The past decade has seen rapid growth of gene discovery for primary immunodeficiencies. With the advent of newborn screening for severe combined immunodeficiency (SCID), coupled with new applications for deep sequencing, genomic analyses, high-throughput cellular screening, and CRISPR gene editing, there is an unprecedented opportunity to establish, by direct testing, how human genetic sequences control immune cell development, and ultimately to treat SCID by genome editing of causal mutations. Our comprehensive program will address the major challenges that must be overcome to capitalize on this transformative opportunity, by integrating clinical data from T cell insufficient patients with basic investigations drawing on the expertise of leaders in immunology, bioinformatics, target validation, and genome editing. The SCID newborn screening assay, pioneered by Dr. Puck and implemented in all 50 states in the USA and an increasing number of countries, employs DNA from infant blood spots to enumerate T cell receptor excision circles (TRECs), a surrogate for thymic output of new T cells. It is highly effective for identifying infants with T cell insufficiency, whose molecular etiologies are often revealed by immune phenotyping and sequencing a panel of known SCID genes. Importantly, however, this unbiased, population-based screening also reveals infants with SCID who lack readily discernable, deleterious causative mutations, as well as other infants belonging to a previously unrecognized group with non- SCID T cell lymphopenia (TCL). We and others have applied whole exome sequencing (WES) to enigmatic cases of SCID and TCL, revealing unanticipated and exciting gene variants that have directly impacted medical care, while revealing new insights into immune mechanisms. Importantly, however, WES fails to identify disease- causing mutations in 60% of these enigmatic cases. This may result from incomplete exome capture, poorly covered exons, or the fact that a disease-causing variant may lie in a non-coding genomic element. This Program will overcome these limitations by combining variant discovery using whole genome sequencing (WGS), T cell RNASeq in patients and parents, and robust high-throughput functional assays to solve these difficult cases. Our work will ultimately usher in an era of novel treatments employing genome editing of autologous hematopoietic progenitors. To identify the mutation(s) responsible for T cell insufficiency from among candidate variants, we will perform functional screening in zebrafish, primary human CD34+ cells and human pluripotent stem cell- derived CD34+ hematopoietic stem and progenitor cells differentiated in vitro on novel Notch-ligand microbeads. Moreover, we will use Perturb-seq, a method for single cell transcriptome comparisons and epistatic analysis to unravel relationships among causal variants and construct a molecular map of human T cell development. Our combined capabilities, encompassing clinical expertise, genomic analysis, gene editing, high-throughput cellular screening, zebrafish and functional immunology, have the power to revolutionize our understanding of how immune cells develop and function while yielding important progress toward new treatment approaches.

HEALTH RELEVANCE: The advent of newborn screening for severe combined immunodeficiency (SCID), coupled with new genome- wide analysis and editing capabilities, constitutes an unprecedented opportunity to establish by direct testing how human genetic sequences control immune cell development, and ultimately to treat SCID by genome editing of causal mutations in patient cells. Our comprehensive program will address the major challenges that must be overcome to capitalize on this transformative opportunity.