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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Program Projects (P01)
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Special Emphasis Panel (ZAI1)
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University of California San Francisco
San Francisco
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