渐进性非综合征型遗传性耳聋的分子和治疗研究

Genetic factors play an important role in progressive hearing loss (HL). Autosomal dominant non-syndromic HL is the most common inheritance mode in progressive HL. Treatment for HL is very limited. There is an urgent need to conduct preclinical gene or cell based therapeutic studies. We have identified P2RX2 as a new gene for autosomal dominant HL DFNA41. P2RX2 receptor is expressed in hair cell (HC)stereocilia and other cell types of the cochlea and plays an important role in the sound transduction and neurotransmission in the inner ear, but its effects on the mechanism of hair cells remains unclear. Moreover, as shown in our preliminary studies for this proposal, we have generated human P2rx2 knockin mouse model and generated the mutant iPSCs from patients with the P2RX2 V61L mutation in the DFNA41 family. The observed deafness phenotype in the ki mice is similar to the pattern of human DFNA41 deafness families and suggest that V61L mutant mice are an ideal model to study human DFNA41 deafness and to conduct preclinical treatment studies. In this study, the electrophysiology and morphological examination of the inner ear of P2rx2 mutant mice will be performed to clarify the effect of P2RX2 on the inner ear function of mice. Importantly, we will use CRISPR /Cas9 gene editing technique to restore the hearing of P2rx2V61L / + deaf mice. In the meantime, we will correct heterozygous P2RX2 mutations in the iPSCs generated from P2RX2 deaf patients by CRISPR/Cas9-mediated genome editing. Our study will provide preclinical experimental basis for the development of new treatments for HL. This is a highly translational research proposal based on strong preliminary data from world class research team.

常染色体显性非综合征型渐进性耳聋与遗传因素密切相关，治疗手段十分有限，故使用动物模型进行病人相关的基因和细胞临床前期治疗研究十分重要。我们前期研究鉴定了第一个与年龄及噪声相关的渐进性耳聋基因P2RX2，体外研究表明其在内耳声音的转导和转化中可能起到重要作用，但其影响毛细胞的具体机制仍不清楚。我们进一步构建了P2rx2V61L突变KI小鼠并表现出了人类相同突变携带者的耳聋特征，据此，我们提出P2rx2小鼠模型是研究人类DFNA41耳聋和寻找治疗方式的理想模型。本研究将完成P2rx2 突变小鼠的内耳电生理与形态检测，以明确P2RX2对内耳功能的影响。更重要的是将运用CRISPR/cas9基因编辑技术，进行P2rx2V61L/+耳聋小鼠的听力恢复和P2RX2突变患者来源的iPSCs基因矫正研究，分别从基因和细胞水平探索渐进性耳聋的基因治疗，为以后临床上开发新的耳聋基因和细胞治疗方式提供实验依据

常染色体显性非综合征型渐进性耳聋与遗传因素密切相关，治疗手段十分有限，故使用动物模型进行病人相关的基因和细胞临床前期治疗研究十分重要。我们前期研究鉴定了第一个与年龄及噪声相关的渐进性耳聋基因P2RX2，相关研究表明其在内耳声音的转导和转化中可能起到重要作用，但其具体致病机制仍不清楚。基于此，本课题通过构建P2rx2 p.V61L突变knock-in小鼠模型，进一步探索P2RX2基因的致聋机制。结果发现P2rx2 p.V61L knock-in小鼠忠实地再现了人类患者的听力表型，即早发的、双侧的、常染色体显性遗传的、渐进性加重的感音神经性耳聋，以及在发病早期具有性别差异，因此该小鼠模型是研究遗传性非综合征型耳聋的理想动物模型。此外，P2rx2 p.V61L knock-in小鼠模型表现出前庭功能障碍和对疼痛的超敏性，因此该小鼠模型也是研究神经系统功能和机制的理想动物模型。内耳形态学研究显示P2rx2 p.V61L knock-in小鼠的内毛细胞退化和带状突触迁移逐渐加重，说明P2rx2对内毛细胞和带状突触的发育和维持至关重要。我们进一步构建P2RX2 p.V60L的iPSCs，突变的iPSCs与正常iPSCs相比在诱导毛细胞样细胞过程中没有显示出差异，这与在动物模型上得到的结论相似。进一步的研究正在进行中。本研究深入分析了P2RX2的致聋机制，为以后开展基因治疗提供研究方式和实验依据。