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Abstract Mammalian cochlear hair cells (HCs) are essential for hearing, and damage to HCs results in severe hearing impairment. Damaged HCs can be regenerated by neighboring supporting cells (SCs), thus the functional regeneration of HCs is the main goal for the restoration of auditory function in vivo. Here, cochlear SC trans‐differentiation into outer and inner HC by the induced expression of the key transcription factors Atoh1 and its co‐regulators Gfi1, Pou4f3, and Six1 (GPAS), which are necessary for SCs that are destined for HC development and maturation via the AAV‐ie targeting the inner ear stem cells are successfully achieved. Single‐cell nuclear sequencing and lineaging tracing results showed that the majority of new Atoh1‐derived HCs are in a state of initiating differentiation, while GP (Gfi1, Pou4f3) and GPS (Gfi1, Pou4f3, and Six1) enhanced the Atoh1‐induced new HCs into inner and outer HCs. Moreover, the patch‐clamp analysis indicated that newborn inner HCs induced by GPAS forced expression have similar electrophysiological characteristics to those of native inner HCs. Also, GPAS can induce HC regeneration in the HC‐damaged mice model. In summary, the study demonstrates that AAV‐mediated co‐regulation of multiple genes, such as GPAS, is an effective means to achieve functional HC regeneration in the mouse cochlea.

Abstract Hereditary deafness is a common neurosensory disorder, and 148 non‐syndromic deafness genes have been identified to date. Gene therapy has been used to treat a variety of genetic diseases, but no gene therapy drug for hereditary deafness has been approved for clinical use. At present, several clinical trials of gene therapy for hereditary deafness are underway. However, few normative documents have been issued to guide the standardization of gene therapy for hearing loss, and this document is the first global gene therapy guideline for hereditary hearing loss. The guidelines were jointly developed and drafted by experienced audiologists, virologists and biologists who are vigorously involved in inner ear gene therapy research in the Hearing, Speech and Communication Subsociety of Biophysical Society of China, Audiology Development Foundation Of China and Audiology Subsociety of Jiangsu Medical Association. These guidelines cover preclinical research and clinical practice of gene therapy for hereditary deafness, including indications, key points of pre‐clinical research, patient selection criteria, pre‐clinical preparation, drug efficacy, drug safety evaluation criteria, ethical review, etc. We hope that the guidelines will promote the standardization of clinical practice related to gene therapy for hereditary deafness in China and around the world.

Abstract Mutations in OTOFERLIN (OTOF) lead to the autosomal recessive deafness 9 (DFNB9). The efficacy of adeno‐associated virus (AAV)‐mediated OTOF gene replacement therapy is extensively validated in Otof‐deficient mice. However, the clinical safety and efficacy of AAV‐OTOF is not reported. Here, AAV‐OTOF is generated using good manufacturing practice and validated its efficacy and safety in mouse and non‐human primates in order to determine the optimal injection dose, volume, and administration route for clinical trials. Subsequently, AAV‐OTOF is delivered into one cochlea of a 5‐year‐old deaf patient and into the bilateral cochleae of an 8‐year‐old deaf patient with OTOF mutations. Obvious hearing improvement is detected by the auditory brainstem response (ABR) and the pure‐tone audiometry (PTA) in these two patients. Hearing in the injected ear of the 5‐year‐old patient can be restored to the normal range at 1 month after AAV‐OTOF injection, while the 8‐year‐old patient can hear the conversational sounds. Most importantly, the 5‐year‐old patient can hear and recognize speech only through the AAV‐OTOF‐injected ear. This study is the first to demonstrate the safety and efficacy of AAV‐OTOF in patients, expands and optimizes current OTOF‐related gene therapy and provides valuable information for further application of gene therapies for deafness.

Abstract OTOF mutations are the principal causes of auditory neuropathy. There are reports on Otof‐related gene therapy in mice, but there is no preclinical research on the drug evaluations. Here, Anc80L65 and the mouse hair cell‐specific Myo15 promoter (mMyo15) are used to selectively and effectively deliver human OTOF to hair cells in mice and nonhuman primates to evaluate the efficacy and safety of OTOF gene therapy drugs. A new dual‐AAV‐OTOF‐hybrid strategy to transfer full‐length OTOF is generated, which can stably restore hearing in adult OTOFp.Q939*/Q939* mice with profound deafness, with the longest duration being at least 150 days, and the best therapeutic effect without difference in hearing from wild‐type mice. An AAV microinjection method into the cochlea of cynomolgus monkeys without hearing impairment is further established and found the OTOF can be safely and effectively driven by the mMyo15 promoter in hair cells. In addition, the therapeutic dose of AAV drugs has no impact on normal hearing and does not cause significant systemic toxicity both in mouse and nonhuman primates. In summary, this study develops a potential gene therapy strategy for DFNB9 patients in the clinic and provides complete, standardized, and systematic research data for clinical research and application.

Sensory hair cells are responsible for detecting and transmitting sound in the inner ear, and damage to HCs leads to hearing loss. HCs do not regenerate spontaneously in adult mammals, which makes the hearing loss permanent. However, hair cells and supporting cells have the same precursors in the inner ear, and in newborn mice, the adjacent SCs can be activated by gene manipulation to differentiate into newly regenerated hair cells. Here, we demonstrate the role of the Ras association domain family member 2 (Rassf2) in supporting cell to hair cell trans-differentiation in the inner ear. Using the AAV vector (AAV-ie) to upregulate Rassf2 expression promoted supporting cell division and hair cell production in cultured cochlear organoids. Also, AAV-Rassf2 enhanced the regenerative ability of Lgr5+ SCs in the postnatal cochlea without impairing hearing, and this might due to the modulation of the Wnt, Hedgehog and Notch signaling pathways. Furthermore, AAV-Rassf2 enhances cochlear supporting cell division and hair cell production in the neomycin injury model. In summary, our results suggest that Rassf2 is a key component in HC regenerative repair, and gene modulation mediated by adeno-associated virus may be a promising gene therapy for hearing repair.

Abstract The cochlea consists of multiple types of cells, including hair cells, supporting cells and spiral ganglion neurons, and is responsible for converting mechanical forces into electric signals that enable hearing. Genetic and environmental factors can result in dysfunctions of cochlear and auditory systems. In recent years, gene therapy has emerged as a promising treatment in animal deafness models. One major challenge of the gene therapy for deafness is to effectively deliver genes to specific cells of cochleae. Here, we screened and identified an AAV-ie mutant, AAV-ie-K558R, that transduces hair cells and supporting cells in the cochleae of neonatal mice with high efficiency. AAV-ie-K558R is a safe vector with no obvious deficits in the hearing system. We found that AAV-ie-K558R can partially restore the hearing loss in Prestin KO mice and, importantly, deliver Atoh1 into cochlear supporting cells to generate hair cell-like cells. Our results demonstrate the clinical potential of AAV-ie-K558R for treating the hearing loss caused by hair cell death.