Using pluripotent stem cells to study gene dosage-dependent immunodeficiency

使用多能干细胞研究基因剂量依赖性免疫缺陷

• 批准号: 9096412
• 负责人: Rene Maehr
• 金额: $ 51.37万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-01 至 2017-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9096412
• 关键词: Address; Affect; Autologous; Binding; Biological Assay; Biological Models; Cell Differentiation process; Cell Line; Cell Therapy; Cell physiology; Cells; Child; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Cues; Data; Defect; Development; DiGeorge Syndrome; Disease; Dose; Embryo; Endoderm; Endoderm Cell; Enhancers; Epigenetic Process; Event; Functional disorder; Gene Dosage; Gene Expression; Gene Targeting; Generations; Genes; Genetic Transcription; Genome; Genomics; Goals; Health; Human; Human Development; Immunologic Deficiency Syndromes; In Vitro; Individual; Investigation; Knowledge; Lead; Link; Methodology; Molecular; Mus; Organogenesis; Pathway interactions; Patients; Phenotype; Pluripotent Stem Cells; Population; Positioning Attribute; Primitive foregut structure; Process; Proximal Enhancer Element; Regulation; Replacement Therapy; Rodent Model; Role; Series; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Staging; Stem cells; Surveys; System; Technology; Testing; Thymic Tissue; Thymic epithelial cell; Thymus Gland; Time; Tissue Transplantation; Transcript; Transcriptional Regulation; Transplantation; Variant; Work; base; chromatin modification; combinatorial; dosage; epigenetic profiling; histone modification; human embryonic stem cell; human stem cells; induced pluripotent stem cell; innovation; insight; mouse model; novel; nuclease; prevent; screening; stem cell differentiation; thymic aplasia; thymic hypoplasia; transcription factor; transcriptomics

DESCRIPTION (provided by applicant): The epigenetic basis for human immunodeficiencies that result from embryonic developmental defects, e.g. thymus hypoplasias, is poorly understood. This is due to sparse disease-relevant patient material available to study and limited rodent models that often differ in genomic organization and phenotype when compared to human. For example, DiGeorge syndrome (DGS), its associated thymus dysfunction, and ensuing immunodeficiency, have been linked to a dose-dependent haploinsufficiency of TBX1 during pharyngeal foregut development. The dose-sensitive regulation and the functional network it operates in remain largely elusive. Advances in the generation of disease-specific human pluripotent stem cells (PSCs) through reprogramming and gene targeting have led to opportunities for disease investigation and ultimately for the development of autologous cell replacement therapies. The primary goal of this project to understand the regulation of TBX1 expression and its dose-dependent function in human stem cell differentiation products relevant to the developmental defect and impending immunodeficiency seen in subjects with DiGeorge syndrome. In aim 1 and 2 we will focus on characterizing factors that we identified to be involved in the regulation of TBX1 expression. Aim 1 will focus on characterizing signaling pathways that control TBX1 expression in thymus developmental defect-relevant differentiation products. We have conducted an innovative combinatorial analysis of signaling pathways that influence TBX1 expression in DGS-relevant cell populations. We will characterize lead pathways for the ability to control cell differentiation and function. In aim 2 we will characteriz epigenetic events that regulate transcriptional control of the tbx1 locus. We have previously identified specific chromatin modifications that are associated with activation of tbx1 gene expression. We will characterize the events and factors that are involved in activation and regulation of tbx1 gene expression. In combination, aim 1 and aim 2 will provide us with a comprehensive insights on how TBX1 expression is regulated. We expect that these mechanistic insights can be applied in vitro to target tbx1-dosage effects that are associated with the immunodeficiency phenotype as seen in subjects with DGS. In aim 3 we will utilize reprogramming, gene targeting and regulated gene expression technology to address the molecular consequence of varying TBX1 expression levels. We expect that this study will not only identify key targets of TBX1 in developmental defect relevant cells, but will also provide unique insights on the functional consequence and the mechanism of tbx1 dosage-dependent activity. The detailed insights on the functional network TBX1 operates in DGS-associated immunodeficiency- relevant cell populations, and how this network is affected by dosage variations in cellular TBX1 levels, will elucidate the underlying mechanism of the developmental defect that results in immunodeficiency. Further, the insights can likely be applied to other dosage-dependent defects in organogenesis and will provide novel targets to overcome such defects in vitro for stem cell-based cell therapies. This work combines the expertise of Dr. Maehr in PSC differentiation, endoderm development and genome manipulation, of Dr. Rando in epigenetic profiling technology, and of Dr. Garber in integrating epigenetic and transcriptomic data to understand cell fate decisions.

描述（由申请人提供）：由胚胎发育缺陷（如胸腺发育不全）引起的人类免疫缺陷的表观遗传学基础尚不清楚。这是由于可用于研究的与疾病相关的患者材料很少，并且与人类相比，啮齿动物模型通常在基因组组织和表型上存在差异。例如，diggeorge综合征（DGS）及其相关的胸腺功能障碍和随后的免疫缺陷，与咽部前肠发育期间TBX1的剂量依赖性单倍不足有关。对剂量敏感的调控及其运作的功能网络在很大程度上仍然难以捉摸。通过重编程和基因靶向产生疾病特异性人类多能干细胞（PSCs）的进展为疾病研究带来了机会，并最终促进了自体细胞替代疗法的发展。本项目的主要目标是了解TBX1表达的调控及其在与DiGeorge综合征受试者的发育缺陷和即将发生的免疫缺陷相关的人干细胞分化产物中的剂量依赖性功能。在目标1和目标2中，我们将重点描述我们确定的参与TBX1表达调控的因素。目的1将重点研究胸腺发育缺陷相关分化产物中控制TBX1表达的信号通路。我们对影响dgs相关细胞群中TBX1表达的信号通路进行了创新的组合分析。我们将描述控制细胞分化和功能的能力的导联途径。在目标2中，我们将描述调节tbx1位点转录控制的表观遗传事件。我们之前已经确定了与tbx1基因表达激活相关的特定染色质修饰。我们将描述参与tbx1基因表达激活和调控的事件和因素。aim 1和aim 2的结合将为我们提供关于TBX1表达如何调控的全面见解。我们期望这些机制见解可以在体外应用于与DGS患者的免疫缺陷表型相关的tbx1剂量效应。在目标3中，我们将利用重编程、基因靶向和调控基因表达技术来解决TBX1表达水平变化的分子后果。我们期望这项研究不仅能确定TBX1在发育缺陷相关细胞中的关键靶点，还能对TBX1剂量依赖性活性的功能后果和机制提供独特的见解。TBX1功能网络在dgs相关免疫缺陷相关细胞群中运作的详细见解，以及该网络如何受到细胞TBX1水平剂量变化的影响，将阐明导致免疫缺陷的发育缺陷的潜在机制。此外，这些见解可能应用于器官发生中的其他剂量依赖性缺陷，并将为体外干细胞治疗提供克服此类缺陷的新靶点。这项工作结合了Maehr博士在PSC分化、内胚层发育和基因组操作方面的专业知识，Rando博士在表观遗传分析技术方面的专业知识，以及Garber博士在整合表观遗传和转录组学数据以了解细胞命运决定方面的专业知识。