Multi-input Drug-Controlled Switches of Mammalian Gene Expression Based on Engineered Nuclear Hormone Receptors

Protein-based switches that respond to different inputsto regulatecellular outputs, such as gene expression, are central to syntheticbiology. For increased controllability, multi-input switches thatintegrate several cooperating and competing signals for the regulationof a shared output are of particular interest. The nuclear hormonereceptor (NHR) superfamily offers promising starting points for engineeringmulti-input-controlled responses to clinically approved drugs. Startingfrom the VgEcR/RXR pair, we demonstrate that novel (multi)drug regulationcan be achieved by exchange of the ecdysone receptor (EcR) ligandbinding domain (LBD) for other human NHR-derived LBDs. For responsesactivated to saturation by an agonist for the first LBD, we show thatoutputs can be boosted by an agonist targeting the second LBD. Incombination with an antagonist, output levels are tunable by up tothree simultaneously present small-molecule drugs. Such high-levelcontrol validates NHRs as a versatile, engineerable platform for programmingmultidrug-controlled responses.