Identifying causal gene programs for vascular disease using high throughput CRISPR genomics

Stanford University, Stanford CA

Investigators

Abstract

PROJECT SUMMARY: Overall Component Vascular diseases of the heart and lung, including coronary artery disease (CAD) and pulmonary arterial hypertension (PAH), are a major health burden. To develop novel preventative and therapeutic strategies, we must identify the genes and pathways that underlie risk for these diseases, and characterize how their functions are shared or distinct across vascular beds and disease contexts. Genetic variants that influence complex traits are thought to regulate genes that work together in biological pathways. However, our knowledge of which genes act in which pathways in specific cell types is incomplete. To address this, we have developed innovative Perturb-seq methods to study hundreds of genes with CRISPR and determine how genes work together in co-regulated pathways (“gene programs”). We have developed statistical methods to test whether many genes for a given disease converge on particular gene programs. Finally, by combining high-throughput CRISPR with in vivo delivery into mouse models, we now have the tools to characterize these gene programs in a native tissue environment across health and disease. Leveraging these advances, the overall aims of our Program are to: 1) Identify and characterize genes that influence risk for vascular disease; 2) Identify convergence of causal genes into gene programs in vascular cells; and 3) Test how gene programs differ across contexts, including cell types, organs, cell-cell interactions, and disease states. Together, these aims will test the overall hypothesis that endothelial cell (EC) and smooth muscle cell (SMC) gene programs have context-specific activities that vary across disease states, tissue beds, and biological sex to mediate genetic risk for different vascular diseases. Our Program is organized into 3 Projects and 3 Cores that will interact in synergy. Project 1 (ECs in CAD) will apply Perturb-seq to systematically dissect the Cerebral Cavernous Malformation (CCM) signaling and other pathways in murine atherosclerosis in vivo, and to understand its pleiotropic effects in the heart, lung, and brain. Project 2 (SMCs in CAD) will leverage Perturb-seq to map the convergence of CAD genes onto SMC gene programs, and dissect the effects of newly discovered genes on SMCs and ECs in vivo. Project 3 (ECs in PAH), will apply Perturb-seq to test whether PAH genes converge on branches of the BMPR2 signaling pathway in pulmonary ECs in vivo, and evaluate how these gene programs differ across disease states or in response to cell-cell interactions. Three Cores will support the Projects and coordinate cross-Center collaborations: (i) the Perturb-seq Core will collect and analyze Perturb-seq data; (ii) the Mouse Core will generate and share mouse models and AAV libraries for in vivo Perturb-seq; and (iii) the Administrative Core will coordinate training and exchange of data and reagents. Thus, our highly synergistic and collaborative studies will illuminate critical gene programs that regulate propensity to vascular disease, leading to novel preventative therapies for CAD and PAH.