Modular pooled discovery of synthetic knockin sequences to program durable cell therapies.

Chronic stimulation can cause T cell dysfunction and limit the efficacy of cellular immunotherapies. Improved methods are required to compare large numbers of synthetic knockin (KI) sequences to reprogram cell functions. Here, we developed modular pooled KI screening (ModPoKI), an adaptable platform for modular construction of DNA KI libraries using barcoded multicistronic adaptors. We built two ModPoKI libraries of 100 transcription factors (TFs) and 129 natural and synthetic surface receptors (SRs). Over 30 ModPoKI screens across human TCR- and CAR-T cells in diverse conditions identified a transcription factor AP4 (TFAP4) construct that enhanced fitness of chronically stimulated CAR-T cells and anti-cancer function in vitro and in vivo. ModPoKI's modularity allowed us to generate an ∼10,000-member library of TF combinations. Non-viral KI of a combined BATF-TFAP4 polycistronic construct enhanced fitness. Overexpressed BATF and TFAP4 co-occupy and regulate key gene targets to reprogram T cell function. ModPoKI facilitates the discovery of complex gene constructs to program cellular functions. [Display omitted] • Pooled knockin of hundreds of TFs/surface receptors combined with different TCRs/CARs • Chronic stimulation screens discover programs to improve T cell persistence • Combinatorial knockin screens with ∼10,000 transcription factor combinations • BATF-TFAP4 dual knockin construct improves CAR-T cell fitness and function Modular pooled knockin screening (ModPoKI) is an adaptable platform that enables the evaluation of hundreds to thousands of different T cell constructs for engineered cellular immunotherapies. [ABSTRACT FROM AUTHOR]