Modeling patient mutations in iPSC-derived neurons to reveal cellular mechanisms of schizophrenia

对 iPSC 衍生神经元中的患者突变进行建模以揭示精神分裂症的细胞机制

基本信息

批准号：
10681311
负责人：
Anna Brosius Sunshine
金额：
$ 18.75万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-15 至 2026-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10681311
关键词：
Award Biological Assay Biology Blood Brain CRISPR correction CRISPR interference CRISPR/Cas technology Case/Control Studies Cell Line Cell model Chromatin Clinic Clinical Clinical Data Clustered Regularly Interspaced Short Palindromic Repeats Collection Complex Consent DNA DNA Sequence Alteration Defect Development Diagnostic Disease Enrollment Epigenetic Process Functional disorder Future Gene Expression Gene Expression Profile Genes Genetic Genetic Heterogeneity Genomics Genotype Goals In Vitro Individual Induced pluripotent stem cell derived neurons Institutional Review Boards Laboratory Study Lead Learning Lesion Measures Mediating Mentorship Modeling Molecular Mutation NR4A2 gene Neurites Neurobiology Neurodevelopmental Disorder Neuronal Differentiation Neurons Oxidative Stress Parents Participant Pathogenesis Pathway interactions Patients Persons Phenotype Point Mutation Program Development RNA Reproducibility Research Resources Schizophrenia Therapeutic Training Transcriptional Regulation Variant career development cell motility chromatin remodeling disease phenotype established cell line exome exome sequencing experimental study genetic association genetic variant improved induced pluripotent stem cell knock-down lymphoblast mutant participant enrollment proband prognostic recruit repaired skills stem cell model synaptic function transcriptome transcriptome sequencing transcriptomics

项目摘要

Schizophrenia is a common and devastating neurodevelopmental disorder that has defied diagnostic and therapeutic advances due to its complex pathogenesis and genetic heterogeneity. Experimental approaches that integrate genetics and biology are necessary to further our understanding of this complex disorder. Both common genetic variants of small effect and rare genetic mutations of severe effect have been implicated in schizophrenia, with the latter more likely to produce phenotypes that can be measured in vitro. This proposal describes a 5-year career development program through which I will develop the conceptual framework and gain the skills necessary to characterize rare, severe mutations in persons with schizophrenia. My project will be to evaluate, using CRISPR-interference (CRISPRi) in iPSCs, genes that share 3 criteria: 1) each gene harbors a damaging mutation in one or more patients from our studies; 2) each gene is significantly associated with schizophrenia per criteria of the SCHEMA consortium; and 3) each gene plays a role in chromatin remodeling or transcriptional regulation. In Aim 1, I will use CRISPRi to knock down expression of each of these genes in iPSC-derived neurons, then compare transcriptional signatures before and after loss of gene expression, and to other iPSC models of schizophrenia. I will also assay for changes in cellular oxidative stress, a cellular phenotype of iPSC models of schizophrenia. In Aim 2, I will generate a patient-derived iPSC line for one of the genes from Aim 1, then create an isogenic iPSC line with the reversion to wild-type of the mutation using CRISPR-mediated homology directed repair. (Lymphoblast lines of all patients from Aim 1 are available for this purpose.) I will compare patient and revertant iPSCs with the same assays as in Aim 1, and also for their ability to form mature neurons, their neurite number, cellular migration defects, transcriptional profiles, and epigenetic effects. In Aim 3, from my clinic, I will enroll additional patient-parent-parent trios; collect blood for DNA, RNA, and the establishment of cell lines; organize the collection of longitudinal clinical data; and carry out exome sequencing and identify damaging variants for my future studies. I have obtained approval of this aim from the UW IRB. This K08 award will provide me with the mentorship and conceptual and experimental training necessary for each of these approaches. My overall goal is to establish my independent laboratory studying the genes responsible for the pathophysiology of schizophrenia.

精神分裂症是一种常见且具有破坏性的神经发育障碍，无法进行诊断和治疗由于其复杂的发病机制和遗传异质性，治疗取得了进展。实验方法为了进一步了解这种复杂的疾病，整合遗传学和生物学是必要的。两者都常见影响较小的基因变异和影响严重的罕见基因突变与精神分裂症有关，后者更有可能产生可在体外测量的表型。该提案描述了一个为期 5 年的职业发展计划，通过该计划我将制定概念框架并获得必要的技能来描述精神分裂症患者罕见、严重的突变。我的项目将是在 iPSC 中使用 CRISPR 干扰 (CRISPRi) 来评估具有 3 个标准的基因：1) 根据我们的研究，每个基因在一名或多名患者身上都存在破坏性突变； 2) 每个基因显着根据 SCHEMA 联盟的标准，与精神分裂症相关； 3）每个基因在染色质中发挥作用重塑或转录调控。在目标 1 中，我将使用 CRISPRi 敲低 iPSC 衍生神经元中每个基因的表达，然后比较基因表达丢失之前和之后的转录特征，以及与其他 iPSC 模型精神分裂症。我还将检测细胞氧化应激的变化，这是 iPSC 模型的一种细胞表型精神分裂症。在目标 2 中，我将为目标 1 中的基因之一生成源自患者的 iPSC 系，然后创建同基因使用 CRISPR 介导的同源定向修复恢复野生型突变的 iPSC 细胞系。（来自目标 1 的所有患者的淋巴母细胞系均可用于此目的。）我将比较患者和回复体 iPSC 的检测与目标 1 相同，并且还具有形成成熟神经元的能力、神经突数量、细胞迁移缺陷、转录谱和表观遗传效应。在目标 3 中，我将在我的诊所招募额外的患者-家长-家长三人组；采集血液以检测 DNA、RNA 和细胞系的建立；组织纵向临床数据的收集；并进行外显子组测序并找出对我未来研究具有破坏性的变异。我已获得华盛顿大学 IRB 对该目标的批准。这个 K08 奖项将为我提供必要的指导以及概念和实验培训这些方法中的每一个。我的总体目标是建立研究基因的独立实验室负责精神分裂症的病理生理学。