PROJECT SUMMARY The ocular surface (OcS) comprises the stratified squamous epithelia of the cornea/conjunctiva, its adnexa, and the overlying tear film. OcS disease disrupts the normal developmental progression, maturation, and turnover of OcS epithelia, causing “epitheliopathy” characterized by cell damage, barrier disruption and programmed cell death. OcS disease initiated by tear dysfunction can be grouped under a syndrome known as dry eye (DE). Studies over the past three decades have defined a mechanism for DE in which epitheliopathy caused by desiccating stress (DS) plays a driving role. Injured cells react by producing innate inflammatory mediators, exacerbating damage and exposing autoantigens. This can lead to an autoimmune-like adaptive T cell-mediated response that is especially prominent in aqueous-deficient DE (ADDE) subtypes. There is an unmet need for therapeutics to treat epitheliopathy due to DE. CLU (clusterin) is an evolutionarily-conserved, homeostatic, secreted glycoprotein, expressed prominently by mucosal epithelia at fluid-tissue interfaces and found in all bodily fluids. CLU has long been recognized to be cytoprotective and anti-inflammatory. The project team discovered that CLU is also proteostatic, acting as both a molecular chaperone and an inhibitor of MMP9, the latter a key mediator of epithelial proteolysis in DE. This raised the hypothesis that CLU present naturally in tears, or supplemented to the OcS via eye drops, can protect the OcS epithelia from damage in DE. Not only was the hypothesis proven, but mechanisms uncovered were unexpectedly novel and interesting. CLU binds selectively to the damaged OcS, sealing the barrier and preventing further damage to cells and their glycocalyces. Its paradigm-changing feature is that it directly targets epitheliopathy, a driver of DE. Simply stated, CLU seals and heals. In ADDE, CLU levels in the tears decrease, making the OcS vulnerable to damage. The goal of this project is to determine whether CLU also dampens inflammation in DE. Recombinant human CLU, expressed using a Current Good Manufacturing Practices (CGMP)-compatible process and formulated as a drug product, will be tested for efficacy in a TSP1 knockout mouse model of ADDE. The anti-inflammatory effect will be quantified via inflammatory markers, and further characterized by quantifying loss of conjunctival goblet cells and corneal nerve fibers, two hallmarks of DE. Direct proof of an anti-inflammatory effect for CLU in DE will strengthen the evidence justifying commercial development.