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）：许多患有 DS（21 三体性）的儿童会出现从轻微到严重的血液学异常。几乎所有 DS 患者都表现出造血紊乱的迹象，包括白细胞减少、淋巴细胞减少和大红细胞增多。大约 10% 的 DS 婴儿会出现短暂性骨髓增生性疾病 (TMD)； 10-30% 随后发展为急性白血病，最常见的是急性巨核细胞白血病 (AMKL)。 GATA1 转录因子的截短突变始终存在于 DS TMD 和 AMKL 中，其影响似乎依赖于 21 体性三体性，而 21 性体性本身会导致多个谱系的过度增殖状态。 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 失活特异性转录本）转基因来沉默 DS 诱导多能性中的整个三体染色体 21 (Chr21)干细胞（iPS）。该系统已经投入使用，可以快速、稳健地沉默 Chr 21 基因，从而可以直接比较其他方面相同的 DS 干细胞的平行培养物，无论是否过度表达 Chr21 基因。具体目的： 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 3 个子目标中解决这个问题：Subaim 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测定来测量人类 DS iPS 细胞的造血祖细胞和成熟细胞的产生，无论是否使用多西环素诱导 Chr21 靶向 XIST 转基因。 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 测序检查校正和未校正 DS 造血细胞的转录组，并分析与 subaim A. Subaim C 中鉴定的造血表型相关的基因表达途径。我们将确定 GATA1s 表达是否诱导 Chr21 依赖性骨髓增殖和基因表达在 DS iPS 细胞中。在具有诱导型 Chr21 沉默的 DS iPS 细胞中，野生型 GATA1 将被敲入 GATA1s 等位基因取代。将在三体性与二体性 GATA1s 半合子造血祖细胞中分析表型和基因表达谱，以确定 21 三体性和 GATA1 联合截短对 subaim B 中确定的网络的影响。这项重点突出、影响力大的研究应该为一种新型“染色体疗法”，可能适用于 DS 的造血和其他并发症。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。