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.

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Triple-transcript and tricistronic expression configurations conserve the DNA packaging capacity of the size-constrained viral transduction systems and enable coordinated and regulated expression of up to three therapeutic transgenes for concerted clinical interventions in future gene therapy scenarios.

Introduction of specific mutations into a synthetic internal ribosome entry site (IRES GTX ) derived from the GTX homeodomain protein revealed additional transcriptional activity. This novel synthetic P GTX promoter exhibited consensus core promoter modules such as the initiator (Inr) and the partial downstream promoter elements (DPE) and mediated high-level expression of a variety of transgenes including the human vascular endothelial growth factor 121 (VEGF 121 ), the human placental secreted alkaline phosphatase (SEAP), and the Bacillus stearothermophilus-derived secreted a-amylase (SAMY) in Chinese hamster ovary cells (CHO-K1) and a variety of other mammalian and human cell lines. The spacing between Inr and DPE modules was found to be critical for promoter performance since introduction of a single nucleotide (resulting in P GTX2 ) doubled the SEAP expression levels in CHO-K1. P GTX2 reached near 70% of P SV40 -driven expression levels and outperformed constitutive phosphoglycerate kinase (P PGK ) and human ubiquitin C (P hUBC ) promoters in CHO-K1. Also, P GTX2 was successfully engineered for macrolide-inducible transgene expression. Owing to its size of only 182 bp, P GTX2 is one of the smallest eukaryotic promoters. Although P GTX2 was found to be a potent promoter, it retained its IRES GTX -specific translation-initiation capacity. Synthetic DNAs, which combine multiple activities in a most compact sequence format may foster advances in therapeutic engineering of mammalian cells.