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.