P2RX2突变致聋患者iPS细胞基因修复及内耳移植治疗P2RX2敲除致聋小鼠的研究

Heredity is the most common cause of hearing loss, the treatment of hereditary hearing loss is still a huge challenge to otolaryngologist. The discovery of iPS cells ushered in a new age in the field of treatment. iPS cells have the ability to differentiate into inner ear cells. Hence, we put forward a hypothesis: The treatment of hereditary hearing loss patients with gene correction and inner ear transplantation of autologous iPS cells is effective. In previous studies, we finished the generation of iPS cells from hereditary hearing loss patients with P2RX2 mutations and the directional differentiation to hair cell and spiral neurons, we discovered the deafness mechanism of P2RX2 mutations from the cellular level, and explored the influence of hearing with the number and transplantation ways of iPS cells in mice. We will complete the gene correction of iPS cells from patients with P2RX2 mutations by CRISPR/Cas9 technology, the differentiation of iPSC cells into inner ear cells after gene correct, and the treatment of P2RX2 knockout mice by inner ear transplantation of this inner ear cells. The completion of this project will reveal preliminarily the biology function of iPS cells after gene repair and inner ear directional differentiation, explore the effectiveness of treatment, and lay the foundation for treatment of hereditary hearing loss with iPS cells.

遗传性耳聋是先天性耳聋的最常见病因，目前从根本上治疗十分困难，而诱导多能干细胞（iPSC）的发现为治疗提供了新思路。iPSC具有分化为内耳细胞的能力，因此我们提出通过基因修复突变患者iPSC，并向内耳细胞定向分化后内耳移植能治疗遗传性耳聋的假说。我们前期完成了P2RX2突变致聋患者iPSC细胞建系及向螺旋神经元及毛细胞定向分化的研究，从细胞层面验证其致聋机制，探索了干细胞内耳移植数目与途径对小鼠听力的影响。本项目将通过CRISPR/Cas9技术基因修复P2RX2突变致聋患者iPSC，并将iPSC向螺旋神经元及毛细胞定向分化，再分别由蜗轴和鼓阶途径移植入P2RX2敲除致聋小鼠内耳，对移植后细胞标记示踪，检测移植后细胞形态功能重塑情况和小鼠听力学指标。本项目的完成将初步揭示iPSC基因修复后内耳定向分化生物学功能恢复情况，探索iPSC内耳移植治疗耳聋的有效性，为iPSC治疗遗传性耳聋奠定基础。

耳聋是人类最常见的感知障碍疾病之一，超过2/3的语前聋患者能找到遗传因素，在当前信息社会中，正常的听觉是人们生活、学习和社会交往必不可少的。目前主要治疗遗传性耳聋的方法是人工耳蜗植入手术，但没有重建损伤的听觉生理结构，仍有部分患者无法通过恢复听力。而诱导多能干细胞（iPSC）的发现为治疗提供了新思路。iPSC具有分化为内耳细胞的能力，因此我们提出通过基因修复突变患者iPSC，并向内耳细胞定向分化后内耳移植能治疗遗传性耳聋的假说。本项目通过CRISPR/Cas9技术基因修复P2RX2突变致聋患者iPSC，并将iPSC向螺旋神经元及毛细胞定向分化，再分别由蜗轴和鼓阶途径移植入P2RX2敲除致聋小鼠内耳，对移植后细胞标记示踪，检测移植后细胞形态功能重塑情况和小鼠听力学指标。通过3年的研究，我们发现：1.BMP4能显著提升iPSC向感觉神经元样细胞诱导分化的效率；2.DFNA41遗传性耳聋致病机制研究为螺旋神经节和毛细胞ATP门控通道障碍，使机体失去对噪声刺激的保护机制，出现噪声相关性感音神经性耳聋症状；3.基因矫正能逆转P2RX2突变导致的毛细胞和螺旋神经节细胞形态和功能缺陷，矫正后干细胞经内耳植入后能定殖于模型小鼠内耳，并恢复部分听力;4.miR-125通过下调CDK2的表达抑制耳蜗前体细胞增殖;5通过激活BMP4信号通路和抑制miR-125/CDK2调动耳蜗内前体细胞增殖，提高遗传性耳聋患者特异性iPSC联合CRISPR/Cas9基因矫正治疗遗传性耳聋动物模型听力水平。本项目的完成初步揭示iPSC基因修复后内耳定向分化生物学功能恢复情况，探索iPSC内耳移植治疗耳聋的有效性，为iPSC治疗遗传性耳聋奠定基础。