Abstract Body: Introduction: Allograft vasculopathy (AV) is the main cause of heart transplant failure. AV occlusive arterial lesions are caused by vascular smooth muscle cell (SMC) dedifferentiation after exposure to host T cell-derived interferon-γ (IFN-γ). We hypothesized that SMC form clonal AV lesions, and that understanding this process may suggest new therapeutic targets. Methods: We employed a minor antigen mismatch mouse aorta graft model of AV, transplanting a tamoxifen-induced Myh11Cre^ERT2-Rainbow-labeled male graft into a wild type female host, conducting sectioning, microscopy, and scRNAseq, as well as in vitro gene analysis after IFN-γ treatment of human coronary artery SMC. Results: We found that AV lesions are oligoclonal, and that lineage-traced SMC can first be detected entering the lumen at day 7 post-transplant. scRNAseq analysis of sections at day 10 post-transplant revealed a small cluster of SMC with a unique signature including genes implicated in migration and development, suggesting that this may represent a “pioneer” SMC population that initiates neointimal lesions. We focused on genes in this cluster with properties in other cell types that could suggest a role in lesion formation and determined that several were significantly upregulated in SMC following IFN-γ treatment in vitro. siRNA knockdown of one of these, Nav2, revealed an upregulation of Acta2, suggesting that Nav2 may contribute to SMC dedifferentiation. Further studies are ongoing to localize these putative pioneer genes in early stage AV lesions and determine their functions in SMC. Conclusion: We determined that AV lesions are clonal in origin and identified genes with potential as therapeutic targets or biomarkers in AV.