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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 on overall production capacity and cell density. CHO-ET1-SEAP1, constitutively expressing SEAP, were exposed to l-arginine concentrations ranging from 0 to 10 000 mg/l. SEAP production (black bars) and maximum cell densities (gray bars). Standard ChoMaster® HTS medium contains 200 mg/l l-arginine. For l-arginine concentration ranging from 0 to 2 g/l, the media is iso-osmotic (280 – 310 mOsm). Above 2 g/l l-arginine, the media shifts into hypersomotic condition (>310 mOsm).
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Figure 1: Impact of l-arginine on overall production capacity and cell density. CHO-ET1-SEAP1, constitutively expressing SEAP, were exposed to l-arginine concentrations ranging from 0 to 10 000 mg/l. SEAP production (black bars) and maximum cell densities (gray bars). Standard ChoMaster® HTS medium contains 200 mg/l l-arginine. For l-arginine concentration ranging from 0 to 2 g/l, the media is iso-osmotic (280 – 310 mOsm). Above 2 g/l l-arginine, the media shifts into hypersomotic condition (>310 mOsm).

Mentions: Since l-arginine plays a central role in the urea cycle, the synthesis of nitric oxide and for the synthesis of cytoplasmic and nuclear proteins (49), it is often the rate-limiting amino acid in fast-growing organisms (50,51). In order to prevent l-arginine becoming a physiologic bottleneck or endogenous l-arginine interfering with ART-controlled transgene expression in mammalian cells, we exposed a CHO-K1-derived SEAP-expressing cell line [CHO-ET1-SEAP1, (10)] to l-arginine concentrations ranging from 0 to 10 000 mg/l and scored SEAP levels and cell density after a 60 h cultivation period. A 10 mg/l l-arginine was sufficient to support robust wild-type-like cell growth with SEAP production levels equivalent to control cultivations in standard ChoMaster® HTS medium containing a default l-arginine concentration of 200 mg/l (Figure 1). This observation is quantitatively comparable with physiologic plasma concentrations of l-arginine found in mice and humans [∼17 mg/l, (52)]. Between 10 mg/l and 2 g/l of l-arginine, SEAP production and cell densities were comparable to populations cultivated in standard ChoMaster® HTS medium. At l-arginine concentrations above 2 g/l the culture media became hyperosmotic (>310 mOsm) which compromised cell viability and titer (Figure 1). Based on this data, we selected 10 mg/l and 1 g/l as low and high l-arginine concentrations in follow-up experiments.Figure 1.


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 on overall production capacity and cell density. CHO-ET1-SEAP1, constitutively expressing SEAP, were exposed to l-arginine concentrations ranging from 0 to 10 000 mg/l. SEAP production (black bars) and maximum cell densities (gray bars). Standard ChoMaster® HTS medium contains 200 mg/l l-arginine. For l-arginine concentration ranging from 0 to 2 g/l, the media is iso-osmotic (280 – 310 mOsm). Above 2 g/l l-arginine, the media shifts into hypersomotic condition (>310 mOsm).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Impact of l-arginine on overall production capacity and cell density. CHO-ET1-SEAP1, constitutively expressing SEAP, were exposed to l-arginine concentrations ranging from 0 to 10 000 mg/l. SEAP production (black bars) and maximum cell densities (gray bars). Standard ChoMaster® HTS medium contains 200 mg/l l-arginine. For l-arginine concentration ranging from 0 to 2 g/l, the media is iso-osmotic (280 – 310 mOsm). Above 2 g/l l-arginine, the media shifts into hypersomotic condition (>310 mOsm).
Mentions: Since l-arginine plays a central role in the urea cycle, the synthesis of nitric oxide and for the synthesis of cytoplasmic and nuclear proteins (49), it is often the rate-limiting amino acid in fast-growing organisms (50,51). In order to prevent l-arginine becoming a physiologic bottleneck or endogenous l-arginine interfering with ART-controlled transgene expression in mammalian cells, we exposed a CHO-K1-derived SEAP-expressing cell line [CHO-ET1-SEAP1, (10)] to l-arginine concentrations ranging from 0 to 10 000 mg/l and scored SEAP levels and cell density after a 60 h cultivation period. A 10 mg/l l-arginine was sufficient to support robust wild-type-like cell growth with SEAP production levels equivalent to control cultivations in standard ChoMaster® HTS medium containing a default l-arginine concentration of 200 mg/l (Figure 1). This observation is quantitatively comparable with physiologic plasma concentrations of l-arginine found in mice and humans [∼17 mg/l, (52)]. Between 10 mg/l and 2 g/l of l-arginine, SEAP production and cell densities were comparable to populations cultivated in standard ChoMaster® HTS medium. At l-arginine concentrations above 2 g/l the culture media became hyperosmotic (>310 mOsm) which compromised cell viability and titer (Figure 1). Based on this data, we selected 10 mg/l and 1 g/l as low and high l-arginine concentrations in follow-up experiments.Figure 1.

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