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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的破坏性神经发育障碍，由于其复杂的发病机制和遗传异质性，治疗进展。实验方法，整合遗传学和生物学是必要的，以进一步我们对这种复杂的疾病的理解。英美影响小的遗传变异和影响严重的罕见遗传突变与精神分裂症有关，后者更可能产生可在体外测量的表型。本提案描述了一个5年职业发展计划，通过该计划，我将制定概念性的框架，并获得必要的技能，以表征罕见的，严重的突变精神分裂症患者。我的项目将是在iPSCs中使用CRISPR干扰（CRISPRi）来评估共有3个标准的基因：1）每个基因在我们的研究中的一个或多个患者中携带一个破坏性突变; 2）每个基因都是显著的根据SCHEMA协会的标准与精神分裂症相关;以及3）每个基因在染色质中起作用重塑或转录调节。在目标1中，我将使用CRISPRi敲低iPSC衍生神经元中这些基因的表达，比较基因表达丧失前后的转录特征，以及与其他iPSC模型的比较。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中的一个基因生成一个患者来源的iPSC系，然后创建一个同基因的使用CRISPR介导的同源性定向修复将突变回复到野生型的iPSC系。（目标1中所有患者的淋巴母细胞系可用于此目的。）我会比较病人和回复突变体使用与Aim 1相同的测定，以及它们形成成熟神经元的能力、它们的神经突数量，细胞迁移缺陷、转录谱和表观遗传效应。在目标3中，从我的诊所，我将招募更多的患者-父母-父母三人组;收集血液用于DNA，RNA和DNA检测。建立细胞系;组织纵向临床数据收集;进行外显子组测序并为我未来的研究找出有害的变异。我已经获得了UW IRB的批准。这个K 08奖将为我提供必要的指导和概念和实验培训，这些方法中的每一种。我的总体目标是建立我的独立实验室，负责精神分裂症的病理生理学。