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 limited therapeutic options but share the
similar sterile inflammatory background and LSCD.
