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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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(A) Validation of PART1 variants containing a different number of ARG-specific operator modules. SEAP expression vector encoding l-arginine-responsive promoters harboring monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) operator modules were co-transfected with pSH91 (PSV40-ARG2-pA) into CHO-K1 and SEAP production was profiled after 60 h. The induction factor is shown on the top of each bar. (B) Dose–response profile of interferon-β expression in CHO-K1. Cells were transiently co-transfected with pSH113 (pSH113, PART1-INF-β-pA) and pSH91 (PSV40-ARG2-pA) and grown for 48 h at different l-arginine concentrations before quantification of the interferon-β production in the supernatant. Fold induction is shown on the top of each bar.
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Figure 4: (A) Validation of PART1 variants containing a different number of ARG-specific operator modules. SEAP expression vector encoding l-arginine-responsive promoters harboring monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) operator modules were co-transfected with pSH91 (PSV40-ARG2-pA) into CHO-K1 and SEAP production was profiled after 60 h. The induction factor is shown on the top of each bar. (B) Dose–response profile of interferon-β expression in CHO-K1. Cells were transiently co-transfected with pSH113 (pSH113, PART1-INF-β-pA) and pSH91 (PSV40-ARG2-pA) and grown for 48 h at different l-arginine concentrations before quantification of the interferon-β production in the supernatant. Fold induction is shown on the top of each bar.

Mentions: Increasing the number of tandem operator modules within a trigger-inducible promoter may increase maximum expression levels as more transactivators can be recruited to the promoter (57). The transcriptional activity of the promoters harboring the operator in a monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) configuration were assessed in CHO-K1 cells co-transfected with pSH91 (pSH91, PSV40-ARG2-pA). In general, increasing the number of operator modules resulted in higher maximum expression levels but also higher basal expression (Figure 4A). Having defined the parameters for optimal transgene regulation, the ART system was further validated for expression of the multiple sclerosis therapeutic interferon-β. Interferon-β was cloned downstream of PART1 (pSH113, PART1-INF-β-pA), which was transactivated by ARG2 (pSH91, PSV40-ARG2-pA). CHO-K1 transiently co-transfected with pSH113 and pSH91 enabled adjustable INF-β expression, when exposed to l-arginine concentrations ranging from 10 mg/l to 1g/l (Figure 4B).Figure 4.


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) Validation of PART1 variants containing a different number of ARG-specific operator modules. SEAP expression vector encoding l-arginine-responsive promoters harboring monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) operator modules were co-transfected with pSH91 (PSV40-ARG2-pA) into CHO-K1 and SEAP production was profiled after 60 h. The induction factor is shown on the top of each bar. (B) Dose–response profile of interferon-β expression in CHO-K1. Cells were transiently co-transfected with pSH113 (pSH113, PART1-INF-β-pA) and pSH91 (PSV40-ARG2-pA) and grown for 48 h at different l-arginine concentrations before quantification of the interferon-β production in the supernatant. Fold induction is shown on the top of each bar.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 4: (A) Validation of PART1 variants containing a different number of ARG-specific operator modules. SEAP expression vector encoding l-arginine-responsive promoters harboring monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) operator modules were co-transfected with pSH91 (PSV40-ARG2-pA) into CHO-K1 and SEAP production was profiled after 60 h. The induction factor is shown on the top of each bar. (B) Dose–response profile of interferon-β expression in CHO-K1. Cells were transiently co-transfected with pSH113 (pSH113, PART1-INF-β-pA) and pSH91 (PSV40-ARG2-pA) and grown for 48 h at different l-arginine concentrations before quantification of the interferon-β production in the supernatant. Fold induction is shown on the top of each bar.
Mentions: Increasing the number of tandem operator modules within a trigger-inducible promoter may increase maximum expression levels as more transactivators can be recruited to the promoter (57). The transcriptional activity of the promoters harboring the operator in a monomeric (pSH117, PARTm1-SEAP-pA; PARTm1, AscI-OARG-MluI-0bp-PhCMVmin), dimeric (pSH119, PARTm2-SEAP-pA; PARTm2, AscI-OARG-7bp-OARG–MluI-0bp-PhCMVmin), trimeric (pSH126, PARTm3-SEAP-pA; PARTm3, AscI-OARG-7bp-OARG-7bp-OARG–MluI-0bp-PhCMVmin) or tetrameric (pSH127, PARTm4-SEAP-pA; PARTm4, AscI-OARG-7bp-OARG-7bp-OARG–7bp-OARG-MluI-0bp-PhCMVmin) configuration were assessed in CHO-K1 cells co-transfected with pSH91 (pSH91, PSV40-ARG2-pA). In general, increasing the number of operator modules resulted in higher maximum expression levels but also higher basal expression (Figure 4A). Having defined the parameters for optimal transgene regulation, the ART system was further validated for expression of the multiple sclerosis therapeutic interferon-β. Interferon-β was cloned downstream of PART1 (pSH113, PART1-INF-β-pA), which was transactivated by ARG2 (pSH91, PSV40-ARG2-pA). CHO-K1 transiently co-transfected with pSH113 and pSH91 enabled adjustable INF-β expression, when exposed to l-arginine concentrations ranging from 10 mg/l to 1g/l (Figure 4B).Figure 4.

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