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

项目摘要 唐氏综合征（DS）或21三体（T21）是世界范围内普遍存在的染色体缺陷，通常与 多种心血管和血液异常先天性心脏病（CHD），影响高达50%的 DS患者是婴儿发病和死亡的主要原因。冠心病主要是由失调引起的， 转录途径。虽然基因组的3D空间组织已经成为一个关键的 调节染色质可及性的机制，其在CHD细胞间变异性和基因调控中的作用 仍然知之甚少。在目标1中，我们将利用3D血管化心脏类器官（3D-vCO）和多个 组学方法来阐明DS相关心血管并发症的机制。我们会委聘 单细胞RNA测序（scRNA-seq）和单细胞ATAC测序（scATAC-seq）来分析细胞内的DNA序列。 血管网络和心肌细胞（CM）特征，从而建立DS-CHD的表型。我们 这项研究将特别侧重于分析血管网络和心肌细胞（CM）的特征， 建立唐氏综合征相关的先天性心脏缺陷（DS-CHDs）的表型。在目标2中，我们 在3D-vCO培养期间的特定时间点（第天）进行Dip-C（二倍体染色体构象捕获） 第3天、第6天和第16天）。我们将分析染色体间相互作用（ICI） 和3D-vCO中基因组的3D结构，即关注CM、CF和EC之间的差异 在DS-CHD和DS-nonCHD患者之间。我们期望获得新的机械见解， DS-CHD的发病机制，并促进旨在减轻 并逆转病变心脏中的细胞异常。

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.