Abstract Th0043: In vivo CRISPR-Screening Identifies <i>Fhl3</i> as a Potential Regulator of Macrophage Accumulation in Atherosclerotic Plaques

Atherosclerotic cardiovascular disease (ASCVD) is the leading cause of death and disability worldwide. Recent clinical trials have shown that inflammation plays a critical role in ASCVD. Plaque inflammation is primarily driven by inflammatory macrophage accumulation and is associated with unstable lesions and poor patient outcomes. scRNA-Sequencing studies have highlighted diverse macrophage populations in atheromas, but fundamental questions persist about how macrophages acquire these transcriptional states and influence pathogenesis. To identify novel pathways that may regulate macrophage function, we leveraged genome-wide association studies (GWAS), which have identified hundreds of loci associated with ASCVD. We hypothesized that novel regulators of macrophage function in plaques can be identified by screening genes associated with atherosclerosis in humans. For this, hematopoietic stem cells (HSCs) from Cas9 transgenic mice were transfected ex vivo with lentiviruses harboring CRISPR knockout guides targeting GWAS-identified genes. Transfected Cas9 + HSCs were transplanted into lethally irradiated Ldlr -/- mice, generating chimeras with multiple genetic alterations within the bone marrow of the same mouse. After the development of atherosclerosis, bone marrow HSCs, blood monocytes, and aortic myeloid cells were isolated, and the abundance of CRISPR-edited cells was quantified. We found an almost two-fold enrichment of Fhl3 deficient myeloid cells in atheromas relative to blood monocytes, suggesting a selective expansion in lesions. FHL3 is a common variant with a 1.05 odds ratio for coronary artery disease, yet few studies have investigated its role in atherosclerosis or macrophage function. In vitro Fhl3 deficient bone marrow-derived macrophages exhibited increased migration, suggesting the increased expansion in lesions may be due to enhanced recruitment. Histological staining of human carotid plaques revealed that FHL3 co-localizes with macrophages, suggesting FHL3 may instill its increased ASCVD risk through macrophage dysfunction in humans. Together we developed a novel in vivo CRISPR screening model and identified Fhl3 as a potential regulator of macrophage accumulation in atheromas. Evaluation of additional risk variants has a high likelihood of uncovering mechanisms of inflammation and identifying drivers of macrophage function in atherosclerosis.