Project Summary Corneal transplants are typically successful in low-risk allograft recipients. However, a vascularized or inflamed host bed is considered high-risk and results in significant increases in graft rejection rates. In addition to vascularization and inflammation, unsuccessful corneal transplants can become fibrotic due to deficiencies in limbal stem cells (LSC) and stromal myofibroblast activation. Because of such deficits in the healing process, it is essential to boost the regenerative capacity of the cornea and limbus in the host bed environment. Ideally, increasing corneal transplantation success will involve mitigating the inflammatory and vascularization response, as well as promoting the LSC regenerative capacity during the healing process. MG53 is a member of the TRIM protein family with multi-cellular functions in tissue-repair and regeneration. While predominantly expressed in muscle cells, native MG53 protein is present in the tear film and aqueous humor, and contributes to maintenance of corneal integrity and preservation of LSC health. Mice with ablation of MG53 are susceptible to corneal injury and develop symptoms of LSC deficiency. Conversely, mice with elevated levels of MG53 in circulation and in ocular tissues are resistant to corneal injury and vascularization. In addition to facilitating tissue repair, MG53 has anti-inflammatory functions associated with chronic injury or infection, and can also modulate TGF-β signaling in corneal stromal fibroblasts. These additional functions of MG53 may contribute to improved tissue remodeling following corneal transplantation. We have obtained preliminary data to show that topical administration of recombinant human MG53 (rhMG53) protein in mice has benefits in facilitating the initial phase of corneal healing, improving the regenerative capacity of the corneal graft, and mitigating vascularization post- corneal transplantation. Based on these findings, we hypothesize that the multi-cellular function of MG53 will have significant benefits on corneal transplantation outcomes. The studies designed in this project are focused on addressing the following fundamental questions: How does muscle-derived MG53 act as a myokine to improve the healing process and regenerative capacity of corneal transplants? What are the risk-benefits of using rhMG53 as a therapeutic agent to enhance the outcomes of corneal transplantation? Can the combination of rhMG53 and an immunosuppressant provide additive benefits to reduce corneal graft failure?