Drug-Inducible Gene Expression Control Using the Hepatitis C NS3/4A Cis-Protease

The ability to tightly control gene expression is a cornerstone of synthetic biology, and drug-inducible platforms have been particularly powerful bioengineering tools as they permit gene expression levels to be precisely regulated via the administration of exogenously-supplied molecular agents. Here, we describe a novel strategy in which user-specified genes can be regulated through the application of FDA-approved anti-viral protease inhibitors that are specific to the NS3/4A cis-protease from Hepatitis C virus (HCV). In this approach, the NS3/4A protease is used to regulate the linkage between genetically fused DNA-binding elements and transcriptional effector domains, which remain linked only in the presence of a NS3/4A protease inhibitor. We describe the application of the strategy in the construction and validation of various “turn-on” transcription factor (TF) systems containing the DNA-binding domains from Gal4 and TetR. Using a combinatorial design strategy, we exploit the modular framework of the system in order to generate customized TFs exhibiting distinct drug-inducible behaviors, as well as those possessing fine-tuned kinetics and/or potencies. A TF system requiring two drug-inputs for activation (i.e., “AND” gate behavior) is also described. Overall, this approach will provide a generalizable framework through which transcription can be precisely regulated in mammalian cells using readily available and FDA-approved anti-viral compounds.