Project 3

项目摘要 脆性X综合征（FXS）是智力残疾的最常见遗传形式，也是一种主要的遗传性疾病。 自闭症谱系障碍（ASD）的原因。FXS是由功能性脆性X缺失引起的智力低下 蛋白（FMRP）。以前的工作集中在FMRP作为翻译调节因子的作用，许多mRNA FMRP的靶基因已被证明是ASD连锁基因。尽管在表征潜在的 动物模型中的疾病机制，已经导致了几项临床试验，包括3期临床试验， 调节代谢型谷氨酸和GABA受体的药物， 不幸的是，患者的结果大多不成功。我们认为，临床前研究的一个主要差距是， FXS的药物开发阶段可以通过开发人FXS诱导的多能干细胞来解决。 干细胞（iPSC）衍生模型，这将使我们能够识别人类特异性治疗靶点，并评估 新的治疗方法。人类iPSC是多能的，能够产生许多不同的细胞类型。 iPSC的三维（3D）类器官培养已经相当忠实地从胚状体培养发展而来。 继人类器官发生，并提供了一个新的平台，以研究人类大脑发育在一个培养皿， 否则无法进行实验。我们开发了FXS iPSC模型，包括2D神经网络模型， 祖细胞（NPC）/皮质神经元和3D皮质类器官，并确定了一些FMRP靶点 人类环境中的mRNA。此外，我们还观察到与FMRP缺失相关的异常。 在分子、细胞和电生理学水平上进行研究。有趣的是，我们的初步数据 表明PI 3 K抑制剂，而不是mGluR 5拮抗剂，可以拯救人FXS iPSC中的细胞表型 衍生的模型系统，可能验证阴性的阳性临床前小鼠研究的失败 人体试验在这项拟议的研究中，我们的目标是使用人类特异性iPSC模型作为翻译工具， FXS的新治疗方法。首先，我们将确定化合物的治疗效果， FXS类器官中的候选途径（目标1）。其次，我们将开发基于CRISPR的基因组和表观基因组 编辑治疗方法以重新激活FXS类器官中的FMR 1表达（Aim 2）。第三，我们将开展 分子表型和基于FMR 1再活化的小分子筛选（Aim 3）。我们建议的工程将 导致新的治疗靶点的鉴定和FXS新治疗策略的开发。

Project Summary Fragile X syndrome (FXS) is the most common inherited form of intellectual disability and a leading genetic cause of autism spectrum disorders (ASD). FXS is caused by the loss of functional fragile X mental retardation protein (FMRP). Previous works have focused on the role of FMRP as a translational regulator, and many mRNA targets of FMRP have been shown to be ASD-linked genes. Despite major progress to characterize underlying disease mechanisms in animal models that has led to several clinical trials, including phase 3 clinical trials of drugs modulating metabotropic glutamate and GABA receptors, improvements of behavioral and cognitive outcomes in patients have unfortunately been largely unsuccessful. We believe that a major gap in the preclinical phase of drug development for FXS can be addressed by the development of human FXS induced pluripotent stem cell (iPSC) derived models, which will enable us to identify human specific therapeutic targets and evaluate novel therapeutic approaches. Human iPSCs are pluripotent and are able to generate many different cell types. Three-dimensional (3D) organoid culture of iPSCs has evolved from embryoid body culture, quite faithfully following human organogenesis, and provides a new platform to investigate human brain development in a dish, otherwise inaccessible to experimentation. We have developed FXS iPSC models, including 2D neural progenitor cells (NPCs)/cortical neurons and 3D cortical organoids, and identified a number of FMRP target mRNAs in the human context. Furthermore, we have observed abnormalities associated with the loss of FMRP at molecular, cellular and electrophysiological levels in FXS iPSC models. Intriguingly, our preliminary data suggest that PI3K inhibitors, but not mGluR5 antagonists, could rescue cellular phenotypes in human FXS iPSC derived model systems, potentially validating the failure of positive preclinical mouse studies with negative human trials. In this proposed study, we aim to use human specific iPSC models as translational tools to develop novel therapeutic approaches for FXS. First, we will determine the therapeutic effects of compounds targeting candidate pathways in FXS organoids (Aim 1). Second, we will develop CRISPR-based genomic and epigenomic editing therapeutic approaches to reactivate FMR1 expression in FXS organoids (Aim 2). Third, we will conduct molecular phenotype and FMR1-reactivation-based small molecule screens (Aim 3). Our proposed works will lead to the identification of novel therapeutic targets and the development of new treatment strategies for FXS.