DESCRIPTION (provided by applicant): A wide variety of physiological and pathological conditions such as infections, inflammations and corneal diseases such as limbal stem cell deficiency (LSCD) and diabetes can affect limbal epithelial stem cells (LESC) functions resulting in serious visual problems. LESC are located within special niches in the limbal basal layer at the corneo-scleral junction of cornea. The health of LESC is a key factor in renewal of healthy and regeneration of wounded corneal epithelium. Altered wound healing process, reminiscent of diabetic cornea, can result in corneal vascularization and loss of transparency, which may lead to corneal blindness. Currently, there is a need to better understand the mechanisms responsible for these abnormalities and develop a specific and efficacious treatment. The mechanisms of LESC proliferation, migration, adhesion, and differentiation in normal cornea homeostasis and wound healing process could be key to understanding corneal diseases. Therefore, understanding of miRNA regulatory mechanisms is critically important in the etiology and treatment of various corneal diseases such as LSCD and diabetic keratopathy, which are the major causes for corneal blindness. We will employ a variety of molecular, functional tests and ex-vivo human organ-cultured corneas to understand the role of microRNAs in both the differentiated epithelial and stem cells of normal and diabetic corneas and their roles in driving peripheral activation of limbal stem cells in normal and diseased cornea. We will focus on determining their targets, and attempt gene therapy with either overexpression or silencing of specific miRNAs to normalize corneal wound healing and LESC marker patterns in diabetic cornea. We will target diabetic corneal epithelial cells by single microRNA or their combinations to improve function of corneal epithelium. This research could lead to the generation of a new cell source for restoring vision in patients with altered LESC. We will use human corneal organ culture in our studies. Normal and diabetic human autopsy eyes will be purchased from the National Disease Research Interchange (NDRI), a national supplier of donor human research tissue. All tissues are obtained in our lab without donor identity.

PUBLIC HEALTH RELEVANCE: This proposal is focused on investigating the role of microRNA (miR-146a, -127- 3p, and -10b), expressed differentially in the stem cell-enriched limbal epithelium vs. central cornea and in diabetic vs. normallimbus, in corneal epithelial homeostasis and wound healing in vitro and in human organ-cultured corneas. MiRNA potential targets will be determined and gene therapy with either overexpression or silencing of specific miRNAs will be used to normalize corneal wound healing and LESC marker patterns in human diabetic corneas. Manipulating the expression of these microRNAs may allow us to uncover miRNA mechanisms of regulation of corneal homeostasis and ameliorate diabetic corneal abnormalities, for future translation to therapy in patients with diabetes and/or limbal stem cell deficiency.