Design of novel synthetic promoters to tune gene expression in T cells

ABSTRACT Transcriptional control of transgene expression can be linked to dynamic changes in cellular states if this is accompanied by differential expression of transcription factors (TFs). Synthetic promoters (SPs) designed to respond to the desired TFs can provide this regulation with compactness, specificity, and orthogonality. T cells display differentially expressed TFs according to the functional state. In solid tumors, the highly immunosuppressive TME and the chronic exposure to antigens lead to a progression of T cells from a functional to a dysfunctional state known as exhaustion (Tex), in which their power against cancer cells is strongly compromised. Importantly, this transition is accompanied by a marked increase of several TFs, among other factors, that drive targeted genetic programs. Strategies to detect and mitigate Tex are extremely needed. Here, we design SPs that respond to TFs differentially expressed in activated and exhausted T cells to enable new classifiers of the functional/dysfunctional states. We developed a library of over 80 SPs responsive to 7 TFs. The SPs showed broad strength of activation of reporter genes or immunomodulatory molecules in HEK293 and Jurkat T cell lines. Moreover, using a transfer learning strategy we show SPs strength predictability. By combining SPs responding to different TFs, we created Boolean logic gates and implemented a feed-forward design that was previously shown to reduce noise in the OFF-state. Finally, as proof of principle, we demonstrate the dynamic activation of the NR4A2-responsive SP according to the T cell state in primary human CD8+ T cells. Collectively we present a sensing platform that provides a versatile tool to study and monitor the dynamic changes occurring in T cells. In perspective the biosensors coupled to therapeutic genes can be used to reprogram the TME and reinvigorate the T cell anti-tumoral functionality, preventing or reverting the exhausted phenotype. Graphical Abstract Created with BioRender.com .