Advances in functional genomics and gene editing have the potential to transform the landscape of cellular immunotherapy and clinical immunology. Harnessing these advances, our group has developed a massively parallel CRISPR base editing platform in primary human T cells, enabling pooled generation and functional assessment of thousands of genetic variants at scale. Using this platform, we mapped the impact of variants in key immune genes on hallmarks of T cell anti-tumor activity, identifying novel gain- and loss-of-function variants that enhance cytokine production, activation, and proliferative capacity. Engineering these variants in CAR- and TCR-engineered T cells improved tumor cell killing in preclinical models of leukemia and immunotherapy-resistant melanoma, establishing a framework for rational genetic “tuning” of T cell therapies. We then extended this base editing platform to address a major diagnostic challenge in clinical immunology - interpretation of variants of uncertain significance (VUS) in inborn errors of immunity (IEI). Focusing on Activated PI3K-Delta Syndrome (APDS), a rare IEI caused by pathogenic gain-of-function variants in PIK3CD and PIK3R1, we coupled base editing screens of these genes in T cells with a clinically relevant readout for PI3K pathway signaling. This enabled definitive classification of thousands of variants, including nearly 100 gain-of-function alleles which were previously uncharacterized or VUS. Functional profiling revealed that these novel variants recapitulate the hyperactivation and exhaustion phenotypes seen in APDS patient T cells and respond favorably to the PI3Kδ inhibitor leniolisib, which is FDA-approved for the treatment of APDS. Integrating our results with large clinical cohorts suggests that APDS prevalence is likely underestimated, and most importantly, variant classifications from our work have already enabled the molecular diagnosis and treatment of a patient with leniolisib. Together, our work establishes a scalable platform for mapping variants to function in human T cells with broad clinical impact, ranging from enabling rational enhancement of immunotherapies to expanding access to precision diagnostics and treatment for patients with rare immune disorders.

— Zachary Walsh