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）的儿童出现轻度至重度的血液学异常。几乎所有DS患者都表现出造血功能紊乱的体征，包括白细胞减少症、淋巴细胞减少症和巨红细胞症。大约10%的DS婴儿发展为暂时性骨髓增生性疾病（TMD）; 10- 30%后来发展为急性白血病，最常见的是急性巨核细胞白血病（AMKL）。GATA 1转录因子的截短突变始终存在于DS TMD和AMKL中，其效应似乎取决于体质性21三体，其本身可导致多个谱系的过度增殖状态。导致DS患者造血缺陷的途径尚不清楚。该提案旨在研究DS中的造血作用，使用一种非常新颖的“染色体治疗”方法，通过位点特异性靶向人XIST（X-失活特异性转录本）转基因来沉默DS诱导的多能干（iPS）细胞中的整个三体21号染色体（Chr 21）。该系统，已经在手，提供快速和强大的沉默的Chr 21基因，从而允许直接比较平行培养的其他相同的DS干细胞，有和没有过表达的Chr 21基因。特定目的：DS iPS细胞中三体Chr 21的全染色体沉默如何纠正DS的造血缺陷？我们将在3个子目标中解决这个问题：Subaim A。我们将确定是否沉默三体Chr 21纠正异常的体外造血表型的DS。我们将使用流式细胞术和造血集落测定法，在有和没有多西环素诱导Chr 21靶向XIST转基因的情况下，测量来自人DS iPS细胞的造血祖细胞和成熟细胞的产生。苏拜姆B。我们将确定三体Chr 21的沉默是否影响Chr 21和非Chr 21基因的表达。我们将通过高通量RNA测序检查校正和未校正DS造血细胞的转录组，并分析与子目标A中鉴定的造血表型相关的基因表达途径。苏拜姆角我们将确定GATA 1 s表达是否诱导DS iPS细胞中的Chr 21依赖性骨髓增殖和基因表达。在具有诱导型Chr 21沉默的DS iPS细胞中，野生型GATA 1将被敲入的GATA 1 s等位基因取代。将在三体与二体GATA 1 s半合子造血祖细胞中分析表型和基因表达谱，以确定21三体和GATA 1截短组合对子目标B中鉴定的网络的影响。这项集中的、高影响力的研究应该为一种可能适用于DS造血和其他并发症的新型“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.