Post-Translational Control: The Next Step in Synthetic Circuits

Strategies to program cellular behaviors are central in synthetic biology. Scientists have developed many synthetic genetic circuits that allow engineered cells to produce therapeutic agents on demand in response to specific stimuli, such as oncoproteins (e.g., mutant KRAS), chemicals (e.g., caffeine), and physical stimuli (e.g., light). However, most circuits are based on gene switches that control transcription from a synthetic expression unit. This delays downstream signal transduction and as a result, transcription-based circuits are too slow for some therapeutic applications.^1-3 

“Killing a virus-infected cell, needing insulin to get your blood glucose to the right level…that all needs to happen extremely fast and gene switches are just too slow…[They go] through transcription, translation, protein secretion, and that takes some time,” explained Martin Fussenegger, professor of biotechnology and bioengineering at the University of Basel. In a study published in NAR, Fussenegger’s team developed a programmable synthetic circuit to control secretion of a protein of interest after translation.¹

Synthetic circuits that rely on post-translational control, mediated by engineered proteins, offer a more rapid approach than transcription-based gene switches. Fussenegger’s team generated a protease-mediated circuit they named POSH (post-translational switch). This circuit involves the transmembrane domain of a cleavable endoplasmic reticulum (ER) retention signal fused to a protein of interest. The system produces the desired protein, which stays in the ER under resting conditions due to the retention signal. The POSH platform depends on a customizable inducer-sensitive protease to release the protein of interest from the ER. The protease is expressed in two parts, which combine in the presence of an inducer to cleave the ER retention signal of the chimeric protein. The protein of interest is then released from the ER and undergoes trafficking to the Golgi for protein secretion. “It's a very clever design,” said Mads Kaern, associate professor in the Department of Cellular and Molecular Medicine at University of Ottawa, who was not involved in the study. “You're using cells as pharmacies, basically…you trick the cells into producing the medicine that the body needs, and then you're controlling when and where and how much is released.”

Killing a virus-infected cell, needing insulin to get your blood glucose to the right level…that all needs to happen extremely fast and gene switches are just too slow. 
- Martin Fussenegger, University of Basel

Fussenegger’s team showed that they could control POSH with a variety of stimuli, including chemical inducers, light, and electrostimulation. They also demonstrated the versatility of this circuit to program protein secretion in several mammalian cell lines. The researchers first characterized POSH control in HEK293T cells, and then established inducible protein secretion in cancer-derived cells lines (HeLa and HEPG2) and a non-human mammalian cell line (COS-7). This array of effective stimuli and applicable systems emphasized that POSH is an adaptable, controllable platform for a range of scientific avenues. “[We were] hoping that others would find what they need to use this technology in their particular research,” Fussenegger explained.

POSH is not the first reported protease-mediated post-translational synthetic circuit. Fussenegger’s study follows publications on programmable systems by research groups at Stanford University and the University of Ljubljana—all three groups established robust synthetic circuits for controlling cellular behavior in culture.^2,3 Fussenegger’s team took their system one step further, into a rodent model of type 1 diabetes. They validated their platform in vivo as a cell therapy by implanting POSH-engineered HEK293T cells into the mice, and as a gene therapy by injecting mice with a plasmid coding for POSH components. The researchers then injected mice with abscisic acid to induce insulin secretion. This led to a prolonged increase of insulin levels in the blood stream, showing that POSH can be used for on-demand secretion of insulin to normalize hyperglycemia in type 1 diabetic mice.

This work positions post-translational circuits like POSH for potential therapeutic avenues in the future treatment of hormone secretion-based conditions such as diabetes. POSH features a faster response than transcription-based gene switches, and is a valuable tool for post-translational control and programming of cell behavior in culture and beyond.