CRISPR/Cas9介导OTOF基因相关听神经病谱系障碍患者特异性iPSC的基因矫正研究

听神经病谱系障碍（ANSD）是近二十余年才为人们所认识的一种特殊类型听力障碍。OTOF基因缺陷是ANSD重要病因之一，我国近半数先天性ANSD患者可检出该基因突变，但其致病机制未完全明确，且缺乏有效对因治疗手段。申请人前期构建了OTOF相关ANSD患者特异性诱导多能干细胞（OTOF/A-iPSC），能体外定向诱导分化为内耳细胞，可望成为OTOF相关ANSD发病机制研究和治疗方法探索的理想人源细胞模型。本项目拟将OTOF/A-iPSC进一步向内耳细胞定向诱导分化，3D培养内耳类器官，体外形成带状突触样结构，模拟人类OTOF相关ANSD发病过程，阐明OTOF基因缺陷引起ANSD的确切机制。结合CRISPR/Cas9基因编辑技术，精确矫正OTOF/A-iPSC中OTOF致病突变，实现iPSC源内耳带状突触的功能修复，为未来OTOF相关ANSD的基因治疗和干细胞移植治疗提供理论依据和技术支撑。

Auditory neuropathy spectrum disorder (ANSD) is a special type of hearing disorder that has received little attention until the last two decades. OTOF gene defect is one of the most common causes of ANSD. In the Chinese population, OTOF gene mutations can be detected in nearly half of the patients with congenital ANSD. However, the exact pathogenesis of OTOF defects that causing ANSD is not completely clear and there is a lack of effective etiological therapy. Previously, we generated OTOF-related ANSD patient-specific induced pluripotent stem cells (OTOF/A-iPSCs). The OTOF/A-iPSCs could be differentiated into inner ear cells in vitro, which was expected to be an ideal human cell model for the elucidation of OTOF-related ANSD and the exploration of therapeutic methods. To clarify the exact pathogenesis of OTOF-related ANSD, we will further differentiate the OTOF/A-iPSCs into inner ear cells, generate inner ear organoids in 3D culture, establish ribbon synapse-like connections, and model human OTOF-related ANSD in vitro. Combining CRISPR/Cas9 gene editing technology, we plan to accurately correct OTOF pathogenic mutation(s) in OTOF/A-iPSCs and rescue the dysfunction of iPSC-derived inner ear ribbon synapses. The implementation of this research will provide a theoretical basis and technical support for future gene therapy and stem cell transplantation therapy of OTOF-related ANSD.