PROJECT SUMMARY/ABSTRACT Systemic sclerosis (SSc) is a multi-system disease with the highest mortality among major rheumatologic diseases. Though certain genetic loci have been robustly linked to SSc susceptility, development of effective therapies for SSc is hampered by a fragmented understanding of mechanisms by which the identified genetic variants lead to SSc. The current proposal is designed to elucidate mechanisms by which a variant in deoxyribonuclease 1-like 3 (DNASE1L3) that results in Arg to Cys substitution at amino acid 206 (R206C) contributes to SSc pathogenesis. The Pl's preliminary data indicate that digestion of circulating genomic DNA is impaired in SSc patients with the DNASE1 L3 R206C variant. Aim 1 of the proposal is designed to further define the circulating DNA profile in SSc patients with DNASE1 L3 R206C. Two specific populations of circulating DNA will be analyzed--nucleosomal DNA associated with apoptosis-derived extracellular vesicles and DNA contained in neutrophil extracellular traps. A potential role of DNASE1 L3 in digestion of mitochondrial DNA will also be examined. Next-generation sequencing will be employed to determine length differences in circulating DNA fragments. An ELISA for measurement of circulating DNASE1L3 will be developed and validated. Aim 2 will elucidate aberrancies in immune cell function that occur in the setting of DNASE1 L3 deficiency, using a dendritic cell functional assay and single cell RNA Sequencing analyses of murine and human immune cells. Together, these studies will shed light on the downstream biochemical and immunologic consequences of DNASE1 L3 dysfunction, strengthening the foundation for development of targeted therapies to treat SSc. The Pl is an MD/PhD Rheumatologist with the long-term goal of becoming an independent investigator focused on SSc pathogenesis. The proposed research and training plan will help the Pl to develop proficiency in analyses of genomic and transcriptomics data and in experimental approaches for the study of adaptive immunology. The work will be performed in an excellent institutional environment with mentorship from Dr. Shervin Assassi, a leader in molecular profiling of SSc, and additional guidance from co-mentors with expertise in bioinformatics and immunology. This award will accelerate the Pl's research as well as career development into an independent researcher.