感音神经性聋主要由内耳毛细胞不可逆损失导致。内耳Lgr5阳性细胞具有干细胞特性，能在多种信号的调控下再生毛细胞。研究表明，单独调控某些基因的表达如Atoh1等并不能促进功能性毛细胞再生。因此，如何使新生毛细胞功能成熟并长期存活是耳科领域核心和亟待解决的关键科学问题。申请人近期改造出一种能安全高效地感染内耳干细胞的新型腺相关病毒AAV-ie，为通过基因治疗促进功能性毛细胞再生提供了有效途径。因此，本项目在前期研究基础上，拟通过AAV-ie协同调控Gfi1，Pou4f3，Atoh1（GPA），Espin和DKK3这5个信号通路促进内耳干细胞再生功能性毛细胞，重建听觉功能。拟实现目标：一、体外研究基因协同调控对内耳干细胞的影响；二、建立新霉素听力损失小鼠模型，协同调控这五个信号通路；三、研究新生毛细胞结构、功能及听觉功能的恢复。本项目将为临床上利用基因治疗和干细胞再生恢复听觉功能奠定研究基础。

Sensorineural hearing loss is mainly caused by irreversible loss of cochlea hair cells. Lgr5+ supporting cells in the sensory epithelia of cochlea are inner ear stem cells, which could be regulated to regenerate hair cells by co-regulation of multiple genes. Studies have shown that the regulation of single alone gene such as Atoh1 does not promote functional hair cell regeneration. Therefore, how to get the functional regenerated hair cells is a key scientific question in the field of hearing research at present. Recently, the applicant modified a new adeno-associated virus (AAV-ie, Nat Comm, 2019) with safe and high efficiency in inner era stem cells, which provides an effective approach to regenerate functional hair cells through gene therapy. Based on the previous study, this project focuses on the co-regulation of Gfi1, Pou4f3, Atoh1 (GPA), Espin and Dkk3 genes in inner ear stem cells by AAV-ie to regenerate functional hair cells and therefore improve hearing ability. In this project we propose to fulfill the following aims: 1. To study the effect of genes co-regulation on inner ear stem cells in vitro; 2. Establish mouse models of hearing loss induced by neomyosin treatment，and further study the effect of five genes co-regulation on the proliferation and differentiation of inner ear stem cells and the maturation and survival of new-born hair cells. 3. To study the function of regenerated hair cells and to evaluate the function recovery of auditory system through gene therapy. This project will lay the foundation for clinical application of gene therapy and hair cell regeneration to restore auditory function.