Genetically engineered human pluripotent stem cells as a platform to define the b

基因工程人类多能干细胞作为定义 b 的平台

基本信息

批准号：
8926472
负责人：
RUDOLF JAENISCH
金额：
$ 54.54万
依托单位：
WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
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：
2014-09-15 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8926472
关键词：
Adult Animals Astrocytes Binding Binding Sites Brain Cell Communication Cells Cerebrum ChIP-seq Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Coculture Techniques DNA-Binding Proteins Development Disease Disease Progression Embryo Epigenetic Process Evaluation Female Gene Activation Gene Expression Gene Expression Regulation Gene Mutation Gene Targeting Genes Genetic Engineering Genomic Segment Genomics Goals Growth Factor Health Human In Vitro Label Lead Maintenance Maps Mediating Mental Retardation Microglia Modification Molecular Mus Mutant Strains Mice Mutation Nervous system structure Neuroglia Neurons Newborn Infant Organoids Patients Phenotype Physiological Physiology Pluripotent Stem Cells Proteins Rett Syndrome Role Site Staging Symptoms Syndrome System Testing Therapeutic Therapeutic Agents Therapeutic Effect Transcription Repressor/Corepressor Transgenes Transgenic Organisms Transplantation Treatment Efficacy Validation X Chromosome base cell type clinically relevant embryonic stem cell gain of function gain of function mutation gene induction gene repression in utero in vivo induced pluripotent stem cell insight interest loss of function loss of function mutation mutant nerve stem cell overexpression postnatal protein complex relating to nervous system research study small molecule

项目摘要

DESCRIPTION (provided by applicant): Rett syndrome (RTT), caused by mutation of the DNA binding protein MECP2, is one of the most common causes for mental retardation in females. Both loss of function mutations of the gene as well as overexpression such as seen in the MECP2 duplication gain of function syndrome, lead to RTT-like syndrome indicating that increased MECP2 levels can be equally detrimental to the nervous system as MECP2 deficiency. While it has been well established that MECP2 deficiency causes RTT in a cell autonomous manner, recent evidence points to an additional cell-non-autonomous mechanism based on therapeutic effects of MECP2 expression in astrocytes or on transplantation with wild type microglia. These observations as well as the recognition that re- expression of MECP2 or treatment with small molecules can halt disease progression and can even revert symptoms in the adult Rett mouse suggest that MECP2 is required for the maintenance of neuronal function. While these results are exciting and suggest a rational treatment in humans, it is crucial to assess therapeutic strategies in well-defined experimental systems using human cells as readout. This project seeks to set up a platform that utilizes human RTT neurons for clarifying the roles of MECP2 in gene expression and that permits the evaluation of candidate treatments in culture as well as under in vivo conditions. Using human iPS cell- derived neuronal cultures, the initial goal is to establish an experimental paradigm that allows defining the molecular role o MECP2 in gene regulation and to provide a robust and quantifiable disease-relevant phenotypic readout in human mutant neurons. We will use molecular approaches such as CHIP-seq to map binding sites of MECP2 to 5mC and 5hmC modified genomic sites and dissect the modes of gene activation and repression. Furthermore, we will identify target genes of MECP2 and MECP2- interacting partners and clarify the deregulation of MECP2 target genes in loss and gain of function RTT. A major focus of the proposal is to establish a platform that allows assessing the efficacy of therapeutic strategies to reverse the RTT phenotype of human neurons under in vitro and in vivo conditions. (i) To overcome limitations of conventional neural 2D culture systems we will use RTT ES or iPS cells as starting point to generate human cerebral organoid cultures. This will enable the analysis of cell-cell interactions and of potentia therapeutic agents in a well-defined 3D test system. (ii) We will transplant GFP-marked neuronal precursors into the developing mouse brain to generate animals that carry human MECP2 mutant neurons incorporated into their brain. By allowing the human neurons to integrate into the intact mouse brain, we seek to establish a clinically relevant platform to perform in vivo validation of growth factors and small molecule compounds that could be beneficial for the treatment of RTT patients.

描述（由申请人提供）：由DNA结合蛋白MECP2突变引起的RETT综合征（RTT）是女性智力低下的最常见原因之一。基因功能突变的丧失以及过表达，例如在MECP2重复综合征中看到的，导致RTT样综合征，表明MECP2水平升高可能同样对MECP2缺乏症对神经系统同样有害。虽然已经很好地确定MECP2缺乏会以细胞自主的方式引起RTT，但最近的证据表明，基于MECP2表达在星形胶质细胞中或对野生型小胶质细胞移植的治疗作用的额外的单个自主机制。这些观察结果以及认识到MECP2或用小分子进行处理可以阻止疾病进展，甚至可以恢复成年Rett小鼠的症状，这表明MECP2维持神经元功能是必需的。尽管这些结果令人兴奋，并暗示了人类的合理治疗方法，但使用人类细胞作为读数来评估定义明确的实验系统的治疗策略至关重要。该项目旨在建立一个使用人类RTT神经元来澄清MECP2在基因表达中的作用的平台，并允许评估培养物中候选治疗以及在体内条件下的评估。使用人IPS细胞造成的神经元培养物，最初的目标是建立一个实验范式，该范式允许在基因调节中定义MECP2的分子作用，并在人类突变神经元中提供强大且可量化的疾病相关的表型读数。我们将使用分子方法（例如chip-seq）将MECP2的结合位点映射到5MC和5HMC修饰的基因组位点，并剖析基因激活和抑制的模式。此外，我们将确定MECP2和MECP2 - 相互作用伙伴的靶基因，并阐明MECP2靶基因在损失和功能RTT的损失和增益中的放松管制。该提案的主要重点是建立一个平台，以评估治疗策略在体外和体内条件下扭转人类神经元的RTT表型的功效。（i）为了克服常规神经2D培养系统的局限性，我们将使用RTT ES或IPS细胞作为产生人类脑器官培养的起点。这将使在明确定义的3D测试系统中对细胞 - 细胞相互作用和Potentia治疗剂进行分析。（ii）我们将将GFP标记的神经元前体移植到发育中的小鼠大脑中，以产生将人类MECP2突变神经元掺入大脑的动物。通过允许人类神经元整合到完整的小鼠大脑中，我们试图建立一个与临床相关的平台，以对生长因子和小分子化合物进行体内验证，这可能有益于治疗RTT患者。