Functional characterization of schizophrenia rare variants using genetically engineered human iPSCs

使用基因工程人类 iPSC 进行精神分裂症罕见变异的功能表征

• 批准号: 10554598
• 负责人: Pan Li
• 金额: $ 69.67万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-01-01 至 2027-11-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10554598
• 关键词: Affect; Autopsy; Biological Assay; Brain; CUL1 gene; Cell Line; Cells; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Copy Number Polymorphism; Development; Diagnostic; Disease; Electrophysiology (science); Excision; Future; Gene Expression; Generations; Genes; Genetic; Genetic Engineering; Genotype; HERC1 gene; Human; Human Engineering; Individual; Interdisciplinary Study; Karyotype determination procedure; Length; Location; Major Mental Illness; Meta-Analysis; Methods; Microscopy; Mutate; Mutation; Mutation Analysis; N-terminal; Neurites; Neurobiology; Neurodegenerative Disorders; Neurodevelopmental Disorder; Neurons; Pathogenesis; Pathway interactions; Phenotype; Point Mutation; Protein Truncation; Proteins; Proteome; Proteomics; Research; Risk; Schizophrenia; Signal Pathway; Signal Transduction; Structure; Synapses; System; Testing; Therapeutic; Variant; Work; chromatin remodeling; comparison control; electrical property; excitatory neuron; exome; experience; experimental study; gene function; genetic architecture; genetic risk factor; genetic variant; homologous recombination; human female; improved; induced pluripotent stem cell; knock-down; loss of function; male; multi-electrode arrays; mutant; new therapeutic target; novel strategies; overexpression; patch clamp; pluripotency; polygenic risk score; protein aggregation; protein expression; proteostasis; rare variant; therapeutic target; transcription factor; transcriptome; transcriptome sequencing

PROJECT SUMMARY Schizophrenia (Scz) is a common and debilitating neurodevelopmental disorder. However, the genetic architecture of Scz is highly complex, impeding research progress. Recently, the Scz Exome Meta-Analysis (SCHEMA) Consortium has identified genes with ultra-rare coding variants that each confer substantial risk for Scz. We hypothesize that the presence of these rare variants in human induced pluripotent stem cells (hiPSCs) will lead to neuronal phenotypes relevant to Scz. The PI, Dr. Pan Li, has extensive experience with gene editing and iPSCs, including development of an improved method for seamless introduction and removal of mutations into hiPSCs. She has previously focused on neurodegenerative disorders, but with this application proposes to change to study of Scz. This transition is facilitated by the location of her lab in the Division of Neurobiology, which has a long track record of research into major mental illnesses and of highly collaborative research using iPSCs (eg the HD iPSC Consortium). In Specific Aim 1, isogenic hiPSC lines with protein truncating variants associated with Scz will be genetically engineered by CRISPR-assisted homologous recombination to generate panels of iPSC lines, with each isogenic line bearing a different N-terminal protein truncating point mutation. Knockdown and overexpression rescue experiments will determine whether there is loss of protein expression and thus loss of gene function. In Specific Aim 2, the effect of mutations on gene and protein expression will be studied using RNA seq and quantitative proteomics. In Specific Aim 3, the effects of mutations on synaptic and electrophysiological phenotypes will be delineated. We hypothesize that understanding the cellular phenotypes and altered signaling pathways evoked by the SCHEMA mutations will clarify the cellular pathogenesis of subtypes of schizophrenia, and provide potential therapeutic targets.

项目摘要 精神分裂症（SCZ）是一种常见且令人衰弱的神经发育障碍。但是，遗传 SCZ的体系结构非常复杂，阻碍了研究进度。最近，SCZ外显元分析 （模式）财团已经确定了具有超稀有编码变体的基因 SCZ。我们假设这些罕见变体在人类诱导的多能干细胞（HIPSC）中存在 将导致与SCZ相关的神经元表型。 PI Pan Li博士在基因编辑方面拥有丰富的经验 和IPSC，包括开发改进的无缝引入和去除突变的方法 进入HIPSC。她以前曾专注于神经退行性疾病，但此应用程序提出了 更改为SCZ的研究。她的实验室在神经生物学系中的位置促进了这种过渡， 这在重大精神疾病和高度协作研究的研究中有很长的记录 IPSC（例如HD IPSC财团）。在特定的目标1中，具有蛋白质截断变体的ISEGENIC HIPSC线 与SCZ相关的SCZ将通过CRIS辅助同源重组进行基因设计 IPSC线的面板，每条同源线带有不同的N末端蛋白截断点突变。 敲低和过表达救援实验将确定是否存在蛋白质表达的损失 从而丧失基因功能。在特定的目标2中，突变对基因和蛋白质表达的影响将 使用RNA SEQ和定量蛋白质组学研究。在特定的目标3中，突变对突触的影响 和电生理表型将被描述。我们假设了解细胞 模式突变引起的表型和改变的信号通路将阐明细胞 精神分裂症亚型的发病机理，并提供潜在的治疗靶标。