity, hyperbilirubinemia, and congenital infections such as cytomegalovirus. Despite the program's success, challenges related to limited parental awareness and disparities in access highlight the need for continuous improvement in screening and follow-up procedures. Additionally, gene therapy is emerging as a promising treatment for hearing loss. While still experimental, gene therapy could become a key complementary treatment option in the future, offering new hope for those with hearing impairments.

The inner ear is a relatively isolated organ, protected by the blood-labyrinth barrier (BLB). This barrier creates a unique lymphatic fluid environment within the inner ear, maintaining a stable physiological state essential for the mechano-electrical transduction process in the inner ear hair cells while simultaneously restricting most drugs from entering the lymphatic fluid. Under pathological conditions, dysfunction of the stria vascularis and disruption in barrier structure can lead to temporary or permanent hearing impairment. This review describes the structure and function of the BLB, along with recent advancements in modeling and protective studies related to the BLB. The review emphasizes some newly developed non-invasive inner ear drug delivery strategies, including ultrasound therapy assisted by microbubbles, inner ear-targeting peptides, sound therapy, and the route of administration of the cerebrospinal fluid conduit. We argue that some intrinsic properties of the BLB can be strategically utilized for effective inner ear drug delivery.

Degeneration of the cochlear spiral ganglion neurons (SGNs) is one of the major causes of sensorineural hearing loss and significantly impacts the outcomes of cochlear implantation. Functional regeneration of SGNs holds great promise for treating sensorineural hearing loss. In this study, we systematically screened 33 transcriptional regulators implicated in neuronal and SGN fate. Using gene expression array and principal component analyses, we identified a sequential combination of Ascl1, Pou4f1 and Myt1l (APM) in promoting functional reprogramming of SGNs. The neurons induced by APM expressed mature neuronal and SGN lineage-specific markers, displayed mature SGN-like electrophysiological characteristics and exhibited single-cell transcriptomes resembling the endogenous SGNs. Thus, transcription factors APM may serve as novel candidates for direct reprogramming of SGNs and hearing recovery due to SGN damages.

Connexins are essential for cellular communication and play a critical role in various physiological processes, including hearing. Connexin26 (Cx26), encoded by the GJB2 gene, is a key component of cochlear gap junctions and is vital for potassium recycling and ATP release-both of which are vital for auditory function. Mutations in GJB2 are the primary cause of sensorineural hearing loss. However, the phenotypic variability observed in individuals with the same mutation suggests the involvement of other complex regulatory factors. While the regulatory mechanisms of Connexin43 have been extensively studied, research on the mechanisms of Cx26 remains limited. This review summarizes the reported regulatory mechanisms of GJB2 from multiple perspectives, both pre- and post-transcription, in an effort to explore ways to regulate connexin expression and provide new insights into gene therapy for diseases caused by alterations in connexin levels.

Mutations in the gap junction β2 (GJB2) gene, which encodes connexin 26, are the leading cause of genetic deafness. These mutations are characterized by the degeneration and fragmentation of gap junctions and gap junction plaques (GJPs) composed of connexin 26. Dominant-negative mutations of GJB2, such as R75W, cause syndromic hearing loss and palmoplantar keratoderma. We previously reported that the R75W mutation, a single-base substitution where C is replaced by T, causes fragmentation of GJPs. Therefore, an adenine base editor (ABE), which enables A-to-G base conversions, can potentially be useful for the treatment of this genetic disease. Here, we report that an all-in-one adeno-associated virus (AAV) vector, which includes a compact ABE (SaCas9-NNG-ABE8e) with broad targeting range, and a sgRNA targeting the R75W mutation in GJB2 corrected this pathogenic mutation and facilitated the recovery of the gap junction intercellular communication network of GJPs. In a transgenic mouse model with the GJB2 R75W mutation, AAV-mediated base editing also restored the fragmented GJPs to orderly outlines in cochlear supporting cells. Our findings suggest that an ABE-based base-editing strategy could be an optimal treatment for the dominant form of GJB2-related hearing loss, GJB2-related skin diseases, and other deafness-related mutations, especially single-base substitutions.

Early intervention for sensorineural hearing loss (SNHL) in childhood is crucial for auditory and language development. In recent years, innovative auditory stimulation techniques and speech therapy strategies, such as middle ear implants, cochlear implants, auditory brainstem implants, and midbrain implants, have provided new avenues for improving patient outcomes. Additionally, basic research advancements in cell reprogramming and regeneration, stem cell therapy, and targeted drug delivery offer promising approaches to meet the individualized needs of children with SNHL. However, many challenges and unresolved issues remain in the treatment of SNHL. This article comments on the case report, which describes a female pediatric patient with SNHL who underwent foot reflexology which led to the normalization of hearing thresholds. Reflexology is considered to have potential benefits in physical rehabilitation, but its efficacy in hearing restoration requires further scientific validation through rigorous clinical trials and large-scale prospective studies.

