By decoding endogenous transcription factor activity into programmable synthetic enhancers, this work transforms undruggable TFs from therapeutic dead-ends into precision logic gates—enabling tumor-selective gene circuits that drive robust antitumor immunity in vivo.
Endogenous transcription factor activity can be systematically decoded through high-throughput functional screening of synthetic enhancers and computationally integrated via protein interaction-based AND-gate circuits to achieve tumor-restricted therapeutic gene expression that drives robust antitumor immune responses in vivo, establishing a generalizable framework for harnessing undruggable TF activity as programmable regulatory signals for precision gene control across cancer and immune cell contexts.
Transcription factors (TFs) regulate gene expression programs that define cellular identity, functional state, and disease progression. Despite their central role in biology and disease, only a small fraction of TFs are directly druggable, leaving most disease-associated TFs beyond pharmacological reach and limiting the development of TF-centric therapeutic strategies. Here we propose a generalizable regulatory framework that repurposes endogenous TF activity not as a therapeutic target, but as an intrinsic trigger for programmable gene control. Using high-throughput functional screening of synthetic enhancers, we decode TF activity with minimal regulatory elements that selectively respond to context-specific transcriptional programs. Applied to ovarian cancer, this framework enables the identification of cancer-selective synthetic enhancers that exploit aberrant TF activity to distinguish malignant cells from normal epithelial cells. By integrating these enhancers with protein interaction-based AND-gate circuits, multiple TF inputs are evaluated through Boolean logic, enabling enhanced tumor specificity and amplified transcriptional output. In murine ovarian cancer models, these circuits selectively drive combinatorial immune effector expression and trigger robust antitumor responses. Extending this framework beyond cancer, we identify activation-responsive synthetic enhancers in primary human T cells that leverage endogenous activation-induced TF programs. These enhancers achieve stronger inducibility with lower basal activity than conventional NFAT-based designs. Together, these results establish TF activity as a programmable regulatory signal that can be decoded, integrated, and deployed to enable precise and context-dependent therapeutic gene regulation.