High Throughput siRNA Screening for Sulfur Mustard‐Induced Corneal Epithelial Cell Injury

Abstract

Chemical injuries to the eyes are true ocular emergencies due to the potential to inflict significant tissue damage that can lead to vision impairment or eventual loss of vision. Sulfur mustard (HD) is a chemical warfare agent that was originally used in World War I and has been used as recently as 2016 in the Syrian conflict. As with many potential chemical threats, no specific treatment for HD ocular exposure is currently available. We previously utilized a high throughput small inhibitory RNA (siRNA) screening model to identify potential therapeutic targets for two different toxicant‐induced ocular injuries: chloropicrin and hydrogen fluoride. Several targets for these injuries are now being evaluated in mouse models of injury. We proposed utilizing the same high throughput screening (HTS) method to identify potential therapeutic targets involved in the response to HD‐induced ocular injury. The model utilized SV40 large T antigen immortalized human corneal epithelial cells (SV40‐HCEC). Selected endpoints were cell viability and IL‐8 expression, as targets of interest should mediate cell health and/or the inflammatory response. Primary screen results were used to down‐select 300 targets from the original 3,027. Deconvolution of these 300 targets narrowed the target list down to 25, which are currently undergoing in vitro validation. Various pathways are implicated by the current target list, including ubiquitination, transcription regulation, signal transduction, and membrane transport. We anticipate that approximately ten validated targets will be transitioned to study in a mouse model to further determine potential therapeutic efficacy for HD‐induced ocular injury. Although work for HD is still in progress, we have compared the target lists that entered in vitro validation studies for all three toxicants evaluated thus far. Only one target was found in common: NR3C1, a glucocorticoid receptor. Additionally, there were two targets in common between chloropicrin and hydrogen fluoride: KMT5A and RELA. These findings show little similarity in the early molecular responses to these toxicants and support the hypothesis that specific therapeutics will be needed to treat different chemical‐related injuries.Support or Funding InformationDisclaimer: The views expressed in this abstract are those of the authors and do not reflect the official policy of the Department of Army, Department of Defense, or the U.S. Government.Funding Sources: This research was supported in part by an appointment to the Postgraduate Research Participation Program at the U.S. Army Medical Research Institute of Chemical Defense administered by the Oak Ridge Institute for Science and Education through an interagency agreement between the U.S. Department of Energy and U.S. Army Medical Research and Materiel Command. Support is also provided by a CounterACT inter‐agency agreement between the NIH/NINDS (Y1‐O6‐9613‐01) and USAMRICD (A120‐B.P2009‐2).