Engineering Gene and Protein Switches for Regulation of Lineage-Specifying Transcription Factors

Human pluripotent stem cells (hPSCs) can be differentiated in vitro to an increasing number of mature cell types, presenting significant promise for addressing a wide range of diseases and studying human development. One approach to further enhance stem cell differentiation methods would be to coordinate multiple inducible gene or protein switches to operate simultaneously within the same cell, with minimal cross-interference, to precisely regulate a network of lineage-specifying transcription factors (TFs) to guide cell fate decisions. Therefore, in this study, we designed and tested various mammalian gene and protein switches responsive to clinically safe small-molecule inhibitors of viral proteases. First, we leveraged hepatitis C virus and human rhinovirus proteases to control the activity of chimeric transcription factors, enabling gene expression activation exclusively in the presence of protease inhibitors and achieving high fold-inductions in hPSC lines. Second, we built single-chain protein switches regulating the activity of three differentiation-related pancreatic TFs, MafA, Pdx1, and Ngn3, each engineered with a protease cleavage site within its structure and having the corresponding protease fused at one terminus. While variants lacking the protease retained most of the unmodified TF activity, the attachment of the protease significantly decreased the activity, which could be rescued upon addition of the corresponding protease inhibitor. We confirmed the functionality of these protein switches for simultaneously controlling the activity of three TFs with a common input molecule, as well as the orthogonality of each protease-based system to independently regulate two TFs. Finally, we validated these very compact systems for precisely controlling TF activity in hPSCs. Our results suggest that they will be valuable tools for research in both developmental biology and regenerative medicine.