title: 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 (P(ART)) 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.
authors.affiliation: Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland.
authors.forename: Shizuka
authors.initials: S
authors.lastname: Hartenbach
authors.forename: Marie
authors.initials: M
authors.lastname: Daoud-El Baba
authors.forename: Wilfried
authors.initials: W
authors.lastname: Weber
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D000818:Animals; D001120:Arginine / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D016466:CHO Cells; D016993:Chlamydophila pneumoniae / Q000235:genetics / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005822:Genetic Vectors; D051379:Mice; D012097:Repressor Proteins / Q000378:metabolism*; D015534:Trans-Activators; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: C087018:ArgR protein, Bacteria; D001426:Bacterial Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D001120:Arginine