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

• 批准号: 8760558
• 负责人: RUDOLF JAENISCH
• 金额: $ 60.6万
• 依托单位: WHITEHEAD INSTITUTE FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 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/8760558
• 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; Genomics; Goals; Growth Factor; 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; public health relevance; relating to nervous system; research study; small molecule

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(申请人提供)：雷特综合征(RTT)，由DNA结合蛋白MECP2突变引起，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度表达，如MECP2复制功能获得综合症中所见，都会导致RTT样综合征，这表明MECP2水平升高对神经系统的损害与MECP2缺乏一样。虽然MECP2缺乏导致RTT的细胞自主性已得到证实，但最近的证据表明，另一种基于MECP2在星形胶质细胞中表达的治疗效果或与野生型小胶质细胞移植的细胞非自主性机制。这些观察结果以及认识到MECP2的重新表达或小分子治疗可以阻止成年Rett小鼠的疾病进展，甚至可以逆转症状，这表明MECP2是维持神经元功能所必需的。虽然这些结果令人兴奋，并建议对人类进行合理的治疗，但在使用人类细胞作为读数的明确定义的实验系统中评估治疗策略是至关重要的。该项目旨在建立一个利用人RTT神经元来阐明MECP2在基因表达中的作用的平台，并允许在培养和体内条件下评估候选治疗方法。利用人iPS细胞来源的神经元培养，最初的目标是建立一个实验范式，允许确定MECP2在基因调控中的分子角色，并在人类突变神经元中提供强大和可量化的疾病相关表型读数。我们将使用CHIP-SEQ等分子方法将MECP2的结合位点定位到5mC和5hmC修饰的基因组位置，并剖析基因的激活和抑制模式。此外，我们将确定MECP2和MECP2相互作用伙伴的靶基因，并阐明MECP2靶基因在功能RTT的丧失和获得中的解除调控。该提案的一个主要焦点是建立一个平台，允许评估在体外和体内条件下逆转人类神经元RTT表型的治疗策略的有效性。(I)为了克服传统神经2D培养系统的局限性，我们将以RTT ES或iPS细胞为起点来产生人脑器官培养物。这将使在一个定义明确的3D测试系统中分析细胞间的相互作用和潜在的治疗剂成为可能。(Ii)我们将把绿色荧光蛋白标记的神经元前体移植到发育中的小鼠大脑中，以产生携带人类MECP2突变神经元的动物。通过允许人类神经元整合到完整的小鼠大脑中，我们寻求建立一个临床相关的平台，对可能有利于RTT患者治疗的生长因子和小分子化合物进行体内验证。