Limits...
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.

Show MeSH

Related in: MedlinePlus

(A) Diagram of the autoregulated l-arginine-inducible SEAP expression vector (pSH122). pSH122 harbors the l-arginine-responsive promoter (PART1) which drives transcription of the dicistronic expression unit encoding the the human placental alkaline phosphatase (SEAP) in the first and the l-arginine-dependent transactivator (ARG2) in the second cistron. Whereas translation of SEAP occurs via a classic cap-dependent mechanism, translation-initation of ARG2 is mediated by an internal ribosome entry site of polioviral origin (IRESPV). pA is the polyadenylation signal. Selected restriction sites are indicated: A, AatII; E, EcoRI; N, NotI; X, XbaI, Xh, XhoI. (B) pSH122 was transiently transfected into CHO-K1, cultivated in media containing 10 mg/l or 1 g/l l-arginine prior to SEAP quantification. Fold induction is indicated on the top of the bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175317&req=5

Figure 6: (A) Diagram of the autoregulated l-arginine-inducible SEAP expression vector (pSH122). pSH122 harbors the l-arginine-responsive promoter (PART1) which drives transcription of the dicistronic expression unit encoding the the human placental alkaline phosphatase (SEAP) in the first and the l-arginine-dependent transactivator (ARG2) in the second cistron. Whereas translation of SEAP occurs via a classic cap-dependent mechanism, translation-initation of ARG2 is mediated by an internal ribosome entry site of polioviral origin (IRESPV). pA is the polyadenylation signal. Selected restriction sites are indicated: A, AatII; E, EcoRI; N, NotI; X, XbaI, Xh, XhoI. (B) pSH122 was transiently transfected into CHO-K1, cultivated in media containing 10 mg/l or 1 g/l l-arginine prior to SEAP quantification. Fold induction is indicated on the top of the bars.

Mentions: The ART system was also validated in an autoregulated positive feedback configuration enabling one-step installation of regulated transgene expression in mammalian cells using a single-vector format. This configuration mediates simultaneous expression of transactivator and transgene, both driven by the transactivator-dependent promoter and was found to be instrumental for the design of noise resistant gene networks (58). Leaky transcripts mediated by the PART1 promoter initiate production of relatively few transactivator molecules which trigger full expression of PART1-driven transgenes in the presence of l-arginine. SEAP expression was assessed in CHO-K1 transiently transfected with pSH122 (pSH122, PART1-SEAP-IRESPV-ARG2-pA, Figure 6A). PART1 transcription mediates basal SEAP and ARG2 production which results in ARG2-triggered auto-induction of the dicistronic expression unit in the presence of high l-arginine concentrations (Figure 6B).Figure 6.


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)

(A) Diagram of the autoregulated l-arginine-inducible SEAP expression vector (pSH122). pSH122 harbors the l-arginine-responsive promoter (PART1) which drives transcription of the dicistronic expression unit encoding the the human placental alkaline phosphatase (SEAP) in the first and the l-arginine-dependent transactivator (ARG2) in the second cistron. Whereas translation of SEAP occurs via a classic cap-dependent mechanism, translation-initation of ARG2 is mediated by an internal ribosome entry site of polioviral origin (IRESPV). pA is the polyadenylation signal. Selected restriction sites are indicated: A, AatII; E, EcoRI; N, NotI; X, XbaI, Xh, XhoI. (B) pSH122 was transiently transfected into CHO-K1, cultivated in media containing 10 mg/l or 1 g/l l-arginine prior to SEAP quantification. Fold induction is indicated on the top of the bars.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: (A) Diagram of the autoregulated l-arginine-inducible SEAP expression vector (pSH122). pSH122 harbors the l-arginine-responsive promoter (PART1) which drives transcription of the dicistronic expression unit encoding the the human placental alkaline phosphatase (SEAP) in the first and the l-arginine-dependent transactivator (ARG2) in the second cistron. Whereas translation of SEAP occurs via a classic cap-dependent mechanism, translation-initation of ARG2 is mediated by an internal ribosome entry site of polioviral origin (IRESPV). pA is the polyadenylation signal. Selected restriction sites are indicated: A, AatII; E, EcoRI; N, NotI; X, XbaI, Xh, XhoI. (B) pSH122 was transiently transfected into CHO-K1, cultivated in media containing 10 mg/l or 1 g/l l-arginine prior to SEAP quantification. Fold induction is indicated on the top of the bars.
Mentions: The ART system was also validated in an autoregulated positive feedback configuration enabling one-step installation of regulated transgene expression in mammalian cells using a single-vector format. This configuration mediates simultaneous expression of transactivator and transgene, both driven by the transactivator-dependent promoter and was found to be instrumental for the design of noise resistant gene networks (58). Leaky transcripts mediated by the PART1 promoter initiate production of relatively few transactivator molecules which trigger full expression of PART1-driven transgenes in the presence of l-arginine. SEAP expression was assessed in CHO-K1 transiently transfected with pSH122 (pSH122, PART1-SEAP-IRESPV-ARG2-pA, Figure 6A). PART1 transcription mediates basal SEAP and ARG2 production which results in ARG2-triggered auto-induction of the dicistronic expression unit in the presence of high l-arginine concentrations (Figure 6B).Figure 6.

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