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

Hartenbach S, Daoud-El Baba M, Weber W, Fussenegger M - Nucleic Acids Res. (2007)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland.

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.

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Impact of l-arginine and its derivatives on ART-controlled gene expression. (A) The structure of the l-arginine derivatives used in this study. (B) Sixty hours after co-transfection with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), SEAP expression of CHO-K1 was measured. Cells were incubated in cell culture media containing 10 mg/l l-arginine and supplemented with 57.4, 574 or 5740 μM of l-arginine, l-ornithine, l-citrulline, agmatine, l-homoarginine, l-arginine methyl ester, l-arginine ethyl ester or l-canavanine (5740 μM correspond to 1 g/l l-arginine).
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Figure 7: Impact of l-arginine and its derivatives on ART-controlled gene expression. (A) The structure of the l-arginine derivatives used in this study. (B) Sixty hours after co-transfection with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), SEAP expression of CHO-K1 was measured. Cells were incubated in cell culture media containing 10 mg/l l-arginine and supplemented with 57.4, 574 or 5740 μM of l-arginine, l-ornithine, l-citrulline, agmatine, l-homoarginine, l-arginine methyl ester, l-arginine ethyl ester or l-canavanine (5740 μM correspond to 1 g/l l-arginine).

Mentions: In order to assess the specificity of ART-controlled transgene expression, we cultivated CHO-K1, co-transfected with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), in the presence of increasing concentrations of l-arginine and several of its derivatives (l-canavanine, l-homoarginine, l-arginine methyl ester and l-arginine ethyl ester) and secondary metabolic products (l-ornithine, l-citrulline, agmatine) and profiled SEAP production after 60 h (Figure 7A). Of all these compounds only l-arginine and its methyl and ethyl esters induced SEAP to significant levels while retaining typical dose–response characteristics (Figure 7B).Figure 7.


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

Hartenbach S, Daoud-El Baba M, Weber W, Fussenegger M - Nucleic Acids Res. (2007)

Impact of l-arginine and its derivatives on ART-controlled gene expression. (A) The structure of the l-arginine derivatives used in this study. (B) Sixty hours after co-transfection with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), SEAP expression of CHO-K1 was measured. Cells were incubated in cell culture media containing 10 mg/l l-arginine and supplemented with 57.4, 574 or 5740 μM of l-arginine, l-ornithine, l-citrulline, agmatine, l-homoarginine, l-arginine methyl ester, l-arginine ethyl ester or l-canavanine (5740 μM correspond to 1 g/l l-arginine).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2175317&req=5

Figure 7: Impact of l-arginine and its derivatives on ART-controlled gene expression. (A) The structure of the l-arginine derivatives used in this study. (B) Sixty hours after co-transfection with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), SEAP expression of CHO-K1 was measured. Cells were incubated in cell culture media containing 10 mg/l l-arginine and supplemented with 57.4, 574 or 5740 μM of l-arginine, l-ornithine, l-citrulline, agmatine, l-homoarginine, l-arginine methyl ester, l-arginine ethyl ester or l-canavanine (5740 μM correspond to 1 g/l l-arginine).
Mentions: In order to assess the specificity of ART-controlled transgene expression, we cultivated CHO-K1, co-transfected with pSH91 (PSV40-ARG2-pA) and pSH93 (PART1-SEAP-pA), in the presence of increasing concentrations of l-arginine and several of its derivatives (l-canavanine, l-homoarginine, l-arginine methyl ester and l-arginine ethyl ester) and secondary metabolic products (l-ornithine, l-citrulline, agmatine) and profiled SEAP production after 60 h (Figure 7A). Of all these compounds only l-arginine and its methyl and ethyl esters induced SEAP to significant levels while retaining typical dose–response characteristics (Figure 7B).Figure 7.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Chemical and Bioengineering, ETH Zurich, Wolfgang-Pauli-Strasse 10, HCI F115, CH-8093 Zurich, Switzerland.

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.

Show MeSH
Related in: MedlinePlus