Scalable generation and functional classification of genetic variants in inborn errors of immunity to accelerate clinical diagnosis and treatment

Next-generation sequencing is pivotal for diagnosing inborn errors of immunity (IEI) but predominantly yields variants of uncertain significance (VUS), creating clinical ambiguity. Activated-PI3Kd syndrome (APDS) is caused by gain-of-function (GOF) variants in PIK3CD or PIK3R1, which encode the PI3Kd heterodimer. We performed massively parallel base-editing of PIK3CD/PIK3R1 in human T-cells and mapped thousands of variants to a clinically important readout (phospho-AKT/S6), nominating >100 VUS and unannotated variants for functional classification, and validated 27 hits. Leniolisib, an FDA-approved PI3Kd-inhibitor, rescued aberrant signaling and dysfunction in GOF-harboring T-cells, and revealed partially drug-resistant PIK3R1 hotspots that responded to novel combination therapies of Leniolisib with mTORC1/2-inhibition. We confirmed these findings in T-cells from APDS patients spanning the functional spectrum discovered in the screen. Integrating our screens with population-level genomic studies revealed that APDS may be more prevalent than previously estimated. This work exemplifies a broadly applicable framework for removing ambiguity from sequencing in IEI. Overall design: Primary human T cells were lentivirally transduced with a custom base editing sgRNA library targeting PIK3CD and PIK3R1, and then base edited with the adenine base editor ABE8e. Base edited T cells were then either re-expanded for 14 days ("proliferation screen") or stimulated acutely for 20 minutes with CD3/CD28 antibody, stained for phosphoproteins pAKT and pS6, and sorted into either [pAKT/pS6 - high] or [pAKT/pS6 - negative] bins by FACS ("AKT/S6 screen"). An additional AKT/S6 screen was performed (with the same conditions and same human T cell donors) using the narrow editing-window adenine base editor ABE9. For all experiments sgRNA sequences were amplified and sequenced using an Element AVITI.