フックス角膜内皮変性症におけるCTGリピート伸長の役割解明

Fuchs角膜内皮营养不良是一种角膜退行性疾病，通常与TCF 4基因中的TGC重复扩增相关。在该项目中，CRISPR/Cas9基因组编辑用于在培养的人角膜内皮细胞中产生TCF 4基因TGC重复序列切除或扩增，以检查TGC重复序列及其周围区域的可能影响。发现不同TGC重复长度的编辑细胞在大体形态和细胞增殖率方面没有显著差异。转录组学和蛋白质组学分析发现差异表达的mRNA和蛋白质，其指向RNA剪接、ER应激、线粒体周转、铁凋亡、上皮细胞分化和雌激素信号通路的变化作为Fuchs内皮角膜营养不良伴TGC重复扩增的疾病发展的可能机制。发现具有TGC重复扩增的细胞系具有RNA FISH的RNA灶和几种新的TCF 4 mRNA同种型，具有长读捕获RNA测序。CRISPR相关的基因组编辑也尝试在小鼠中使用不同的方法，尽管尚未产生具有TCF 4基因TGC重复扩增的成功小鼠模型。

Fuchs endothelial corneal dystrophy is a degenerative disease of the cornea that is commonly associated with TGC repeat expansion in the TCF4 gene. In this project, CRISPR/Cas9 genome editing was used to produce TCF4 gene TGC repeat excision or expansion in cultured human corneal endothelial cells to examine the possible effects of TGC repeats and the surrounding region. Edited cells of different TGC repeat lengths were found to have no significant difference in gross morphology and cell proliferation rates. Transcriptomic and proteomic analyses found differentially expressed mRNAs and proteins that pointed to RNA splicing, ER stress, mitochondrial turnover, ferroptosis, epithelial cell differentiation and changes in the estrogen signaling pathway as possible mechanisms for the development of disease in Fuchs endothelial corneal dystrophy with TGC repeat expansion. The cell line with TGC repeat expansion was found to have RNA foci with RNA FISH and several novel TCF4 mRNA isoforms with long-read capture RNA-sequencing. CRISPR-related genome editing was also attempted in mice using different approaches, although a successful mouse model with TCF4 gene TGC repeat expansion has yet to be generated.