Project summary Diabetic foot ulcers (DFUs) are a widespread, serious, and common complication of diabetes. Diabetes-related amputations are associated with disability, drastic reduction in quality of life, high morbidity and resulting alarming mortality rate of 35% over 5 years. Non-invasive detectable biomarkers that correlate with healing outcomes of DFU are critically needed for early targeting of hard-to-heal ulcers for advanced interventions beyond standard of care (SOC) therapy, as well as for timely monitoring of response to treatment. Preliminary evidence developed in our laboratory supports circulating urinary microRNAs (miRNAs) as detectable biomarkers for DFUs and other diabetes-associated complications; these miRNAs can be isolated and quantified from urine in a technically robust and reproducible manner. Utilizing an R61/R33 funding mechanism in response to RFA-DK-21-001, this project will capitalize upon patient enrollment in the existing protocols and infrastructure of the Diabetic Foot Consortium to develop a practical, quantitative, non-invasive, cost-effective prognostic biomarker comprised of a urinary microRNA subset that, when assessed in patients at baseline, accurately and reliably differentiates DFUs that have the capacity to heal with SOC from those that do not. Specifically, in R61/ Exploratory phase, a combined data reduction and prognostic modeling approach will be applied to narrow the determine miRNome of top 10-20 miRNA parameters that predict DFU clinical healing outcomes at Week 12. Sensitivity, specificity, and accuracy of predictions will be evaluated and with successful completion of R61 milestones the model will progress to the R33/Validation phase, in which the custom miRNA subset biomarker panel will be interrogated for its ability to predict healing outcomes in a prospectively enrolled cohort of 100 additional patients with open DFUs. Results of the Validation phase will yield a biomarker ready for submission for FDA biomarker qualification approval and subsequent direct clinical implementation as a point-of-care assessment to guide clinical management decisions and improve wound closure outcomes, thereby reducing associated DFU morbidity and mortality.