PROJECT SUMMARY Down syndrome (DS) or Trisomy 21 (T21) is a prevalent chromosomal defect worldwide, often associated with multiple cardiovascular and hematological anomalies. Congenital heart disease (CHD), affecting up to 50% of DS patients, is a leading cause of morbidity and mortality in infants. CHD is primarily caused by dysregulated transcriptional pathways. Although the 3D spatial organization of the genome has emerged as a crucial mechanism for regulating chromatin accessibility, its role in cell-to-cell variability and gene regulation within CHD remains poorly understood. In Aim 1, we will utilize 3D-vascularized cardiac organoids (3D-vCOs) and multi- omics approaches to elucidate the mechanisms of DS-associated cardiovascular complications. We will employ single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) to analyze the vascular network and cardiomyocyte (CM) characteristics, thereby establishing phenotypes of DS-CHDs. Our study will specifically focus on analyzing the vascular network and cardiomyocyte (CM) characteristics to establish phenotypes of Down syndrome-associated congenital heart defects (DS-CHDs). In Aim 2, we will perform Dip-C (Diploid Chromosome Conformation Capture) at specific timepoints during 3D-vCO culture (Day 3, Day 6, and Day 16) using DS patients iPSC lines. We will analyze the inter-chromosomal interactions (ICIs) and 3D architecture of the genome in 3D-vCOs, namely focusing on differences among the CMs, CFs, and ECs between DS-CHD versus DS-nonCHD patients. We anticipate gaining new mechanistic insights into the pathogenesis of DS-CHDs and facilitating the development of novel therapeutic strategies aimed at mitigating and reversing cellular abnormalities in the diseased heart.

PROJECT NARRATIVE The study aims to investigate the molecular mechanisms of Down syndrome-associated congenital heart disease (DS-CHD), which affects approximately 50% of individuals with Down syndrome (DS) or Trisomy 21 (T21). DS- CHD is a significant contributor to infant morbidity and mortality. To achieve this, the study will utilize 3D- vascularized cardiac organoids, multi-omics analysis, and 3D genome architecture analysis to examine differences in gene expression and developmental pathways between individuals with DS and CHD and those without CHD. The anticipated outcome of this research is to gain new mechanistic insights into the development of DS-CHDs and potentially facilitate the development of novel therapeutic strategies to address cellular abnormalities in the affected heart.