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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、毁灭性的神经发育障碍，它一直无法被诊断和治疗