Hematopoiesis in Down Syndrome iPS cells: Correction by Chromosome 21 Silencing

描述（由申请人提供）：许多患有DS（21三体）的儿童会出现从轻微到严重的血液异常。几乎所有DS患者都有造血功能紊乱的症状，包括白细胞减少症、淋巴细胞减少症和巨噬细胞增多症。大约10%的退行性滑移婴儿会发展为一过性骨髓增生性疾病（TMD）；10- 30%后来发展为急性白血病，最常见的是急性巨核母细胞白血病（AMKL）。GATA1转录因子的截断突变一直存在于DS - TMD和AMKL中，其影响似乎依赖于本体质21三体，而21三体本身会在多个谱系中引起超增殖状态。导致退行性椎体滑移患者造血缺陷的途径尚不清楚。本研究旨在研究DS的造血功能，使用一种高度新颖的“染色体治疗”方法，通过位点特异性靶向人类XIST （X-inactive specific transcript）转基因来沉默DS诱导的多能干细胞（iPS）中的整个三体21号染色体（Chr21）。该系统，已经掌握，提供快速和强大的沉默Chr21基因，从而允许直接比较平行培养的其他相同的干细胞，有或没有Chr21基因的过表达。具体目的：在DS iPS细胞中，三体Chr21的全染色体沉默如何纠正DS的造血缺陷？我们将在三个子目标中解决这个问题：子目标A.我们将确定三体Chr21的沉默是否可以纠正DS的异常体外造血表型。我们将使用流式细胞术和造血集落试验，测量在强力霉素诱导和不诱导chr21靶向XIST转基因的情况下，人类DS - iPS细胞产生的造血祖细胞和成熟细胞。Subaim B.我们将确定三体Chr21的沉默是否影响Chr21和非Chr21基因的表达。我们将通过高通量RNA测序来检测校正和未校正的DS造血细胞的转录组，并分析与subaim A. subaim c中发现的造血表型相关的基因表达途径。我们将确定GATA1s的表达是否诱导了DS iPS细胞中chr21依赖性的骨髓增殖和基因表达。在诱导Chr21沉默的DS iPS细胞中，野生型GATA1将被敲入的GATA1s等位基因所取代。我们将分析三体和二体GATA1半合子造血祖细胞的表型和基因表达谱，以确定21三体和GATA1联合截断对b项中确定的网络的影响。这项重点突出、影响深远的研究将为一种可能适用于DS的造血和其他并发症的新形式的“染色体治疗”提供原理证明。拟议的实验还应确定治疗或预防DS造血缺陷的潜在治疗靶点。

DESCRIPTION (provided by applicant): Many children with DS (trisomy 21) develop hematological abnormalities, ranging from mild to severe. Virtually all individuals with DS show signs of disordered hematopoiesis, including leukopenia, lymphopenia, and macrocytosis. Approximately 10% of DS infants develop a transient myeloproliferative disorder (TMD); 10- 30% later develop acute leukemia, most often acute megakaryoblastic leukemia (AMKL). Truncation mutations of the GATA1 transcription factor are consistently present in DS TMD and AMKL and its effects appear dependent upon constitutional trisomy 21 which itself causes a hyperproliferative state in multiple lineages. The pathways that underlie hematopoietic defects in DS patients are poorly understood. This proposal seeks to investigate hematopoiesis in DS, using a highly novel method of "chromosome therapy" by site-specific targeting of a human XIST (X-inactive specific transcript) transgene to silence the entire trisomic chromosome 21 (Chr21) in DS induced pluripotent stem (iPS) cells. This system, already in hand, provides rapid and robust silencing of Chr 21 genes, thus allowing direct comparison of parallel cultures of otherwise identical DS stem cells, with and without over-expression of Chr21 genes. Specific Aim: How does whole chromosome silencing of the trisomic Chr21 in DS iPS cells correct the hematopoietic defects of DS? We will address this question in 3 subaims: Subaim A. We will determine whether silencing of trisomic Chr21 corrects the abnormal in vitro hematopoietic phenotype of DS. We will measure production of hematopoietic progenitor and mature cells from human DS iPS cells, with and without doxycycline induction of a Chr21-targeted XIST transgene, using flow cytometry and hematopoietic colony assays. Subaim B. We will determine whether silencing of trisomic Chr21 affects expression of Chr21 and non- Chr21 genes. We will examine the transcriptome of corrected and uncorrected DS hematopoietic cells by high-throughput RNA sequencing and analyze gene expression pathways associated with the hematopoietic phenotype identified in subaim A. Subaim C. We will determine whether GATA1s expression induces Chr21-dependent myeloproliferation and gene expression in DS iPS cells. Wild type GATA1 will be replaced by a knock-in GATA1s allele in DS iPS cells with inducible Chr21 silencing. The phenotype and gene expression profile will be analyzed in trisomic versus disomic GATA1s hemizygous hematopoietic progenitors to determine the effect of combined trisomy 21 and GATA1 truncation on the networks identified in subaim B. This focused, high-impact study should provide a proof of principle for a novel form of "chromosomal therapy" potentially applicable to hematopoietic and other complications of DS. The proposed experiments should also identify potential therapeutic targets for treatment or prevention of DS hematopoietic defects.