PROJECT SUMMARY:The COVID-19 pandemic has dramatically highlighted serious unmet needs of ARDS including the absence ofeffective FDA-approved pharmacologic interventions that address ARDS mortality. ARDS phenotypeheterogeneity the complexity of dysregulated inflammation and the absence of predictive biomarkers have allcontributed to failed ARDS therapeutic clinical trials. Predictive biomarkers either protein- or genomic-basedthat identify specific ARDS sub-phenotypes and likely responders to specific ARDS therapeutics couldsignificantly influence effective clinical trial designs to assess novel therapeutics and therefore benefit theoutcomes of ARDS trials. Our group has long championed the utility of genomic-intensive approaches to identifymultiple novel ARDS therapeutic targets. We previously identified eNAMPT (extracellular nicotinamidephosphoribosyl transferase) as a novel ARDS therapeutic target/gene which serves as a damage-associatedmolecular pattern protein (DAMP) and ligand for Toll-like receptor 4 (TLR4). NAMPT SNPs predict ARDSseverity and eNAMPT amplifies dysregulated lung/systemic inflammatory responses that contribute to multi-organ injury/failure. We demonstrated the utility of a humanized eNAMPT-neutralizing mAb as a therapeuticstrategy in ARDS and other inflammatory conditions. Plasma eNAMPT along with IL-6 IL-8 IL-1RA MIF andAng-2 was highly predictive of 28-day ARDS mortality. We also identified variants in selectin P ligand gene(SELPLG) encoding P-selectin glycoprotein ligand 1 (PSGL1) and P-selectin gene (SELP) as associated withincreased susceptibility to ARDS in Blacks. PSGL1/P-selectin interactions are critical to lung inflammation vialeukocyte trafficking platelet aggregation and thrombosis. Plasma PSGL1 and P-selectin levels are significantlyelevated in sepsis ARDS and COVID-19 pneumonia patients and PSGL1 inhibition (mAb TSGL-Ig) significantlyattenuates preclinical lung injury in ARDS. This R-21 application will utilize over 900 plasma samples andgenotyping results available from the NHLBI Prevention and Early Treatment of Acute Lung Injury (PETAL)Network Reevaluation of systemic Early neuromuscular blockade (ROSE) study (see NHLBI BioLINCC letter).We will validate two highly novel stratification tools to improve patient stratification in the design of future ARDSclinical trials targeting eNAMPT/TLR4 and PSGL1/P-Selectin interactions. Specific Aim (SA) #1 will develop agenotype-based biomarker assay combining: i) carefully selected SELPLG/SELP variants with plasmaPSGL1/P-selectin levels and ii) NAMPT variants with plasma eNAMPT levels. These genotypes will identifyARDS subjects as candidates for future clinical trials targeting PSGL-1/P-selectin interactions and theeNAMPT/TLR4 signaling pathway. SA #2 will validate the predictive capacity of a seven-biomarker panel(eNAMPT IL-6 IL-8 IL-1RA PSGL-1 IL-1 Ang-2) for ARDS mortality. Successful completion of this highlyinnovative R21 grant will generate a novel point of care pharmacogenetic enrichment tools to be leveragedin designing human ARDS clinical trials targeting PSGL1/P-selectin and eNAMPT/TLR4 interactions.