Systematic variant-to-pathway, phenotype, and precision treatment response evaluation in genetic disorders of the immune system (GDIS) - Genetic diseases of the immune system (GDIS) encompass a wide range of clinical manifestations—spanning infection, inflammation, autoimmunity, and cancer—and are driven by both rare and common genetic variants. GDIS cause distinct and quantifiable dysregulation of cellular signaling pathways providing exceptional opportunities to advance our biological understanding of and precision medicines for these diseases. The growing catalog of variants identified by next-generation sequencing includes many variants of uncertain significance (VUS), introducing diagnostic ambiguity and limiting the use of precision therapeutic intervention. Furthermore, there is substantial clinical phenotypic overlap of different GDIS suggesting crosstalk and convergence of dysregulated pathways. We hypothesize that scalable functional variant mapping will clarify GDIS-gene variant effects across molecular, individual, and population levels, and that phenotypic convergence across distinct GDIS will reveal therapeutic targets for precision medicine. We have established a massively parallel, high-efficiency CRISPR-base editing framework to generate and functionally map GDIS-gene variants to relevant phenotypic readouts in primary human T cells. Using Activated PI3Kδ Syndrome (APDS), caused by PIK3CD or PIK3R1 gain-of-function (GOF) variants, as a model disease, we performed forward saturation screens, identified hundreds of novel GOF and loss-of-function (LOF) variants, demonstrate their sensitivity to Leniolisib, an FDA-approved PI3Kδ inhibitor for APDS, and identify partly drug- resistant variants that responded to precision combination therapies. Through immunophenotype wide association studies (IPheWAS), we find that ADPS may be orders of magnitude more common than previously estimated. To begin testing our convergence hypothesis, we performed large-scale screens testing variants in >100 GDIS genes involved in key T cell pathways. We identified several unexpected interactions between GDIS- genes and non-canonical pathway activities, including convergence of CARD11, PI3KCD, PIK3R1 and CTLA4 variants. We validated these interactions in patient cells with corresponding variants and demonstrate that pathway crosstalk can be leveraged as a unified dependency for precision drug interventions. We now propose to: (1) comprehensively identify disease-causing variants of PIK3CD and PIK3R1 in key cell types (T and B cells) and dissect the biochemical underpinnings of partial Leniolisib-resistance, and (2) generate and functionally map GDIS-gene variants in additional genes (including in CARD11, MALT1, NFKB1 and CTLA4) and their crosstalk across key genes and T cell pathways to identify opportunities for precision therapy interventions. We will test a range of therapies, including inhibitors of PI3Kδ, mTOR, and MALT1, and agonist treatment of CTLA4 using a fusion antibody. This work will produce high-resolution functional variant maps, uncover new genotype-phenotype relationships at the molecular, patient and population level, and establish a framework for variant-driven precision medicine across the spectrum of immune diseases.
