Orthogonalized human protease control of secreted signals

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. While protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. Here, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins. We sourced a specific human protease and its FDA-approved inhibitor. We engineered cytokines whose activities can be activated and abrogated by proteolytic cleavage. We utilized species specificity and re-localization strategies to orthogonalize the cytokines and protease from the human context that they would be deployed in. hDIRECT should enable local cytokine activation to support a variety of cell-based therapies such as muscle regeneration and cancer immunotherapy. Our work offers a proof of concept for the emerging appreciation of humanization in synthetic biology for human health. ### Competing Interest Statement The board of trustees of the Leland Stanford Junior University have filed a patent on behalf of the inventors (C.A.A. and X.J.G.) of the small-molecule control of membrane and secreted proteins using human proteases platform described (US provisional Application No. 63/458833). An additional provisional patent application has also been filed on behalf of the inventors (C.A.A., X.J.G., J.P, and Q.C.) for the orthogonalization of human renin. X.J.G. is a co-founder and serves on the scientific advisory board of Radar Tx.