Functional Analysis of Variants Underlying T Cell Defects

T 细胞缺陷变异的功能分析

基本信息

批准号：
10462634
负责人：
DAVID L. WIEST
金额：
$ 51.77万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-08 至 2025-08-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10462634
关键词：
Address Affect Bioinformatics CD34 gene CD8B1 gene CRISPR screen Cell Differentiation process Cell model Cells Clustered Regularly Interspaced Short Palindromic Repeats Code DNA Sequence Defect Development Diagnosis Diagnostic Disease Elements Embryo Embryonic Development Essential Genes Etiology Evaluation Expression Profiling Flow Cytometry Gene Cluster Gene Expression Profile Genes Genetic Epistasis Genomics Hematopoietic Hematopoietic stem cells Human IL2RG gene IL7R gene Impairment In Vitro Individual Inherited Knowledge Ligands Link Maps Messenger RNA Methods Microspheres Modeling Molecular Mus Neonatal Screening Organ Orthologous Gene PTPRC gene Pathogenesis Pathogenicity Patients Play Process Research Personnel Resolution Role Severe Combined Immunodeficiency System T cell differentiation T-Cell Development T-Cell Receptor T-Lymphocyte Testing Transcend Umbilical Cord Blood Untranslated RNA Validation Variant Zebrafish base exome sequencing gene product genetic variant genome editing genome sequencing high throughput analysis human model in vivo insight knock-down loss of function member mutant notch protein novel novel therapeutics personalized approach population based programs screening single-cell RNA sequencing stem cells transcriptome whole genome

项目摘要

The overall aim of this Program Project is to integrate the expertise of its members in a comprehensive effort to exploit bioinformatic and genomic advances to enable not only identification of disease-causing variants discovered through population-based newborn screening for severe combined immunodeficiency (SCID), but also to develop genome editing as a personalized approach to treatment. Whole exome sequencing (WES) and whole genome sequencing (WGS) identify multiple candidate variants (Project 1; Cores B and C) that must then be screened to identify the pathogenic variant(s) responsible for T cell insufficiency. After Project 2 employs CRISPR-based screening in normal human hematopoietic progenitor cells to identify genes that are important for T cell development, Project 3 will integrate all of the findings from the program into a unifying model of human T cell development. Investigators Brenner, Puck, and Wiest have already collaborated to integrate bioinformatic variant calling with functional validation in zebrafish and human hematopoietic cells to identify BCL11B as a novel SCID gene and investigate its mode of action (Punwani et al, NEJM, 2016). This approach will be amplified to perform high-throughput analysis of hundreds of variants. Project 3 Aim 1 will establish a molecular map of human T cell development by characterizing the differentiation of primary human hematopoietic stem and progenitor cells (HSPC) in vitro using single-cell RNASeq. The molecular map will then be enriched by using loss-of-function analysis to assess the role in T cell development of known SCID genes and additional, novel genes determined by Project 2 to play an essential role in human T cell development. We will do so using Perturb-seq, a novel method that links loss-of-function of individual genes to single cell expression signatures at sequential stages of differentiation. This approach provides not only a precise definition of the developmental stage of arrest based on the expression signature, but also insight into the mechanism of arrest in a manner that transcends the limited resolution afforded by flow cytometry analysis of the heterogeneous hematopoietic intermediates (Adamson et al, Cell, 2016). Indeed, Perturb-seq will enable us to establish groups of genes that are co-expressed during T cell development, and to test the epistatic relationships between these genes at each developmental stage. In Aim 2, we will perform functional analysis on candidate disease-causing coding variants using both the zebrafish and human HSPC models. We will employ the zebrafish embryo model to determine if a particular coding variant actually damages the function of a gene product sufficiently to block T cell development in vivo, and whether other organs are also affected. In addition, we will perform in depth mechanistic analysis on the 3-4 highest priority variants, as insight gained from this analysis will help to inform the variant nomination process in Project 1. Collectively, these efforts will markedly advance our understanding of human T cell development, which will drive optimization of the discovery, mechanistic understanding and treatment of human SCID and related diseases.

该计划项目的总体目标是整合其成员的专业知识，全面努力利用生物信息学和基因组学的进步不仅能够识别致病变异通过基于人群的新生儿严重联合免疫缺陷 (SCID) 筛查发现，但是还开发基因组编辑作为个性化的治疗方法。全外显子组测序 (WES) 和全基因组测序 (WGS) 识别出多个候选变异（项目 1；核心 B 和 C），这些变异必须然后进行筛选以确定导致 T 细胞不足的致病变异。项目2采用后基于 CRISPR 的正常人类造血祖细胞筛选，以确定重要基因对于 T 细胞开发，项目 3 将将该项目的所有发现整合到一个统一的人类模型中。 T 细胞发育。研究人员 Brenner、Puck 和 Wiest 已经合作整合生物信息学在斑马鱼和人类造血细胞中进行变异调用和功能验证，将 BCL11B 识别为新型 SCID 基因并研究其作用模式（Punwani 等人，NEJM，2016）。这种方法将扩增以对数百种变体进行高通量分析。项目 3 目标 1 将建立一个分子通过表征初级人类造血干的分化绘制人类 T 细胞发育图使用单细胞 RNASeq 进行体外祖细胞 (HSPC) 分析。然后将通过使用丰富分子图谱功能丧失分析，以评估已知 SCID 基因和其他新基因在 T 细胞发育中的作用项目 2 确定的基因在人类 T 细胞发育中发挥重要作用。我们将使用 Perturb-seq，一种将单个基因功能丧失与单细胞表达特征联系起来的新方法分化的连续阶段。这种方法不仅提供了发展的精确定义基于表达签名的逮捕阶段，还可以通过以下方式洞察逮捕机制：超越了异质造血细胞流式细胞术分析所提供的有限分辨率中间体（Adamson 等人，Cell，2016）。事实上，Perturb-seq 将使我们能够建立基因组，在 T 细胞发育过程中共表达，并在每个阶段测试这些基因之间的上位关系发育阶段。在目标 2 中，我们将对候选致病编码变体进行功能分析使用斑马鱼和人类 HSPC 模型。我们将利用斑马鱼胚胎模型来确定是否特定的编码变体实际上会损害基因产物的功能，足以阻断 T 细胞体内的发育，以及其他器官是否也受到影响。此外，我们还将进行深入的机制对 3-4 个最高优先级变体进行分析，因为从该分析中获得的见解将有助于为变体提供信息项目 1 的提名过程。总的来说，这些努力将显着增进我们对人类的理解 T 细胞开发，这将推动 T 细胞的发现、机制理解和治疗的优化人类 SCID 和相关疾病。