Elucidating CHD in Down Syndrome with Cardiac Organoids and 3D Genome Architecture

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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.