PROJECT SUMMARY Corneal endothelial dystrophies are a common cause of vision loss and are characterized by a slowly progressive, bilateral dysfunction of the corneal endothelium. The main therapeutic option to restore vision in these patients remains corneal transplantation, as medical management is usually inadequate. Mutations in the SLC4A11 gene are associated with several endothelial dystrophies, including Fuchs endothelial corneal dystrophy (FECD), congenital hereditary endothelial corneal dystrophy (CHED), and Harboyan syndrome. SLC4A11 is a transporter protein that functions to maintain osmotic balance in corneal endothelium, and many point mutations in SLC4A11 found in disease lead to misfolding of the full-length protein. There is recent evidence that assisting mutant SLC4A11 to regain proper folding is a promising therapeutic approach. A prior small-scale, high throughput chemical screen identified the non-steroidal anti-inflammatory drug glafenine for its ability to correct folding defects in misfolded SLC4A11, and following correction the mutant SLC4A11 protein regained functional activity. Though glafenine is not a candidate for clinical use due to anaphylaxis and renal toxicity, its efficacy supports an approach using other small molecule folding correctors to repair defective SLC4A11 in corneal endothelial dystrophies. Our long-term objective is to develop first-in-class therapeutics for patients with vision impairment due to protein misfolding in the cornea. We propose to test the hypothesis that compounds identified through phenotypic screening assays will promote the correct folding of mutant SLC4A11 and restore corneal endothelial cell function. The goals of this proposal are to: Aim 1) Perform focused screening to identify correctors of SLC4A11 folding; Aim 2) Perform large, unbiased chemical library and genetic screening to identify novel targets and mechanisms; and Aim 3) Assess cytotoxic effects of NSAIDs on corneal cells. Using advanced experimental methods, including high-content microscopy-based screening assays and novel CRISPR-based genetic screens, the proposed studies will provide insight into the correction of protein folding defects as a therapeutic strategy for corneal endothelial dystrophies. Results from the study will be used to develop a new, non-surgical treatment option for patients with vision loss due to corneal endothelial dystrophies and establish a novel therapeutic approach to corneal disease.