Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo.

Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation of functional proteins by tunable proteolytic activity. In the absence of inhibitor, the co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop of the protein of interest, irreversibly fragmenting the protein structure and destroying its functionality. We selected three proteases and demonstrated the versatility of the iCROP framework by validating it to regulate the functional activity of ten different proteins. iCROP switches can be delivered either as mRNA or DNA, and provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed in the off state without inhibitor. iCROPs for effectors of the NF-κB and NFAT signaling pathways were assembled and confirmed to enable precise activation/inhibition of downstream events in response to protease inhibitors. In lipopolysaccharide-treated mice, iCROP-sr-IκBα suppressed cytokine release ("cytokine storm") by rescuing the activity of IκBα, which suppresses NF-κB signaling. We also constructed compact inducible CRISPR-(d)Cas9 variants and showed that iCROP-Cas9-mediated knockout of the PCSK9 gene in the liver lowered blood LDL-cholesterol levels in mice. iCROP-based protein switches will facilitate protein-level regulation in basic research and translational applications.

Design of programmable post-translational switch control platform for on-demand protein secretion in mammalian cells.

The development of novel strategies to program cellular behaviors is a central goal in synthetic biology, and post-translational control mediated by engineered protein circuits is a particularly attractive approach to achieve rapid protein secretion on demand. We have developed a programmable protease-mediated post-translational switch (POSH) control platform composed of a chimeric protein unit that consists of a protein of interest fused via a transmembrane domain to a cleavable ER-retention signal, together with two cytosolic inducer-sensitive split protease components. The protease components combine in the presence of the specific inducer to generate active protease, which cleaves the ER-retention signal, releasing the transmembrane-domain-linked protein for trafficking to the trans-Golgi region. A furin site placed downstream of the protein ensures cleavage and subsequent secretion of the desired protein. We show that stimuli ranging from plant-derived, clinically compatible chemicals to remotely controllable inducers such as light and electrostimulation can program protein secretion in various POSH-engineered designer mammalian cells. As proof-of-concept, an all-in-one POSH control plasmid encoding insulin and abscisic acid-activatable split protease units was hydrodynamically transfected into the liver of type-1 diabetic mice. Induction with abscisic acid attenuated glycemic excursions in glucose-tolerance tests. Increased blood levels of insulin were maintained for 12 days.

Regulation of protein secretion through chemical regulation of endoplasmic reticulum retention signal cleavage.

Secreted proteins, such as hormones or cytokines, are key mediators in multicellular organisms. Response of protein secretion based on transcriptional control is rather slow, as it requires transcription, translation and transport from the endoplasmic reticulum (ER) to the plasma membrane via the conventional protein secretion (CPS) pathway. An alternative regulation to provide faster response would be valuable. Here we present two genetically encoded orthogonal regulatory secretion systems, which rely on the retention of pre-synthesized proteins on the ER membrane (membER, released by a cytosolic protease) or inside the ER lumen (lumER, released by an ER-luminal protease), respectively, and their release by the chemical signal-regulated proteolytic removal of an ER-retention signal, without triggering ER stress due to protein aggregates. Design of orthogonal chemically-regulated split proteases enables the combination of signals into logic functions. Its application was demonstrated on a chemically regulated therapeutic protein secretion and regulated membrane translocation of a chimeric antigen receptor (CAR) targeting cancer antigen. Regulation of the ER escape represents a platform for the design of fast-responsive and tightly-controlled modular and scalable protein secretion system for mammalian cells.

Design of modular autoproteolytic gene switches responsive to anti-coronavirus drug candidates.

The main (Mpro) and papain-like (PLpro) proteases encoded by SARS-CoV-2 are essential to process viral polyproteins into functional units, thus representing key targets for anti-viral drug development. There is a need for an efficient inhibitor screening system that can identify drug candidates in a cellular context. Here we describe modular, tunable autoproteolytic gene switches (TAGS) relying on synthetic transcription factors that self-inactivate, unless in the presence of coronavirus protease inhibitors, consequently activating transgene expression. TAGS rapidly report the impact of drug candidates on Mpro and PLpro activities with a high signal-to-noise response and a sensitivity matching concentration ranges inhibiting viral replication. The modularity of the TAGS enabled the study of other Coronaviridae proteases, characterization of mutations and multiplexing of gene switches in human cells. Mice implanted with Mpro or PLpro TAGS-engineered cells enabled analysis of the activity and bioavailability of protease inhibitors in vivo in a virus-free setting.