Jieyu Qi

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.

Irreversible damage to hair cells (HCs) in the cochlea leads to hearing loss. Cochlear supporting cells (SCs) in the murine cochlea have the potential to differentiate into HCs. Neuron membrane glycoprotein M6B (Gpm6b) as a four‐transmembrane protein is a potential regulator of HC regeneration according to our previous research. In this study, we found that AAV‐ie‐mediated Gpm6b overexpression promoted SC‐derived organoid expansion. Enhanced Gpm6b prevented the normal decrease in SC plasticity as the cochlea develops by supporting cells re‐entry cell cycle and facilitating the SC‐to‐HC transformation. Also, overexpression of Gpm6b in the organ of Corti through the round window membrane injection facilitated the trans‐differentiation of Lgr5⁺ SCs into HCs. In conclusion, our results suggest that Gpm6b overexpression promotes HC regeneration and highlights a promising target for hearing repair using the inner ear stem cells combined with AAV.

Sensorineural hearing loss (SNHL), a highly prevalent sensory impairment, results from a multifaceted interaction of genetic and environmental factors. As we continually gain insights into the molecular basis of auditory development and the growing compendium of deafness genes identified, research on gene therapy for SNHL has significantly deepened. Adeno-associated virus (AAV), considered a relatively secure vector for gene therapy in clinical trials, can deliver various transgenes based on gene therapy strategies such as gene replacement, gene silencing, gene editing, or gene addition to alleviate diverse types of SNHL. This review delved into the preclinical advances in AAV-based gene therapy for SNHL, spanning hereditary and acquired types. Particular focus is placed on the dual-AAV construction method and its application, the vector delivery route of mouse inner ear models (local, systemic, fetal, and cerebrospinal fluid administration), and the significant considerations in transforming from AAV-based animal model inner ear gene therapy to clinical implementation.

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 OTOF^{p.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.

Hair cells (HCs) in mammals cannot spontaneously regenerate after damage. Atoh1 overexpression can promote HC regeneration in the postnatal cochlea, but the regenerated HCs do not possess the structural and functional characteristics of HCs in situ. The stereocilia on the apical surface of HCs are the first‐level structure for sound conduction, and regeneration of functional stereocilia is the key basis for the reproduction of functional HCs. Espin, as an actin bundling protein, plays an important role in the development and structural maintenance of the stereocilia. Here, we found that the upregulation of Espin by AAV‐ie was able to induced the aggregation of actin fibres in Atoh1‐induced HCs in both cochlear organoids and explants. In addition, we found that persistent Atoh1 overexpression resulted in impaired stereocilia in both endogenous and newly formed HCs. In contrast, the forced expression of Espin in endogenous and regenerative HCs was able to eliminate the stereocilia damage caused by persistent Atoh1 overexpression. Our study shows that the enhanced expression of Espin can optimize the developmental process of stereocilia in Atoh1‐induced HCs and can attenuate the damage to native HCs induced by Atoh1 overexpression. These results suggest an effective method to induce the maturation of stereocilia in regenerative HCs and pave the way for functional HC regeneration via supporting cell transdifferentiation.

Sensorineural hearing loss a result from hair cell damage, which is irreversible in mammals owing to the lack of hair cell regeneration, but recent researches have shown that Lgr5⁺ supporting cells are progenitors capable of regenerating hair cells. RPS14 (ribosomal protein S14) is a 40S ribosomal subunit component and is associated with erythrocyte differentiation, and in this study, we used a novel adeno‐associated virus‐inner ear system to upregulate Rps14 expression in cultured hair cell progenitors and observed an enhancement on their ability to proliferate and differentiate into hair cells. Similarly, Rps14 overexpression in the mice cochlea could promote supporting cells proliferation by activating the Wnt signalling pathway. In addition, over‐expressing Rps14 induced hair cells regeneration in the organ of Corti, and lineage tracing showed that the new hair cells had transformed from Lgr5⁺ progenitors. In conclusion, our analysis reveals the potential role of Rps14 in driving hair cell regeneration in mammalian.

Mechanosensitive hair cells (HCs) in the cochlear sensory epithelium are critical for sound detection and transduction. Mammalian HCs in the cochlea undergo cytogenesis during embryonic development, and irreversible damage to hair cells postnatally is a major cause of deafness. During the development of the organ of Corti, HCs and supporting cells (SCs) originate from the same precursors. In the neonatal cochlea, damage to HCs activates adjacent SCs to act as HC precursors and to differentiate into new HCs. However, the plasticity of SCs to produce new HCs is gradually lost with cochlear development. Here, we delineate an essential role for the guanine nucleotide exchange factor Net1 in SC trans-differentiation into HCs. Net1 overexpression mediated by AAV-ie in SCs promoted cochlear organoid formation and HC differentiation under two and three-dimensional culture conditions. Also, AAV-Net1 enhanced SC proliferation in Lgr5-EGFP^CreERT2 mice and HC generation as indicated by lineage tracing of HCs in the cochleae of Lgr5-EGFP^CreERT2/Rosa26-tdTomato^loxp/loxp mice. We further found that the up-regulation of Wnt/β-catenin and Notch signaling in AAV-Net1-transduced cochleae might be responsible for the SC proliferation and HC differentiation. Also, Net1 overexpression in SCs enhanced SC proliferation and HC regeneration and survival after HC damage by neomycin. Taken together, our study suggests that Net1 might serve as a potential target for HC regeneration and that AAV-mediated gene regulation may be a promising approach in stem cell-based therapy in hearing restoration.