Abstract Vesicant agents, such as sulfur mustard (SM), causes immediate pain, inflammation, burns, and/or blisters, and corneal lesions often resolve clinically, however, in some cases, vesicant exposure cause chronic and delayed mustard gas keratopathy (MGK), leading to impaired vision and vision loss. MGK can proceed from the prolonged epithelial cell death-induced sterile inflammatory responses and subsequent delayed-onset limbal stem cell deficiency (LSCD). Anti-inflammatory drugs, MMP inhibitors or anti-VEGF therapy have shown beneficial in ameliorating the severity of the pathology, but a single agent or combined therapy of these agents is not sufficient to inhibit MGK. Therefore, the goal of this project is to develop an effective countermeasure against MGK with extracellular vesicles (EVs) produced from mesenchymal stem cells (MSCs). To achieve the goal, the proposed study will test the central hypothesis that MSC-derived EVs (MSC-EVs) exert sustained effects in suppressing SM-induced pathological changes in the eyes by delivering therapeutic proteins and miRNAs that target apoptosis, sterile inflammatory responses and NV, thereby preventing the development of MGK. MSCs have shown to promote corneal wound healing in several pre-clinical models by protecting cell death and suppressing sterile inflammatory responses. Emerging evidence indicates that MSC-derived EVs (MSC-EVs) are a promising alternative to MSC therapy as they recapitulate a broad range of the therapeutic effects shown by MSC treatment while being devoid of safety risks and infrastructure challenges associated with live MSC treatment. Moreover, MSC-EVs have many desirable features of an ideal gene delivery system as they deliver proteins and miRNAs from their parent cell to recipient cells and thereby, they can provide long lasting effects than a single agent targeting inflammatory cytokines, MMPs or VEGF. Importantly, MSC-EVs produced under microcarrier cultures have superior anti-inflammatory function and also carry a significant amount of anti- apoptotic, anti-inflammatory and anti-angiogenic miRNAs compared to those from conventional monolayer cultures. Therefore, MSC-EVs laden with therapeutic proteins and miRNAs can be produced with a scalable microcarrier-culture system without viral engineering. Aim 1 will optimize EV-based intervention conditions for the treatment of vesicant-induced ocular injury. Aim 2 will test the therapeutic potential of MSC-EVs in a rabbit model of SM-induced ocular injury. Successful completion of the proposed study will provide valuable preclinical data of MSC-EVs in SM-induced ocular injury and determine the mechanism(s) underlying the MSC-EVs action in MGK prevention, contributing as the first step in a continuum of research that focuses on the deployment of the safe and clinically feasible EV-based therapy. Ultimately, it will provide major therapeutic advances for patients with SM injuries as well as a broad range of disorders have limit...