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 uncov...