Laying a Foundation for Precision Medicine for Fuchs' Dystrophy

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PROJECT SUMMARY Fuchs’ endothelial corneal dystrophy (FECD) is an age-related degenerative disorder resulting in corneal edema and loss of vision. FECD occurs in 4% of whites over the age of 40 years and is the leading indication for corneal transplantation in the U.S. Over 70% of cases are caused by a CTG triplet repeat expansion in the TCF4 gene. Expanded CUG repeat RNA (CUGexp) transcripts expressed from this gene locus accumulate as nuclear foci in the corneal endothelium of patients. The CUGexp foci bind and functionally sequester the splicing factors MBNL1 and MBNL2 to trigger mis-splicing in FECD endothelial tissue. To examine the endothelial cell-type specificity for FECD, we will determine if somatic mutations in the post-mitotic corneal endothelium of FECD patients results in a larger triplet repeat expansion than in their blood. We will examine if other anterior segment cell-types including corneal epithelium, stromal keratocytes, and trabecular meshwork cells are prone to accumulation of CUGexp foci and associated molecular defects. To test the MBNL sequestration hypothesis, we will examine if the knockdown of MBNLs in healthy donor endothelial tissue is sufficient to recapitulate the mis-splicing and upregulation of extracellular matrix (ECM) genes found early in the disease course. In early and late FECD, we observed a marked overexpression of the cochlin gene that produces a secreted ECM protein also capable of recruiting immune cells. The cochlin protein was previously detected in the trabecular meshwork of patients with primary open angle glaucoma (POAG), and our preliminary data indicate that the protein is present in the aqueous humor and trabecular meshwork of FECD subjects. We will examine levels of the cochlin protein in corneal tissue, aqueous humor samples, and trabecular meshwork tissue of FECD patients for its possible contribution to corneal disease findings and the increased risk for glaucoma in these patients. Additionally, we will examine the prevalence of POAG in our large UTSW FECD cohort and determine if there is a correlation with the triplet repeat length. We will use single-cell RNA sequencing combined with IHC to identify the immune cells and their contribution to late-stage FECD. We have characterized the efficacy of 45 antisense oligonucleotides (ASOs) and duplex RNAs that block disease-causing CUGexp foci. Using patient-derived cells and tissue, we will examine their potency based on their ability to block foci formation and their specificity based on transcriptome-wide assessment of on- and off-target events. We will examine delivery, length of action, and safety using wild-type mice. To enable studies of disease biology and in vivo drug testing, we have generated what we believe is the first mouse model with a knock-in of expanded CTG repeats. In preliminary studies, we show that our knock-in strategy successfully recapitulates CUGexp foci formation in corneal endothelium. We will further characterize this murine model by aging these mice to examine for the transcriptional and histologic alterations of FECD disease, and then use these mice to test our lead CUG-repeat targeting oligonucleotides for therapeutic development.