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

唐氏综合症 iPS 细胞的造血作用：通过 21 号染色体沉默进行校正

基本信息

批准号：
9069836
负责人：
JEANNE Bentley LAWRENCE
金额：
$ 29.15万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-07-01 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9069836
关键词：
Abbreviations Acute Lymphocytic Leukemia Acute Megakaryocytic Leukemias Acute Myelocytic Leukemia Acute leukemia Address Affect Alleles Biological Assay Biological Models Blood Cells Child Chromosomes Chromosomes, Human, Pair 21 Complement Constitutional Defect Development Disease Dosage Compensation (Genetics)Down Syndrome Doxycycline Erythroid Female Fetal Liver Flow Cytometry Foundations GATA1 gene Gene Expression Gene Expression Profile Genes Genetic Variation Hand Health Hematological Disease Hematology Hematopoiesis Hematopoietic High-Throughput RNA Sequencing Human In Vitro Individual Infant Investigation Knock-in Leukopenia Lymphopenia Measures Mediating Megakaryocytes Methods Molecular Mutation Myeloproliferation Myeloproliferative disease National Institute of Diabetes and Digestive and Kidney Diseases Pathology Pathway interactions Patients Phenotype Prevention Production Protein Isoforms RNA Sequence Analysis Research Risk Site Stem cells System Technology Therapeutic Transcript Transgenes Trisomy Untranslated RNA X Chromosome autosome base epigenetic variation high risk induced pluripotent stem cell innovation insight interest knock-down leukemia leukemogenesis male novel overexpression progenitor research study therapeutic target therapy development transcriptome transient myeloproliferative disorder

项目摘要

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.

描述（由申请人提供）：许多DS儿童（三体术）患有血液学异常，范围从轻度到重度。几乎所有患有DS的人都均显示出造血无序的迹象，包括白细胞减少症，淋巴细胞减少症和大细胞增多症。大约10％的DS婴儿患有短暂的骨髓增生性疾病（TMD）； 10-30％后来患有急性白血病，最常见的是急性巨核细胞白血病（AMKL）。 DS TMD和AMKL中始终存在GATA1转录因子的截短突变，其效果似乎取决于21宪法三体构造，该宪法三体术本身在多个谱系中引起了过度增殖状态。 DS患者的造血缺陷构成的途径知之甚少。该提案旨在通过使用人Xist（X-Inactive特异性转录本）转基因的位点特异性靶向DS的DS中的造血作用，以使整个Trisomic染色体21（ChR21）在DS诱导的Pluripotent stem（IPS）细胞中静音。该系统已经掌握在手上，提供了CHR 21基因的快速而稳健的沉默，从而可以直接比较其他相同的DS干细胞的平行培养物，并具有CHR21基因过表达的情况。具体目的：DS IPS细胞中Trisomic CHR21的整个染色体沉默如何纠正DS的造血缺陷？我们将在3个Subiaims中解决这个问题：SubaimA。我们将确定Trisomic CHR21沉默是否纠正DS的体外造血表型的异常。我们将使用流式细胞仪和造血菌落测定法测量和不具有强力霉素诱导ChR21靶向的XIST转基因的造血祖细胞和成熟细胞的产生，并具有有和不具有强力霉素的诱导。 Subaim B.我们将确定trisomic 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测序进行校正和未矫正的DS造血细胞的转录组，并分析与Subaim A. A. Subaim C中鉴定的造血表型相关的基因表达途径。野生型GATA1将被带有诱导CHR21沉默的DS IPS细胞中的敲入GATA1S等位基因所取代。将分析表型和基因表达谱图在三化与动态性GATA1s中半细胞造血祖细胞中，以确定21组合三体切开术和GATA1截断的作用，而GATA1截断对Subaim B中确定的网络对subaim B中确定的网络B.这种高度施加的临时型号应为“临床”的临床施加，以便为“ hempopy sirdect”提供了一个更加典型的“ hem poff”，以使其具有更大的外形式旋转式，以使其具有新颖的形式性的纯种，' DS。提出的实验还应确定用于治疗或预防DS造血缺陷的潜在治疗靶标。