The role of autism susceptibility genes in the 16p11.2 locus on the development and function of human stem cell-derived neural cells

项目摘要/摘要 最近的报告估计，美国每6名儿童中就有1名符合诊断标准 神经发育障碍，如自闭症谱系障碍(ASD)、注意缺陷多动障碍 (ADHD)和智力残疾(ID)。自闭症的流行，其特点是持续的社会 在过去的15年里，障碍、语言缺陷和重复行为增加了120%， ASD背后的疾病机制在很大程度上尚不清楚，这一事实进一步加剧了这个问题 不存在有针对性的治疗干预措施。人类基因组测序的最新进展已经开始 通过识别几个遗传风险因素来阐明疾病的途径，最常见的 即16p11.2位点(16p11.2del)的缺失。初步研究已经提名了16p11.2中的特定基因 神经元功能障碍的基因座，尽管这些发现是建立在小鼠和斑马鱼模型上的，而不是人类 神经细胞类型。这一建议旨在阐明16p11.2del表型背后的疾病机制。 使用体外诱导的多能干细胞(IPSC)来源的人脑细胞。在目标1(K99)中，人类IPSC- 使用新方案产生的神经前体细胞和神经元将与人类胚胎进行比较 使用单细胞rna测序(scrna-seq)技术验证这些体外细胞模型的脑组织 以备将来研究之用。该提案的目标2(K99)将采用一种创新的“盘中人口”战略，其中 来自许多不同神经型和16p11.2del患者的干细胞株将被汇集到一个培养瓶中 询问表型差异。Aim 3(R00)将利用这些scRNA-seq技术来破译这一角色 在对神经发育重要的特定途径中的16p11.2基因。这些目标的圆满实现 可能导致确定用于治疗干预的遗传靶点，同时也显著改变 该领域对人类大脑疾病进行体外建模的方式。这些实验将提供新的 对本提案的首席调查员(PI)进行scRNA-seq和生物信息学方法方面的培训 作为一个独立研究实验室的基础，该实验室将使用干细胞来源的神经细胞和大型 转录组数据集，结合动物模型，阐明细胞和分子机制 管理神经发育障碍。这项工作将在布罗德研究所和哈佛大学完成 大学，在那里有无数的技术和智力增长的机会。私家侦探将出席 定期与导师和合作者会面，听取对实验设计和职业生涯的反馈 决定。PI将参加哈佛大学的拨款撰写和项目管理课程，同时还将提高他的 在国际科学会议上展示数据的沟通技巧。作为一个整体，这个职业生涯 发展计划将帮助PI在一个研究型学术机构建立自己的小组，并 成为神经发育疾病模型领域的领导者。

PROJECT SUMMARY/ABSTRACT Recent reports estimate that 1 out of every 6 children in the United States meet the diagnostic criteria for neurodevelopmental disorders such as autism spectrum disorders (ASD), attention-deficit hyperactivity disorder (ADHD), and intellectual disability (ID). The prevalence of ASDs, which are characterized by persistent social impairments, language deficits, and repetitive behaviors, has increased by 120% over the past 15 years, a problem further exacerbated by the fact that the disease mechanisms underlying ASDs are largely unknown and no targeted therapeutic interventions exist. Recent progress in human genome sequencing has begun to illuminate pathways to disease through the identification of several genetic risk factors, the most common of which is the deletion of 16p11.2 locus (16p11.2del). Initial studies have nominated specific genes in the 16p11.2 locus in neuronal dysfunction, though these findings are built on mouse and zebrafish models rather than human neural cell types. This proposal aims to elucidate the disease mechanisms underlying 16p11.2del phenotypes using in vitro induced pluripotent stem cell (iPSC)-derived human brain cells. In Aim 1 (K99), human iPSC- derived neural progenitor cells and neurons generated using novel protocols will be compared to human fetal brain tissue using single-cell RNA sequencing (scRNA-seq) techniques to validate these in vitro cellular models for future studies. Aim 2 of this proposal (K99) will employ an innovative “population-in-a-dish” strategy in which stem cell lines from many different neurotypical and 16p11.2del patients will be pooled into one culture flask to interrogate phenotypic differences. Aim 3 (R00) will leverage these scRNA-seq techniques to decipher the role of 16p11.2 genes in specific pathways important for neurodevelopment. The successful completion of these aims could lead to the identification of genetic targets for therapeutic intervention, while also dramatically changing the way the field conducts in vitro modeling of human brain disorders. These experiments will provide new training for the principal investigator (PI) of this proposal in scRNA-seq and bioinformatics methods that will serve as the foundation of an independent research laboratory that will use stem cell-derived neural cells and large transcriptome datasets, combined with animal models, to elucidate the cellular and molecular mechanisms governing neurodevelopmental disorders. This work will be completed at the Broad Institute and Harvard University, where the opportunities for technical and intellectual growth are innumerable. The PI will attend regular meetings with mentors and collaborators to receive feedback on experimental design and career decisions. The PI will attend grant writing and project management courses at Harvard, while also improving his communication skills by presenting data at international scientific conferences. As a whole, this career development plan will help the PI establish his own group at a research-oriented academic institution and become a leader in the field of neurodevelopmental disease modeling.