Modulation of Macrophage Function through Alternative Splicing in Cardiometabolic Diseases

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PROJECT SUMMARY This application is for Dr. Jennie Lin to develop her career as an academic physician-scientist focusing on the functional genomics of cardiometabolic diseases. The career development plan includes training in mRNA processing, macrophage biology, genome editing, and animal models of human disease, with formal mentoring by Dr. Kiran Musunuru and Dr. Daniel Rader along with a multi-disciplinary committee of distinguished scientists. The proposed studies and structured mentored activities will take place at the Perelman School of Medicine at the University of Pennsylvania, which offers a remarkably rich research and training environment that will foster Dr. Lin's professional development. The proposed research will focus on how key alternative splicing (AS) events contribute to the pathophysiology of cardiometabolic diseases, which continue to incur significant morbidity and mortality worldwide. Associated with chronic systemic inflammation, cardiometabolic diseases such as atherosclerosis involve the activation of macrophages to shift toward a causally implicated pro-inflammatory phenotype, but the mechanisms underlying this shift remain incompletely understood. Although transcript-level changes have previously been characterized, a missing link in our understanding of macrophage plasticity and function is whether post-transcriptional regulation is involved.! With the advent of RNA sequencing (RNA-seq), AS − the generation of multiple different mRNA isoforms from a single gene − is emerging as a pervasive cell-specific mechanism that can lead to human diseases if dysregulated. Because AS plays an impactful role in cellular differentiation, it may contribute to the phenotypic plasticity of activated macrophages and hence the pathogenesis of cardiometabolic diseases. Although few studies describe isoform-level differences in the extremes of macrophage phenotypes, our preliminary RNA-seq studies of activated and resting human monocyte-derived macrophages have identified more than 200 AS events for genes associated with inflammation and cell survival. This proposal outlines a plan to study how AS events in macrophages for two specific genes contribute to inflammatory and metabolic pathways relevant to cardiometabolic diseases and, in particular, atherosclerosis. The first gene is PLD1, which has known roles in lipid processing and undergoes increased inclusion of exon 16 in pro-inflammatory macrophages. The second gene is ZC3HC1, which houses in its alternative exon 8 a genetic variant associated with coronary artery disease. The proposed studies include two parallel aims that will investigate for each of these genes how AS alters (1) macrophage inflammatory phenotype, (2) metabolic pathways relevant to gene function and atherogenesis, and (3) the development and progression of atherosclerosis in vivo. In undertaking the proposed studies and training plan, the Dr. Lin will elucidate novel mechanisms of macrophage function in the cardiometabolic context, mature as an independent translational investigator in functional genomics, and prepare to compete successfully for R01 funding for future projects aimed at precision medicine.