CRISPR/Cas9介导的母系遗传性聋病基因突变矫正研究

Deafness is one of the major public health problems worldwide. The mitochondrial 12S rRNA 1555A>G (m.1555A>G) gene mutation has been associated with both aminoglycoside-induced and maternally inherited nonsyndromic deafness. The m.1555A>G mutation is a primary factor underlying the development of deafness but itself insufficient to produce deafness phenotype. Nuclear modifier genes and aminoglycosides acting as modifier factors modulate the phenotype manifestation. Our previous data showed that the nuclear modifier gene TRMU affected the phenotype manifestation of the deafness-associated m.1555A>G mutation. The overall aim of this investigation is to elucidate the pathogenesis of maternally inherited deafness. Firstly, three lymphoblastoid cell lines (carrying both m.1555A>G and homozygous TRMU A10S mutations, carrying only m.1555A>G mutation and contol) are induced to iPSCs and their ability to differentiate into inner ear hair cells is evaluated. Secondly, the effects of TRMU A10S and m.1555A>G mutation on the morphology and function of inner ear hair cells are investigated. Finaly, the A10S mutation will be corrected using CRISPR/Cas9 technique to explore the function renovation strategy of inner ear hair cells. Success of this research will lead to the deep understanding of the pathogenesis of maternally inherited deafness and provide valuable information and technology for the therapeutic approaches for hearing loss.

聋病是全球性重大公共卫生问题。线粒体12S rRNA 1555A>G（m.1555A>G）突变是导致母系遗传性聋病的重要原因，然而该突变自身不足以造成聋病表型，核修饰基因等其它修饰因子在聋病表型的表达中起协同作用。我们前期工作表明核修饰基因TRMU调控m.1555A>G突变相关聋病表型的表达。本项目在此基础上进一步研究TRMU A10S突变与m.1555A>G突变互作导致聋病的机制。首先构建携带突变的疾病特异性iPSCs，评估两突变互作对iPSC向内耳毛细胞分化的影响；其次通过检测内耳毛细胞的形态功能，研究突变组织特异性致病的机理；最后利用CRISPR/Cas9技术矫正TRMU突变，探讨内耳毛细胞功能缺陷的修复策略。本项目的实施将有利于全面阐明母系遗传性聋病的致病机制，为聋病的干预治疗提供科学依据和技术支撑。

耳聋是全球性重大公共卫生问题。线粒体12S rRNA 1555A>G（m.1555A>G）突变是导致母系遗传性聋病的重要原因。m.1555A>G突变自身尚不足以造成聋病表型，核修饰基因TRMU调控m.1555A>G突变相关聋病表型的表达。本研究构建了TRMU A10S纯合突变和m.1555A>G突变、仅m.1555A>G突变及正常对照三株永生化淋巴细胞源的iPSCs，并定向分化为内耳毛细胞样细胞，发现TRMU A10S纯合突变iPSCs分化为内耳毛细胞样细胞的效率下降。扫描电镜观察到携带TRMU A10S纯合突变的毛细胞样细胞纤毛结构存在明显缺陷。电生理功能检测表明TRMU A10S纯合突变的内耳毛细胞样细胞功能异常。线粒体功能实验显示TRMU A10S纯合突变的毛细胞样细胞氧耗量与ATP产量降低，活性氧产生增加。通过CRISPR/Cas9系统对TRMU A10S突变的iPSCs进行矫正并定向分化为内耳毛细胞样细胞。实验发现与矫正前相比，毛细胞分化效率得到提升，纤毛结构及电生理功能以及线粒体功能得到恢复。本研究表明遗传矫正核修饰基因TRMU突变对内耳毛细胞功能缺陷起到修复作用，为线粒体基因突变相关耳聋的干预治疗提供了新的视角。