Transcription Factor 4 (TCF4) Regulation of Corneal Health

项目总结 Fuchs内皮性角膜营养不良(FECD)是一种双侧可遗传的角膜内皮变性 这影响了美国约4%的40岁以上人口。FECD是世界上最大的 角膜移植的常见适应症。基因组学的重大进展极大地增加了我们的 对FECD的了解，并确定了不同的遗传和表观遗传原因和关联。特别是， 传统的连锁研究和GWAS研究发现了一个扩展的CTG三核苷酸重复序列(TNR) 扩增最多的是转录因子4(TCF4)基因的非编码区(命名为CTG18.1 与晚发性FECD密切相关的基因改变。内含子在TNR中的扩展 在欧洲人群中，约70%的FECD病例与TCF4基因有关。尽管有这些实质性的 在识别参与癌症发生的危险等位基因和生物途径方面的进展 FECD，FECD的最终治疗仍以角膜移植为主。有效 缺乏对FECD的医疗治疗。FECD研究领域的一个主要挑战是缺乏 小鼠模型TCF4重复扩张。其他相关基因的小鼠模型已经被开发出来 与FECD(COL8A2，SLC4a11)有关，但这些基因只对少数病例负责。因此，当前 研究TCF4重复扩增FECD的模型包括体外标本、原代细胞培养和 从晚期FECD患者的组织标本中获得永生化细胞系 角膜移植。需要TCF4重复扩增的体内模型，特别是可以 可用于早期FECD的研究。主要的障碍是建立动物模型的挑战 由于技术困难，具有大量不间断的重复。在初步研究中，我们开发了一种 建立三核苷酸重复扩增障碍敲入小鼠模型的新方法。我们独一无二的 该方法利用CRISPR和高效的微同源介导的末端连接(MMEJ)来 将三核苷酸重复序列敲入基因位点。这种方法使我们能够将338个CTG重复序列敲入 并成功构建了TCF4-CTG338敲入杂合子小鼠。我们的总体目标是 确定TCF4重复扩增在FECD发病机制中的作用，以便新的治疗方法可以 被开发出来。在我们的具体目标中，我们采取了两个主要策略：用短TCF4重复来表征小鼠 (338个重复)，并培育出具有长TCF4三核苷酸重复(1,346)的小鼠 重复)，因为重复扩展长度与角膜疾病相关。我们假设携带TCF4的小鼠 重复扩增可作为CTG扩增介导的FECD的体内模型。我们预计这将是 该项目最终将对TCF4重复扩增如何导致FECD疾病建立新的见解 并发现有希望的治疗靶点。更广泛地说，这部作品为小说的发展搭建了一个平台 研究三核苷酸重复扩张性疾病的小鼠模型。

PROJECT SUMMARY Fuchs endothelial corneal dystrophy (FECD) is a bilateral, heritable degeneration of the corneal endothelium that affects roughly 4% of the population older than 40 years in the United States. FECD is one of the most common indications for corneal transplantation. Major advances in genomics have dramatically increased our understanding of FECD, and identified diverse genetic and epigenetic causes and associations. In particular, traditional linkage studies and GWAS studies identified an expanded CTG trinucleotide repeat (TNR) expansion (named CTG18.1) in the non-coding region of transcription factor 4 (TCF4) gene as the most strongly associated genetic alteration associated with late-onset FECD. The intronic TNR expansion within the TCF4 gene is responsible for about 70% of FECD cases in European populations. Despite these substantial advances made in identifying risk alleles and biological pathways that are involved in the development of FECD, definitive treatment of FECD has mainly remained surgical with corneal transplantation. Effective medical treatments for FECD are lacking. A major challenge in the field of FECD research is the absence of a mouse model of the TCF4 repeat expansion. Mouse models have been developed for other genes associated with FECD (COL8A2, Slc4a11) but these genes are responsible for only a minority of cases. Thus, current models for studying TCF4 repeat expansion FECD include ex-vivo specimens, primary cell cultures, and immortalized cell lines derived from tissue specimens obtained from patients with advanced FECD undergoing corneal transplantation. An in vivo model of the TCF4 repeat expansion is needed, in particular one that could be used to study early stage FECD. The main roadblock has been the challenge of creating animal models with large uninterrupted repeats due to the technical difficulties. In preliminary studies, we have developed a novel method for generating knock-in mouse models of trinucleotide repeat expansion disorders. Our unique approach takes advantage of CRISPR and the highly efficient microhomology-mediated end joining (MMEJ) to knock in trinucleotide repeats into gene loci. This approach has enabled us to knock in 338 CTG repeats into the Tcf4 locus and successfully generate Tcf4-CTG338 knock-in heterozygous mice. Our overall objective is to determine how the TCF4 repeat expansion contributes to FECD pathogenesis so that new therapeutics may be developed. In our Specific Aims, we take two major strategies: characterize mice with a short TCF4 repeat (338 repeats) that have already been generated and develop mice with a long TCF4 trinucleotide repeat (1,346 repeats) as repeat expansion length is correlated with corneal disease. We hypothesize that mice with a TCF4 repeat expansion can serve as an in vivo model for CTG expansion-mediated FECD. We anticipate that this project will ultimately establish new insight into how a TCF4 repeat expansion contributes to FECD disease and uncover promising therapeutic targets. More broadly, this work establishes a platform to develop novel mouse models for the study of trinucleotide repeat expansion diseases.