Transcription Factor 4 (TCF4) Regulation of Corneal Health

项目摘要 Fuchs角膜内皮营养不良（FECD）是一种遗传性角膜内皮细胞变性 这影响了美国40岁以上人口的大约4%。FECD是世界上 角膜移植的常见适应症。基因组学的重大进展极大地提高了我们的 了解FECD，并确定了不同的遗传和表观遗传原因和协会。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）， 在转录因子4（TCF 4）基因的非编码区中的扩增（命名为CTG18.1）是最常见的 与晚发型FECD密切相关的遗传改变。TNR基因内含子的扩增， 在欧洲人群中，TCF 4基因负责约70%的FECD病例。尽管这些重大 在确定风险等位基因和生物途径方面取得的进展， FECD，FECD的确定性治疗主要是通过角膜移植进行手术。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一个 TCF 4重复扩增的小鼠模型。已经为其他相关基因开发了小鼠模型 与FECD（COL 8A 2，Slc 4a 11），但这些基因是负责只有少数情况下。因此，当前 用于研究TCF 4重复扩增FECD的模型包括离体标本、原代细胞培养物和 来源于组织样本的永生化细胞系，所述组织样本获自经历了以下过程的晚期FECD患者： 角膜移植需要TCF 4重复扩增的体内模型，特别是可以 用于研究早期FECD。主要的障碍是创造动物模型的挑战 由于技术上的困难而具有大的不间断重复。在初步研究中，我们开发了一种 用于产生三核苷酸重复扩增病症的敲入小鼠模型的新方法。我们独特 这种方法利用CRISPR和高效的微同源介导的末端连接（MMEJ）， 将三核苷酸重复敲入基因座。这种方法使我们能够敲入338个CTG重复序列， Tcf 4基因座，并成功产生Tcf 4-CTG 338敲入杂合小鼠。我们的总体目标是 确定TCF 4重复扩增如何有助于FECD发病机制，以便新的治疗方法可以 发展。在我们的具体目标中，我们采取了两个主要策略：用短TCF 4重复序列表征小鼠 (338重复序列），并开发出具有长TCF 4三核苷酸重复序列（1，346 重复），因为重复扩增长度与角膜疾病相关。我们假设TCF 4基因的小鼠 重复扩增可用作CTG扩增介导的FECD的体内模型。我们预计， 该项目将最终建立新的见解，了解TCF 4重复扩增如何导致FECD疾病 发现有希望的治疗靶点。更广泛地说，这项工作建立了一个平台， 用于研究三核苷酸重复扩增疾病的小鼠模型。

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.