PROJECT SUMMARY Coronary artery disease (CAD) remains a global public health burden despite advances in risk factor targeted therapies. Meta-analyses across ancestrally diverse populations have identified >300 genetic loci associated with CAD risk. We and others have investigated candidate genes that are dysregulated in the coronary vascular wall; however, the causal genes and regulatory mechanisms remain mostly unknown. Recent advances in single-cell genomics profiling have begun to unravel the cellular diversity during atherosclerosis. Molecular quantitative trait (QTL) mapping and gene regulatory network approaches in disease-relevant tissues have refined some of the regulatory mechanisms, however the specific cell types and phenotypic states driving these changes remain unclear. We have leveraged a large biobank of coronary artery tissues from ancestrally diverse individuals to perform bulk and single-cell profiling of genetic variation and cell states. Using single-nucleus profiling of chromatin accessibility in healthy and subclinical atherosclerotic coronary arteries we identified cis-regulatory elements to explain transitions of smooth muscle cells (SMC) to fibroblast-like cell types. By meta-analyzing single-cell gene expression data in atherosclerosis, we have also identified etiologic SMC phenotypic states underlying CAD as well as coronary artery calcification (CAC). Here we propose to extend our previous efforts to apply multimodal single-cell profiling to capture QTLs and gene regulatory mechanisms in both subclinical and advanced atherosclerotic coronary segments. Building on our holistic framework and resources, this proposal will generate unprecedented insights into the hierarchical regulatory networks that influence dysregulated vascular wall processes. By enriching these high-resolution data with large-scale population genetics, clinical imaging, and genomic and histologic cardiometabolic tissue biobanks we will provide context for translating these findings to patients at various stages of disease. We have already identified several disease stage and cell state-specific markers for atherosclerosis. We will validate and discover new candidate causal genes and functional regulatory elements using perturbation assays coupled to high-throughput phenotyping in SMC, along with spatial imaging in intact tissues across the disease trajectory. Together with our interdisciplinary and experienced collaborators and unique established resources, we are well-positioned to carry out this work and discover fine-grained molecular features and hallmarks of CAD initiation and progression in the coronary vessel wall. Ultimately, these studies will enable the next generation of multimodal treatment strategies for risk stratification and eradication of this debilitating disease.