Hearing loss represents a highly prevalent and debilitating sensory disorder affecting roughly one in five people worldwide. In a majority of patients with congenital hearing loss, genetic mutations cause the disease. Up until recently, therapeutic options for individuals with hearing loss were limited to hearing aids and different types of auditory implants. However, after numerous years of intensive basic and translational research, gene therapy strategies are now being investigated in clinical trials. First results show significant hearing improvement in treated patients, highlighting gene therapy's role as a promising treatment for certain forms of genetic hearing loss. In this article, we provide an overview of genetic hearing loss and inner ear gene therapy research including relevant strategies that have been established in animal models and will likely be investigated in human patients soon. Furthermore, we summarize and contextualize the novel findings of recently completed and ongoing clinical trials, and discuss future hurdles needed to be overcome to allow for a broad and safe clinical application of inner ear gene therapy.

Auditory neuropathy (AN) is an under-recognized form of hearing loss characterized by lesions in inner hair cells (IHCs), ribbon synapses and spiral ganglion neurons (SGNs). The lack of a targeted therapy for AN has increased the need for a better understanding of the pathogenic mechanism of AN. As mitogen-activated protein kinase (MAPK) signaling is ubiquitous in many biological processes, its alteration may facilitate the pathogenesis of multiple sites in AN. Here, we summaries the characteristics of AN under different molecular bases and first explore the mechanism of MAPK at different lesion sites. Alterations of extracellular signal-regulated kinase (ERK)/MAPK occur in IHCs and SGNs, whereas modulations of p38 and c-Jun NH2-terminal kinase (JNK) were found in ribbon synapses and SGNs. In conclusion, inductive MAPK alterations in the pathogenesis and development of AN are likely to represent a potential therapeutic target to guide the development of treatments.

Achieving cell-specific gene expression is crucial in the design of safe and efficacious gene therapies for the treatment of sensorineural hearing loss. Although a variety of adeno-associated virus (AAV) serotypes have been used to deliver genes to inner ear hair cells, few serotypes currently allow specific targeting of supporting cells. We sought to specifically target supporting cells by combining an AAV serotype with high tropism for the inner ear with enhancer sequences from the supporting cell-specific gene Lunatic Fringe (Lfng). We identified three candidate Lfng enhancer sequences using bioinformatic analysis to identify accessible chromatin and histone marks associated with active transcription of the Lfng locus in supporting cells. Candidate Lfng enhancers or the ubiquitous CBh promoter driving an EGFP reporter gene were packaged into the AAV-ie capsid, and the virus was introduced into the inner ear of neonatal mice. AAV-CBh-EGFP transduced multiple sensory and non-sensory inner ear cell types, as well as cells in the brain. One of the three Lfng enhancers gave robust EGFP expression in border cells, inner phalangeal cells, pillar cells, and all three rows of Deiters' cells along the entire cochlear duct, as well as in vestibular organ supporting cells. Significantly, no fluorescently labeled cells were detected in the brains of mice injected with this virus. We further designed an AAV-Lfng-CreERT2 vector that drove strong recombination in Cre reporter mice supporting cells after tamoxifen treatment. Our results provide a tool to specifically target supporting cells of the juvenile and adult inner ear.

Hearing loss is the most common sensory deficit in humans, and roughly half of childhood-onset sensorineural hearing loss is genetic. Advances in gene therapy techniques have led to the first clinical trials for OTOF-associated hearing loss DFNB9. Therapies for other hearing loss genes are in various stages of development, and therefore a comprehensive evaluation of potential candidate genes can help to prioritize and guide these efforts.

A list of 93 nonsyndromic hearing loss genes with consensus support was generated. Critical factors for evaluation were identified as gene size, timing of cochlear degradation, cell type(s) of primary expression, availability of mouse models and efficacy of adeno-associated virus experiments in those mice, and human hearing loss severity, onset, and prevalence. Each factor was addressed with gene-specific PubMed searches for applicable studies.

Each gene was evaluated according to the above factors, with favorable results indicating the most promising candidates for gene therapy. Genes that satisfied all the above conditions included TMPRSS3, PCDH15, and TMC1. Other genes, such as LOXHD1 and MYO6, had not yet had gene replacement attempts in a mouse model but otherwise satisfied all conditions and were likewise identified as promising candidates.

Based on this analysis, hearing loss genes vary widely in terms of their favorability for treatment by gene therapy approaches. Targeting development efforts to promising candidates will ensure the highest likelihood of clinical success. Several genes were identified as appealing next targets, signaling an increasing role of gene therapies in hearing loss care moving forward.

For more than 20 years there has been speculation about a future in which newborns are routinely screened at birth for genetic disorders using genome sequencing, but prospective large-scale studies assessing this vision have only recently begun. Genome sequencing may provide a means of expanding the scope of conditions included in newborn screening programs and improving the positive predictive value of traditional newborn screening. However, the use of genome sequencing for newborn screening has also raised concerns including acceptability, equity, and scalability. By reviewing the initial results of the GUARDIAN study and contrasting them with other pilot studies investigating the use of genome sequencing for large-scale newborn screening, we highlight how the lessons learned from these studies are shaping the future for the implementation of truly universal and equitable newborn genomic screening.