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MECP2突变引起，是女性智力迟钝的最常见原因之一。该基因的功能突变缺失和功能综合征中MECP2重复获得的过表达都会导致rtt样综合征，这表明MECP2水平的升高与MECP2缺乏对神经系统同样有害。虽然已经确定MECP2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或野生型小胶质细胞移植中MECP2表达的治疗作用的额外细胞非自主机制。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疾病进展，甚至可以恢复成年Rett小鼠的症状，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，并提示了人类的合理治疗，但在使用人类细胞作为读数的定义良好的实验系统中评估治疗策略至关重要。本项目旨在建立一个利用人类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。使用人类iPS细胞衍生的神经元培养，最初的目标是建立一个实验范例，允许定义MECP2在基因调控中的分子作用，并在人类突变神经元中提供可靠和可量化的疾病相关表型读数。我们将使用CHIP-seq等分子方法来绘制MECP2与5mC和5hmC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。进一步，我们将鉴定MECP2和MECP2-相互作用伙伴的靶基因，并阐明MECP2靶基因在功能RTT丧失和获得中的调控作用。该提案的一个主要重点是建立一个平台，允许评估治疗策略在体外和体内条件下逆转人类神经元RTT表型的功效。(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类大脑类器官培养。这将使分析细胞-细胞相互作用和潜在的治疗剂在一个明确定义的3D测试系统。（ii）我们将把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，来进行生长因子和小分子化合物的体内验证，这些生长因子和小分子化合物可能对RTT患者的治疗有益。

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.