PROJECT SUMMARY/ABSTRACT Fuchs endothelial corneal dystrophy (FECD) is a polygenic disease that affects 6.1 million Americans over 40 years of age and is the leading indication for corneal transplant surgery in the U.S. FECD is diagnosed based on visible damage to corneal endothelial cells (CECs) and degenerative extracellular matrix deposits on the inner cornea (guttae). FECD pathogenesis is characterized by CEC dysfunction, endothelial-to-mesenchymal transition (EMT), extracellular matrix (ECM) degeneration, and CEC death that lead to vision loss. Although it can be diagnosed early, FECD requires corneal transplantation for vision loss because there are no therapies to prevent its progression. We found previously that the Rho-associated kinase inhibitor (RKI) ripasudil decreases FECD phenotypes, warranting an exploration of its potential as a possible therapeutic option to prevent transplantation. Currently, two RKIs (netarsudil and ripasudil) are being used as adjuvant therapies to accelerate endothelial wound healing in Descemetorhexis without endothelial keratoplasty (DWEK). Studies have not yet been performed comparing these two RKIs head-to-head. Problems with RKI treatments include ocular irritation and frequent dosing requirements. Additionally, RKIs have not been tested as a preventative for FECD progression. The overall objectives of the proposed research are to determine the relative efficacy of two RKIs (netarsudil and ripasudil) for use in two distinct applications for the treatment of FECD (DWEK, primary drug therapy to prevent FECD progression). Our central hypothesis is that netarsudil and ripasudil will have equivalent efficacy in wound healing and FECD phenotype reversal, and that packaging RKIs into targeted nanoparticles (NPs) will decrease off-target side effects and permit reduced dosing frequency. The rationale for the proposed research includes the following: i) head-to-head RKI comparisons are needed because if netarsudil works then clinical use may be achieved more quickly; ii) dose-reduction and sustained-release strategies are needed because ocular irritation limits RKI use; and iii) testing RKIs for suppression of FECD phenotypes is needed to determine if future clinical trials are warranted. Guided by strong preliminary data, our hypothesis will be tested through the following specific aims: 1) Determine the optimum dosing of RKIs in a FECD DWEK treatment model; and 2) Determine the clinical efficacy and toxicity of RKIs with and without targeted NP packaging in a FECD disease prevention mouse model. The approach is innovative in its use of a new cell culture model that makes it possible to delineate the pathological events related to disease progression in early-onset FECD, as well as novel nanoparticles that are capable of delivering drugs to FECD cells with more efficiency and less toxicity. The proposed research is significant because RKI use would permit FECD treatment alternatives that do not rely on donor co...