An engineered L-arginine sensor of Chlamydia pneumoniae enables arginine-adjustable transcription control in mammalian cells and mice

Abstract

For optimal compatibility with biopharmaceutical manufacturing and gene therapy, heterologous transgene control systems must be responsive to side-effect-free physiologic inducer molecules. The arginine-inducible interaction of the ArgR repressor and the ArgR-specific ARG box, which synchronize arginine import and synthesis in the intracellular human pathogen Chlamydia pneumoniae, was engineered for arginine-regulated transgene (ART) expression in mammalian cells. A synthetic arginine-responsive transactivator (ARG), consisting of ArgR fused to the Herpes simplex VP16 transactivation domain, reversibly adjusted transgene transcription of chimeric ARG box-containing mammalian minimal promoters (PART) in an arginine-inducible manner. Arginine-controlled transgene expression showed rapid induction kinetics in a variety of mammalian cell lines and was adjustable and reversible at concentrations which were compatible with host cell physiology. ART variants containing different transactivation domains, variable spacing between ARG box and minimal promoter and several tandem ARG boxes showed modified regulation performance tailored for specific expression scenarios and cell types. Mice implanted with microencapsulated cells engineered for ART-inducible expression of the human placental secreted alkaline phosphatase (SEAP) exhibited adjustable serum phosphatase levels after treatment with different arginine doses. Using a physiologic inducer, such as the amino acid l-arginine, to control heterologous transgenes in a seamless manner which is devoid of noticeable metabolic interference will foster novel opportunities for precise expression dosing in future gene therapy scenarios as well as the manufacturing of difficult-to-produce protein pharmaceuticals.

Arginase-Negative Mutants of Arabidopsis Exhibit Increased Nitric Oxide Signaling in Root Development

“…A test of this is to use mitochondrial (and other organelle)-specific fluorescent probes for Arg. Arg-induced reporter genes developed in animal cells (Hartenbach et al, 2007) may be modified to elucidate cellular differences in plant Arg content.…”

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

“…Inducer-triggered modulation of the affinity of the transactivator to its cognate promoter results in adjustable and reversible transcription control of the specific target gene (13)(14)(15)(16). In recent years, a panoply of such heterologous transcription control modalities have been developed, which are responsive to various inducer molecules such as antibiotics (13,14,17), steroid hormones and their analogs (18,19), quorum-sensing molecules (20,21), immunosuppressive and antidiabetic drugs (22,23), biotin (24), L-arginine (25), as well as volatile acetaldehyde (16). Apart from gaseous acetaldehyde, which can simply be inhaled, all other inducers need to be either taken up orally or be administered by injection in any future gene therapy application.…”

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…Because the ligandbinding domain is part of the protein, the binding of the ligand to the regulator dimer leads to the dissociation of the operator-dimer complex, thus influencing gene-expression rates. Based on this design strategy, mammalian cells have been engineered with various synthetic systems to sense different ligand inputs (e.g., antibiotics, Gossen and Bujard 1992;Fussenegger et al 2000;Urlinger et al 2000;Weber et al 2002Weber et al , 2003and Weber et al 2006Weber et al , 2009Hartenbach et al 2007;Gitzinger et al 2009Gitzinger et al , 2012Kemmer et al 2010;Bacchus et al 2013) and proven to be useful tools for diverse biological applications. Moreover, a synthetic system that enables dual-input gene-expression control was developed using a single engineered prokaryotic regulator protein fused to the transrepressor domain KRAB, termed KRAB-CbaR (Xie et al 2014).…”