Abstract Body: The G allele of human single nucleotide polymorphism (SNP) rs217727 is associated with reduced systolic, diastolic, and pulse pressure, compared to the A allele. rs217727 is located in exon 5 of the long non-coding RNA H19. H19 also encodes miR-675 from exon 1. The nearest protein-coding gene, MRPL23, is 48.3 kb away from rs217727. In whole blood analysis, rs217727 has been identified as an expression quantitative trait locus for MRPL23 and troponin T3 fast skeletal type (TNNT3, 76.1kb away). The goal of this study was to identify the effector genes and pathways of rs217727 in endothelial cells, which play a critical role in blood pressure (BP) homeostasis. We differentiated human induced pluripotent stem cells (iPSCs) into endothelial cells (iECs), followed by VE-cadherin-based purification. Chromatin conformation analysis in iECs using Micro-C revealed that rs217727 shares chromatin loops with several genes in a genomic region from 220.7 kb upstream to 179.3 kb downstream of the rs217727. Using CRISPR-Cas9, we generated isogenic iPSC lines homozygous for either the low BP (G) or high BP (A) allele. After six rounds of iEC differentiation, gene expression was assessed using targeted RT-qPCR. iECs with the high BP allele showed significantly reduced expression of H19 (0.58±0.06-fold relative to iECs with the low BP allele), TNNT3 (0.44±0.04-fold), and lymphocyte specific protein 1 (LSP1, 142.6 kb away; 0.12±0.02-fold), and a slight increase in CD81 (380.5 kb away; 1.09±0.03-fold) (n≥34, p<0.01). Additionally, miR-675-5p was significantly downregulated in iECs with the high BP allele (0.31±0.10-fold, n≥15, p<0.01). No significant expression differences were observed for MRPL23, insulin-like growth factor 2 (IGF2,141.6 kb away), Synaptotagmin 8 (SYS8, 169.9 kb away), CTSD (231.8 kb away), or MRPS6 (on a different chromosome). LSP1 has been reported to promote eNOS expression. Reduced LSP1 expression in iECs with the high BP allele was accompanied by decreased eNOS level measured by RT-qPCR (0.63±0.04-fold, n≥27, p<0.01) and western blot (0.66±0.15-fold, n=4, p=0.06). H19 is known to modulate apoptotic pathways. TUNEL assays indicated a two-fold increase in apoptosis in iECs with the high BP allele. These findings suggest that rs217727 modulates local gene expression which may, in turn, influence endothelial function relevant to BP regulation.