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

描述（由申请人提供）：Rett综合征（RTT）由DNA结合蛋白MECP 2突变引起，是女性智力低下的最常见原因之一。该基因的功能缺失突变以及过度表达（如在MECP 2功能复制获得综合征中所见）均导致RTT样综合征，表明增加的MECP 2水平可能与MECP 2缺陷一样对神经系统有害。虽然已经确定MECP 2缺陷以细胞自主方式引起RTT，但最近的证据指出了基于星形胶质细胞中MECP 2表达的治疗效果或野生型小胶质细胞移植的额外细胞非自主机制。这些观察结果以及认识到MECP 2的再表达或用小分子治疗可以停止疾病进展，甚至可以逆转成年Rett小鼠中的症状，表明MECP 2是维持神经元功能所必需的。虽然这些结果令人兴奋，并表明在人类中的合理治疗，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至关重要。该项目旨在建立一个平台，利用人类RTT神经元来阐明MECP 2在基因表达中的作用，并允许在培养和体内条件下评估候选治疗。使用人iPS细胞衍生的神经元培养物，最初的目标是建立一种实验范式，其允许定义MECP 2在基因调控中的分子作用，并在人突变神经元中提供稳健且可量化的疾病相关表型读数。我们将使用诸如CHIP-seq的分子方法来映射MECP 2与5 mC和5 hmC修饰的基因组位点的结合位点，并剖析基因激活和抑制的模式。此外，我们将确定MECP 2和MECP 2相互作用的伴侣的靶基因，并阐明MECP 2靶基因在功能RTT的丧失和获得中的失调。该提案的一个主要重点是建立一个平台，允许评估治疗策略的疗效，以逆转在体外和体内条件下的人神经元的RTT表型。(i)为了克服传统神经2D培养系统的局限性，我们将使用RTT ES或iPS细胞作为起点来产生人脑类器官培养物。这将使细胞间的相互作用和潜在的治疗药物的分析在一个明确的三维测试系统。(ii)我们将GFP标记的神经元前体移植到发育中的小鼠大脑中，以产生携带人类MECP 2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，以进行生长因子和小分子化合物的体内验证，这可能有利于RTT患者的治疗。

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.