Abstract
Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. Current designs rely on engineered receptors that are limited to single inputs, and often suffer from high leakiness and low fold induction. Here, we present Generalized Engineered Activation Regulators (GEARs) that overcome these limitations by being pathway-specific rather than input-specific. GEARs consist of the MS2 bacteriophage coat protein fused to regulatory or transactivation domains, and work by rerouting activation of the NFAT, NFκB, MAPK or SMAD pathways to dCas9-directed gene expression from genomic loci. This system enables membrane depolarization-induced activation of insulin expression in β-mimetic cells and IL-12 expression in activated Jurkat cells, as well as IL-12 production in response to the immunomodulatory cytokines TGFβ and TNFα in HEK293T cells. Engineered cells with the ability to reinterpret the extracellular milieu have potential for applications in immunotherapy and in the treatment of metabolic diseases.
Highlights
Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering
GEARNFAT enables rewiring of intracellular calcium signaling
We first focused on nuclear factor of activated T-cells (NFAT), which is dephosphorylated upon activation of a calcium-dependent calmodulin–calcineurin cascade, and designed GEARNFAT, which consists of MS2 bacteriophage coat protein (MCP) fused to the transactivation domains of p65 and HSF1 (p65TA–HSF1TA), and the regulatory domain of NFAT (NFATreg)
Summary
Rewiring cellular sensors to trigger non-natural responses is fundamental for therapeutic cell engineering. GEARNFAT translocates to the nucleus (Supplementary Fig. 1) and activates sgRNA-specified gene expression in response to an increase of the intracellular calcium level. Incorporating GEARNFAT into these cells enabled membrane depolarization-dependent expression of a reporter gene controlled by an insulin promoter (Fig. 2b).
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