MITF基因突变致Waardenburg综合征耳聋的基因治疗研究

Waardenburg综合征（WS）是临床上常见的遗传性综合征型耳聋，是由神经嵴发育不全导致。其临床表型、致病基因及发病机制等研究已取得公认的进展，然而如何实施有效的治疗仍是亟待解决的问题。基于基因编辑和iPSCs的技术为WS耳聋基因治疗提供了新思路。MITF是神经嵴分化黑色素细胞的总调控因子，该基因突变与WS患者耳聋密切相关；故本研究选择MITF基因作为WS耳聋基因治疗研究的靶基因。前期研究基础上开展如下工作：构建Mitf p.R217del KI小鼠，观察小鼠内耳病理生理改变；构建携带MITF p.R217del突变等WS患者特异性iPSCs，CRISPR/Cas9体外基因修复后定向诱导黑色素细胞；基因修复后细胞导入Mitf突变小鼠内耳以挽救其毛细胞功能和恢复听力。本研究有望为WS耳聋基因治疗选择合适的靶基因和内耳靶细胞提供实验依据，并为下一步直接内耳导入iPS源毛细胞打下基础。

Waardenburg syndrome (WS) is a rare genetic condition characterized by syndromic hereditary hearing loss and caused by hypoplasia of neural crest (NC) in cochlea. Great progress has been made in identification the phenotype, pathogenic mutations and pathogenesis. However it is still a challenge to treat this disease effectively. Recently, CRISPR/Cas9 mediated gene editing in induced pluripotent stem cells (iPSCs) has shed a light on gene therapy of hearing loss in WS. MITF is a major regulator involved in differentiation and development of NC cells, and it plays a very important role in the survival, migration, differentiation and development of melanocytes. The majority of WS2 patients carry mutations in MITF gene. So we choose MITF as a model to study the pathogenesis and gene therapy of WS in this proposal. Based on our previous results, we will create Mitf p.R217del Knockin mice and study the pathogenesis of inner ear in mouse model. In addition, we will generate MITF p.R217del and other WS patients' iPSCs from patients separately, repair them with CRISPR/Cas9 and induce them to melanocytes in vitro. Finally, we will try to restore the function in inner ear by injection repaired melanocytes in mouse cochlear. This project is expected to give us ideas on how to choose target genes and corresponding cells in inner ear and will be the fundamental for transplantation iPSCs in future.