氯离子通道基因RP32突变致视网膜色素变性的机制研究

Retinitis pigmentosa (RP), one of the most severe forms of inherited retinal dystrophy, can be transmitted usually as an autosomal dominant, recessive, or X-linked trait. We recently demonstrated that a c.75C>A mutation in novel Chloride channel gene, RP32, causes autosomal recessive retinitis pigmentosa. Here we propose using Clustered Regularly Interspersed Short Palindromic Repeats/Cas9 nuclease-null (CRISPR/Cas9), a powerful new approach for mouse genome editing, to develop a RP32 gene knockout model of frame shift mutation. Initially, we will evaluate on eye development, photoreceptor morphology and ERG records of a RP32 knockout mouse, following this model with a targeted knock-in of c.75C>A variant in both alleles. Followed by immunocytochemistry study to analyze gene expression between WT and mutant RP32 cDNA transfect cells, in situ hybridization analysis between human and mouse eyes, RNA sequencing, channel activity test and proteomics analysis. These studies and the use of a mammal animal model are instrumental in understanding the molecular and developmental role of RP32 in the retina and in retinitis pigmentosa. Such information will be useful in designing gene and stem cell therapy as possible interventions for RP in the near future.

视网膜色素变性(RP)是一组严重的遗传性眼底病，可呈常染色体显性、隐性和X连锁遗传。2013年我们在3个常染色体隐性RP家系，定位和克隆了新致病基因，氯离子通道蛋白RP32。本课题拟利用CRISPR/Cas9新技术敲除致病基因RP32，构建小鼠RP模型。同时在基因敲除基础上敲入含有点突变基因序列，体内研究错义突变导致RP的表型：观察小鼠眼球发育、视网膜光感受器细胞结构和视觉电生理学改变；同时利用免疫荧光染色、免疫组化、离子通道活性测定、RNA测序和蛋白质组学等研究从细胞分子与蛋白水平分析该基因的功能；分析总结哺乳动物和人类RP基因型和表型的异同。本课题对探索RP发病机制有重要的科学意义，结果将为基因或干细胞治疗该疾病奠定理论基础。

视网膜色素变性(Retinitis pigmentosa, RP)是一组严重的遗传性眼底病，可呈常染色体显性、隐性和X性连锁遗传。2013年课题组在3个常染色体隐性RP家系，定位和克隆了新致病基因氯离子通道蛋白RP32(CLCC1)。本课题利用CRISPR/Cas9技术条件性敲除致病基因Clcc1构建了小鼠RP模型。研究发现Clcc1 CKO小鼠的视网膜切片染色可见光感受器细胞稀疏，视网膜电图（electroretinogram, ERG)实验中光感受器细胞（视锥细胞和视杆细胞），与双极细胞的反应明显下降，离子通道活性测定发现Clcc1基因突变可通过内质网通透性的变化，引起细胞膜的氯离子通道关闭，进而导致视网膜光感受器细胞的发育异常，从而形成RP。同时我们也在利用RNA测序、蛋白质组学和电镜等方法期望从更深层次理解在细胞分子与蛋白水平的功能。本课题对探索RP发病机制有重要的科学意义，结果将为基因或干细胞治疗该疾病奠定理论基础。