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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 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.