PROJECT SUMMARY Fuchs' endothelial corneal dystrophy (FECD) is a degenerative ocular disease affecting 4% of people over 40 and more that >300 million people globally. A hallmark of FECD is the accelerated loss of corneal endothelial cells (CECs), which are required for maintaining corneal transparency and visual clarity. This study will elucidate the underexplored regenerative potential of the corneal endothelium and use this knowledge to devise innovative therapeutic interventions for FECD. Our hypothesis, grounded in preliminary experiments, is that CECs, although naturally dormant, harbor an innate capacity for regeneration. Using innovative live imaging approaches, genetic mouse models, and modified mRNA technology to manipulate CECs in vivo, we identified a prospective therapeutic pathway: stimulating CEC proliferation in vivo to renew the corneal endothelium, thus restoring corneal transparency and vision in FECD patients. To test this, we propose a two- pronged approach utilizing relevant animal models. Aim 1 centers on elucidating the proliferative potential of CECs in normal physiology and during FECD progression. Using established mouse models that recapitulate the main features of FECD, we will perform real-time analysis of CEC dynamics by two-photon microscopy. Moreover, we will investigate the molecular blueprint of a pro-regenerative endothelial cell identity, that can be targeted for therapeutic intervention in FECD. For this we will perform in vivo assays of precision corneal endothelial ablation combined with single-cell genomic analyses to elucidate the molecular programs that influence the proliferation of CECs in vivo. To further illuminate the signaling pathways responsible for regulating the cell cycle of CECs in response to changes of their biomechanical environment during FECD disease progression, we will test the requirement of YAP, the downstream effector of the Hippo pathway, for corneal endothelial function and regenerative ability. Aim 2 builds on our preliminary data demonstrating effective control of CECs with injectable mRNA-encoded growth factors to induce their proliferation in vivo. We propose a new mRNA-based therapy for FECD by directly targeting CECs to prompt the regeneration of a diseased corneal endothelium, restoring its functionality. Toward this, we will systematically test the optimal composition and delivery of our mRNA therapeutic factors to develop a robust treatment protocol. We will comprehensively evaluate the effectiveness and safety of our mRNA treatment in two complementary mouse models of early- and late-onset FECD, then scale up to pre-clinical trials in canine and non-human primate models. Our team of investigators with deep expertise in regenerative biology and FECD disease modeling is optimally equipped to carry out this research. With this study, we are aiming to not only revolutionize our understanding of corneal endothelial physiology and FECD pathophysiology, but also develop groundb...