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

Protein-based switches that respond to different inputs
to regulate
cellular outputs, such as gene expression, are central to synthetic
biology. For increased controllability, multi-input switches that
integrate several cooperating and competing signals for the regulation
of a shared output are of particular interest. The nuclear hormone
receptor (NHR) superfamily offers promising starting points for engineering
multi-input-controlled responses to clinically approved drugs. Starting
from the VgEcR/RXR pair, we demonstrate that novel (multi)drug regulation
can be achieved by exchange of the ecdysone receptor (EcR) ligand
binding domain (LBD) for other human NHR-derived LBDs. For responses
activated to saturation by an agonist for the first LBD, we show that
outputs can be boosted by an agonist targeting the second LBD. In
combination with an antagonist, output levels are tunable by up to
three simultaneously present small-molecule drugs. Such high-level
control validates NHRs as a versatile, engineerable platform for programming
multidrug-controlled responses.