Sense-and-response to intracellular signaling via nucleocytoplasmic localization control of a synthetic transcription factor platform

Many therapeutically relevant signaling pathways are sensed inside the cell, yet a modular synthetic biology platform for responding to intracellular signaling at the post-translational level is lacking. Signaling pathways control the nuclear localization and activity of native transcription factors via phosphorylation of nuclear localization/export or cytoplasmic anchor sequences. Here, we apply this concept to develop a platform of nucleocytoplasmic localization tags (NCtags) that control the nuclear translocation of a synthetic transcription factor (synTF) for transgene induction in engineered cells. We identify human-based synTF components that are basally cytoplasmic and induce transgenes under the control of NCtags with minimal background. We demonstrate the modular nature of this platform through transcriptional reporter induction in response to a calcium-inducible NFAT-derived NCtag, and development of novel NCtags responsive to hyperosmolarity, cAMP, or logical combinations of cAMP and calcium. Multiple factors in the tumor microenvironment elevate cAMP in T cells, leading to reduced function. We demonstrate that NCtag-synTF fusions expressed in T cells can respond to endogenous sources of cAMP or calcium in breast cancer spheroids to induce transgene expression. Our use of human components and an all-in-one lentiviral vector encoding the NCtag-synTF-transgene system establishes this as a novel platform for sense-and-response inside therapeutic cells.