Project 3

项目摘要 脆性X综合征(FXS)是最常见的遗传性智力残疾形式，也是一种主要的遗传性疾病 自闭症谱系障碍(ASD)的病因。功能性脆性X智力低下引起的FXS 蛋白质(FMRP)。以前的工作主要集中在FMRP作为翻译调节因子的作用，以及许多mRNA FMRP的靶点已被证明是与ASD相关的基因。尽管在表征潜在特征方面取得了重大进展 动物模型的疾病机制导致了几项临床试验，包括3期临床试验 调节代谢性谷氨酸和GABA受体的药物，改善行为和认知 不幸的是，患者的结果很大程度上并不成功。我们认为，临床前的一个主要缺口是 FXS的药物开发阶段可以通过开发人FXS诱导的多能性来解决 干细胞(IPSC)衍生模型，这将使我们能够识别人类特定的治疗靶点并评估 新的治疗方法。人类的IPSCs是多能性的，能够产生许多不同类型的细胞。 IPSCs的三维(3D)器官培养是从胚状体培养演变而来的，非常忠实 跟踪人类器官发生，并提供了一个在培养皿中研究人脑发育的新平台， 否则无法进行实验。我们已经开发了FXS IPSC模型，包括2D神经 祖细胞/皮质神经元和3D皮质器体，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丢失相关的异常 FXS IPSC模型的分子、细胞和电生理水平。有趣的是，我们的初步数据 提示PI3K抑制剂，而不是mGluR5拮抗剂，可以挽救人FXS IPSC的细胞表型 衍生的模型系统，潜在地验证阳性的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定的IPSC模型作为翻译工具来开发 FXS的新治疗方法。首先，我们将确定靶向化合物的治疗效果 FXS类有机化合物的候选途径(目标1)。第二，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有机体中FMR1的表达(目标2)。第三，我们将进行 分子表型和基于FMR1再激活的小分子筛选(目标3)。我们建议的工程将会 导致FXS新的治疗靶点的确定和新治疗策略的开发。

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.