Human Isogenic Organoid Models of Genetic Forms of Autism to Identify Convergent and Divergent Pathomechanisms in Autism

项目摘要 自闭症谱系障碍（ASD）是一种临床上复杂的异质性疾病， 美国在多种形式的遗传和特发性ASD中识别共同病因将 极大地推进了诊断生物标志物发现和治疗开发。细胞调节异常 翻译已经成为至少一部分ASD形式所共有的病理生理机制。 然而，对ASD表型的细胞机制的系统研究已经被证明是一种新的研究方法。 由于缺乏可靠和可重复的人类ASD细胞模型和可扩展的实验工具， 用于在翻译水平上的细胞类型分辨表征。为了解决这些瓶颈问题， 为了解决翻译失调作为ASD中的共同特征的作用，我们（1）使用先进的基因组 工程工具，以产生一个广泛验证的，等基因系列的诱导多能干细胞（iPSC） 线模拟15综合征形式的ASD引起的高度渗透基因和基因组变异，代表 约占ASD总人口的10%（据我们所知，这是迄今为止创建的最大的此类小组），（2）建立了一个 强大的人类iPSC衍生的大脑发育的皮质类器官模型，以及（3）开发的ribo-STAMP， 用于异质细胞群体中单个细胞的翻译谱分析的方法，这是第一个也是唯一一个 方法使翻译被测量在单细胞分辨率。在这个项目中，我们确定共同的和 在基因组工程化的基于等基因干细胞的ASD类器官模型中， 使用单细胞转录组学和翻译组学方法。我们使用细胞和 功能表型测定和患者来源的iPSC模型。如果成功，我们的研究将确定共同的 和独特的预防意识的单细胞分辨基因表达签名，预测细胞和功能 结果。我们预计，我们的数据集和对基因表达中细胞类型特异性缺陷的见解 遗传形式的自闭症将极大地加速一个统一框架的发展， 遗传和特发性病例的分类，促进生物标志物的鉴定和 靶向疗法的开发。

PROJECT SUMMARY Autism spectrum disorder (ASD) is a clinically complex, heterogeneous condition affecting 1 in 44 children in the U.S. The identification of common etiologies across multiple forms of genetic and idiopathic forms of ASD will critically advance diagnostic biomarker discovery and therapeutic development. Dysregulation of cellular translation has emerged as a pathophysiological mechanism common to at least a subset of ASD forms. However, systematic investigation of the cellular mechanisms that converge onto the ASD phenotype has been hampered by a paucity of robust and reproducible human ASD cellular models and scalable experimental tools for cell-type resolved characterization at the level of translation. To address these bottlenecks and to directly address the role of translational dysregulation as a common feature in ASD, we have (1) used advanced genome engineering tools to generate an extensively validated, isogenic series of induced pluripotent stem cell (iPSC) lines modeling 15 syndromic forms of ASD caused by highly penetrant gene and genome variants, representing ~10% of the total ASD population (the largest such panel created to date, to our knowledge), (2) established a robust human iPSCs-derived cortical organoid model of brain development, and (3) developed ribo-STAMP, a method for translational profiling of individual cells in heterogeneous cell populations, which is the first and only method enabling translation to be measured at single- cell resolution. In this project, we identify common and divergent pathological mechanisms in genome- engineered isogenic stem cell based organoid models of ASD, using single-cell transcriptomic and translatomic approaches. We validate our findings using cellular and functional phenotypic assays and in patient-derived iPSC models. If successful, our study will identify common and unique translation-aware single-cell resolved gene expression signatures that predict cellular and functional outcomes. We anticipate that our datasets and insights into cell-type specific deficits in gene expression of genetic forms of autism will critically accelerate the development of a unified framework that enables molecular categorization of both genetic and idiopathic cases, facilitating the identification of biomarkers and the development of targeted therapies.