Identification of smooth muscle cell genes causal in atherosclerotic plaque stability and cardiovascular disease risk - despite effective LDL-c therapies, cardiovascular disease (CVD) risk remains a…

Identification of smooth muscle cell genes causal in atherosclerotic plaque stability and cardiovascular disease risk - despite effective LDL-c therapies, cardiovascular disease (CVD) risk remains a major unmet clinical need. We and others have identified >300 loci for coronary artery disease (CAD). Genes that function in vascular smooth muscle cells (SMC) are causal at several loci yet the causal genes at most loci remain unknown. Using single cell profiling and SMC lineage-tracing in mouse models, we found that SMC transition through an intermediate SMC-derived cell (SDC) state into protective or harmful phenotypes that modulate disease. We hypothesize that SMC genes play a prominent causal role in plaque instability and CVD risk independent of lipoprotein genes. To address this, we will leverage unique mouse model and human resources, including the pakistan genomics resource (PGR, n=250,000 for study) that includes the largest global cohort of human gene knockout "KOS" (complete KOS >5000; heterozygous KOS >18,000 genes) as well as the munich vascular biobank (MVB) with >2,000 human plaques and clinical, histology, transcriptomics and genetic data. In aim 1, we will integrate SMC lineage tracing in mouse models with analyses of >1 million participants with gwas SNP, whole-exome (WES) and whole-genome (WGS) data, eliminating all loci/genes associated with plasma lipoproteins. Implementing the largest rare variant and gene burden testing for CAD to date, we will prioritize likely causal SMC/SDC genes and reveal predicted loss of function (plof) variant directional effects. To operationalize call-back studies, we will limit to genes with at least 5 plof carriers in PGR. Gene priority will be refined by multiethnic fine-mapping, CO-localization analyses and biological plausibility. We expect to prioritize ~30 SMC/SDC genes. All will undergo large phewas for pleiotropy and safety. For the top 5 genes, call-back studies will validate causality and directionality and assess safety through deep phenotyping of atherosclerosis traits, safety and pleiotropy markers in PGR families (n=200 per family). Preliminary work prioritized 15 SMC/SDC genes, all strong causal candidates, and initial call-back in PGR expanded large pedigrees for the most promising genes (e.g., PDE3A, SERPINH1, HHIPL1, ZEB2). In aim 2, we will use RNA in situ sequencing (hybriss), RNA-scope, proximity ligation assays (MYH11-H3K4ME2 SMC/SDC mark) and histology to define SMC/SDC gene expression and location for ~30 prioritized genes in stable VS. Unstable MVB plaques. Change in allele specific expression for genes in SDC types in stable VS. Unstable MVB plaques and CO-localization of their CIS-eqtls to CAD snps will inform causal and directional effects on plaque stability. For at least 2 top genes, we will use a TET-on DRE/CRE dual inducible recombinase system for SMC gene deletion at late time points to test effects and mechanisms on features of plaque stability in advanced lesions and disease regression. We are poised to test in mouse models if PDE3A, one compelling example, promotes SMC proliferation, senescence and vascular remodeling. Overall, we propose a unique integrative platform, validated by human genetics, to fine-map loci, discover causal genes and elucidate safe therapeutic targets in SMC/sdcs causal pathways for atherosclerosis stability and CVD risk.