权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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：
10024573
负责人：
DAVID L. WIEST
金额：
$ 48.95万
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/10024573
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.

本计划项目的总体目标是将其成员的专业知识综合起来，以全面的努力