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MOLECULAR CONSEQUENCES OF TELOMERASE DYSFUNCTION DURING HEMATOPOIETIC DEVELOPMENT

造血发育过程中端粒酶功能障碍的分子后果

基本信息

批准号：
10187638
负责人：
Luis Francisco Zirnberger Batista
金额：
$ 38.13万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10187638
关键词：
Adult Affect Age Aplastic Anemia CRISPR/Cas technology Cell Differentiation process Cell Survival Cells ChIP-seq Clinical Complex DNA Damage Data Degradation Pathway Development Disease Dyskeratosis Congenita Engineering Event Failure Functional disorder Gene Expression Gene Expression Regulation Genes Genetic Genome engineering Goals Hematopoiesis Hematopoietic Homeostasis Human Impairment In Vitro Inherited Knowledge Length Maintenance Mammalian Cell Mediating Methods MicroRNAs Modeling Molecular Mutation Output Pancytopenia Pathogenesis Pathway interactions Patient-Focused Outcomes Patients Pattern Phosphodiesterase I Pluripotent Stem Cells Population Positioning Attribute RNA Decay RNA Degradation RNA Interference RNA-Directed DNA Polymerase Regulation Research Rest Ribonucleoproteins Role Signal Transduction Source Specific qualifier value Syndrome System TP53 gene Telomerase Telomerase RNA Component Telomere Maintenance Telomere Shortening Treatment outcome biochemical tools bone marrow failure syndrome cell type cytopenia differentiation protocol disease phenotype exosome experimental study hematopoietic differentiation human disease human embryonic stem cell human pluripotent stem cell improved knock-down mutant novel patient population programs response stem cell biology stem cell differentiation stem cell function stem cells telomere therapy development tool transcriptome sequencing

项目摘要

PROJECT SUMMARY/ABSTRACT The overarching theme of our research is to decipher the molecular events responsible for bone marrow failure (BMF) in patients with impaired telomere maintenance, with the goal of improving treatment outcomes in these patients. The focus of this proposal is to use human pluripotent stem cells (hPSCs) as a novel platform to understand the molecular regulation of definitive hematopoietic impairment in cells with disease-associated mutations in telomerase, and to find novel alternatives to increase hematopoietic output in cells with dysfunctional telomeres. Dyskeratosis congenita (DC) is an inherited BMF syndrome where patients have very short telomeres for their age, typically below the first percentile length when compared to the rest of the population. All mutations discovered in DC are in genes that affect telomere homeostasis, including mutations in the telomerase components TERT, TERC, and dyskerin (DKC1). Difficulties in isolating relevant cell populations from DC patients have precluded the development of therapies against this disease. We recently overcame this limitation and combined genome engineering in hPSCs with in vitro human hematopoietic differentiation methods to create a novel and robust platform to study the mechanisms of hematopoietic failure caused by telomere dysfunction. In this submission, three specific aims are proposed that utilize this system to understand the molecular consequences of telomerase impairment during hematopoietic development, and to decipher novel targets to rescue hematopoietic output in cells harboring eroded telomeres. In aim 1, we will decipher the role of DNA damage responses (DDR) in the progression of definitive hematopoietic failure from cells with low levels of TERC. In particular, we will focus on the role of the p53 pathway in the hematopoietic impairment of cells with short telomeres, decipher the roles of miR-34a and miR-145 during abnormal hematopoiesis and decipher the genetic consequences of silencing DDR to restore hematopoietic output in cells harboring DC- associated mutations. In aim two we will decipher if the modulation of RNA decay in cells harboring mutations in DKC1 is a viable strategy to restore hematopoietic output in these cells. We will modulate TERC posttranscriptional processing and degradation by inhibition of specific RNA decay pathways. Finally, in aim 3, we will investigate if novel, non-canonical roles of TERC outside telomere maintenance can influence hematopoietic output and potentiate disease phenotypes in patients harboring mutations that reduce TERC levels, since these are commonly afflicted with severe disease phenotypes. Collectively, the studies proposed will allow us to decipher novel targets for directed therapies in patients suffering with telomere-syndromes. Our unique cellular and molecular tools, combined with our expertise in telomerase, stem cell biology and hematopoiesis puts us in an ideal position to make a significant impact in this field.

项目总结/摘要我们研究的首要主题是破译负责骨骼的分子事件端粒维持受损患者的骨髓衰竭（BMF），目的是改善治疗这些患者的结果。该提案的重点是使用人类多能干细胞（hPSC）作为一个新的平台，以了解细胞中确定性造血功能障碍的分子调控，疾病相关的端粒酶突变，并找到新的替代方案，以增加造血输出，端粒功能失调的细胞先天性角化不良（DC）是一种遗传性BMF综合征，患者的端粒非常短对于他们的年龄，与其他人群相比，通常低于第一个百分位数的长度。所有在DC中发现的突变是在影响端粒稳态的基因中，包括端粒酶组分TERT、TERC和dyskerin（DKC 1）。分离相关细胞群的困难来自DC患者的疾病已经阻碍了针对这种疾病的治疗的发展。我们最近克服了这种限制和hPSC中的基因组工程与体外人造血分化的组合方法来创建一个新的和强大的平台，研究造血功能衰竭的机制，端粒功能障碍在这份提交文件中，提出了三个具体目标，利用这一系统来了解造血发育过程中端粒酶损伤的分子后果，并破译新的目的是拯救含有受损端粒的细胞中的造血输出。在目标1中，我们将破译 DNA损伤反应（DDR）在低分化造血干细胞的确定性造血功能衰竭进展中的作用 TERC的水平。特别是，我们将重点关注p53通路在造血损伤中的作用，短端粒的细胞，破译miR-34 a和miR-145在异常造血过程中的作用，破译沉默DDR的遗传后果，以恢复携带DC的细胞中的造血输出，相关突变在目标二中，我们将破译是否在携带突变的细胞中RNA衰减的调制是恢复这些细胞中造血输出的可行策略。我们将调整TERC 通过抑制特定的RNA衰变途径，抑制转录后加工和降解。最后，在目标3中，我们将研究TERC在端粒维持之外的新的、非经典的作用是否会影响携带降低TERC突变的患者的造血输出和增强疾病表型水平，因为这些通常患有严重的疾病表型。总的来说，提出的研究将使我们能够破译定向治疗的新靶点，患有端粒综合症的患者。我们独特的细胞和分子工具，结合我们的在端粒酶，干细胞生物学和造血方面的专业知识使我们处于一个理想的位置，在这个领域的影响。