Diabetes is the most widespread blinding disease in working-age adults. Up to 70% of diabetic patients suffer from corneal problems including neuropathy and epithelial keratopathy (delayed wound healing, recurrent erosions, ulcers) that impair vision and cause pain and discomfort. Diabetic keratopathy is underdiagnosed, and therapy remains symptomatic. We identified markers altered in human diabetic corneas and restored their normal-like levels in corneal organ cultures by gene and nano therapy, which also restored normal stem cell phenotype and corneal wound healing. Epigenetic changes also appear to contribute to diabetic complications. In the previous funding period, we designed new approaches to normalize diabetic corneas (1) to avoid toxicity and (2) to remove stable epigenetic changes in diabetic cells. We developed nontoxic nanobioconjugates (NBC) that efficiently normalized stem cell marker expression and wound healing in cultured LEC and organ- cultured corneas, based on antisense modulation of c-Met, MMP-10, and cathepsin F expression. We also discovered a new marker, Wnt5a, reduced in diabetic corneas by dual DNA methylation and microRNA-203 epigenetic suppression. Addition of Wnt5a or demethylating agent Decitabine to diabetic cells or corneas restored wound healing kinetics and stem cell marker expression. In another approach, we produced induced pluripotent stem cells (iPSC) from diabetic LEC to remove diabetic epigenetic signatures and differentiated them back to potentially normalized limbal-like epithelium. Such formerly diabetic cells expressed several stem cell markers similar to normal, as we expected. In this proposal, we will optimize normalization of human diabetic corneas structurally (by stem cell marker expression) and functionally (by wound healing kinetics). We hypothesize that (1) a combination of NBC targeting several diabetic markers with DNA demethylating agents would provide more efficient diabetic corneal normalization than single therapies, and (2) iPSC-derived LEC-like cells could be differentiated into transplantable corneal epithelial cells using air-lifting and natural corneal surface support. We aim to understand mechanistically how the normalizing treatments change gene and protein expression levels and affect corneal cell populations using validated single-cell RNA-seq approach. Aim 1. To develop combined nano and epigenetic therapy for diabetic corneal wound healing and progenitor cells using novel NBCs and DNA demethylating agents. Aim 2. To unravel changes in gene expression and cell populations in diabetic corneal epithelium by scRNA-seq following treatment with combined pharmacological agents. Aim 3. To produce and characterize differentiated corneal epithelium with non- diabetic properties in air-lifted cultures using diabetic iPSC-derived limbal-like epithelial cells. Our aims fit well major initiatives of the NEI Strategic Plan 2021: (1) A game-changing advance – gaining single-cell resolution in ...