PROJECT SUMMARY Pulmonary arterial hypertension (PAH), a deadly cardiopulmonary disease that develops following a genetic or environmental insult, is characterized by pulmonary endothelial cell dysfunction and occlusive vascular remodeling. The loss or mutation of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) function, which regulates the growth and differentiation of pulmonary vascular cells, plays an important role in PAH. Switch-Independent 3a (SIN3a) is a master transcriptional scaffold that plays an essential role in regulating gene transcription and maintaining chromatin structure, and its absence or inappropriate recruitment has been associated with aberrant gene silencing. We have recently shown that SIN3a plays a central role in the hypermethylation of the BMPR2 promoter in pulmonary artery smooth muscle cells and the pathogenesis of PAH. Interestingly, BMPR2 is highly expressed in pulmonary artery endothelial cells (PAECs) and is vital to their function and survival. The molecular mechanisms of SIN3a epigenetic regulation underlying BMPR2 gene alteration in PAH-PAEC are not fully elucidated, thus, identifying the mechanism(s) that restore SIN3a expression/function in PAECs in the setting of PAH will offer new therapeutic opportunities. Our objectives in this proposal are to elucidate the role and the downstream mechanisms of SIN3a on the regulation of BMPR2 and EC dysfunction in PAH and to evaluate the therapeutic effects of targeting PAECs using lipoprotein nanoparticles encapsulating modified RNA encoding SIN3a (SIN3a.modRNA-LNP) and AAV6.hSIN3a gene therapy. While investigating how SIN3a regulates BMPR2 in PAECs, we discovered a novel molecular pathway by which SIN3a overexpression inhibits the Enhancer of Zeste Homolog 2 (EZH2) and reverses FOXK2 promoter region histone methylation and restores FOXK2 and consequently upregulates BMPR2 by increasing FOXK2 binding to the BMPR2 promoter. Based upon these findings, we hypothesize that SIN3a plays a critical role in PAEC homeostasis; mechanistically, disrupting SIN3a expression contributes to FOXK2 promoter histone modifications and thus to BMPR2 dysregulation and EC dysfunction, resulting in PAH. In this proposal, the hypothesis will be tested by pursuing the following specific aims: Aim1) To define the role of SIN3a and the epigenetic mechanisms in regulating the BMPR2 gene and PAH-PAEC dysfunction. Aim 2) To characterize SIN3a/FOXK2 interplay in BMPR2 regulation in the setting of PAH in human lung tissues and SIN3a KO mice. Aim 3) To evaluate the therapeutic efficacy of targeted SIN3a gene transfer to lung endothelial cells in small animal models of PAH. The proposed study presents the novel concept that modulation of SIN3a is essential for endothelium function in PAH disease and applies a systematic approach to integrate sophisticated functional validation assays, modified mRNA, and AAV6 gene therapy. Significantly, the proposed research is expected to vertically advance and...