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

• 批准号: 10369266
• 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
• 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/10369266
• 关键词: Award; Biological Assay; Biology; Blood; Brain; 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; Neurons; Oxidative Stress; Parents; Participant; Pathogenesis; Pathway interactions; Patients; Persons; Phenotype; Play; Point Mutation; Program Development; RNA; Reproducibility; Research; Resources; Role; Schizophrenia; Therapeutic; Training; Transcriptional Regulation; Variant; base; 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; proband; prognostic; recruit; repaired; skills; stem cell genes; stem cell model; synaptic function; transcriptome; transcriptome sequencing; transcriptomics

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毁灭性的神经发育障碍，尚未诊断和 由于其复杂的发病机理和遗传异质性，治疗性进步。实验方法 综合遗传学和生物学对于进一步的理解是必要的。两者都是常见的 精神分裂症已与小小作用的遗传变异和罕见的严重效应遗传突变有关， 后者更有可能产生可以在体外测量的表型。 该提案描述了一项为期5年的职业发展计划，我将通过该计划开发概念 框架并获得精神分裂症患者中罕见，严重突变所必需的技能。 我的项目将是在IPSC中使用CRISPR Interference（CRISPRI）评估，这些基因共享3个标准：1） 每个基因在我们的研究中都有一个或多个患者的破坏突变； 2）每个基因显着 与架构财团标准的精神分裂症相关； 3）每个基因在染色质中起作用 重塑或转录调节。 在AIM 1中，我将使用CRISPRI击倒IPSC衍生的神经元中每个基因的表达，然后 比较基因表达丧失之前和之后的转录特征，以及其他IPSC模型 精神分裂症。我还将测定细胞氧化应激的变化，这是IPSC模型的细胞表型 精神分裂症。 在AIM 2中，我将为AIM 1中的一个基因生成一个患者来源的IPSC线，然后创建一个等源性 IPSC使用CRISPR介导的同源性修复将突变的野生型重新转换为野生型。 （AIM 1的所有患者的淋巴细胞系可用于此目的。）我将比较患者和恢复。 IPSC具有与AIM 1相同的测定法，也是为了形成成熟神经元的能力，其神经元数， 细胞迁移缺陷，转录谱和表观遗传效应。 在AIM 3中，我将从我的诊所中注册其他患者父母的三重奏；收集DNA，RNA和RNA的血液 建立细胞系；组织纵向临床数据的收集；并进行外显子组测序 并确定未来研究的破坏性变体。我已从UW IRB获得了此目标的批准。 这个K08奖将为我提供所需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我的独立实验室研究基因 负责精神分裂症的病理生理学。