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Macrolide-based transgene control in mammalian cells and mice

Nature Biotechnology volume 20, pages 901–907 (2002)Cite this article

Abstract

Heterologous mammalian gene regulation systems for adjustable expression of multiple transgenes are necessary for advanced human gene therapy and tissue engineering, and for sophisticated in vivo gene-function analyses, drug discovery, and biopharmaceutical manufacturing. The antibiotic-dependent interaction between the repressor (E) and operator (ETR) derived from an Escherichia coli erythromycin-resistance regulon was used to design repressible (EOFF) and inducible (EON) mammalian gene regulation systems (E.REX) responsive to clinically licensed macrolide antibiotics (erythromycin, clarithromycin, and roxithromycin). The EOFF system consists of a chimeric erythromycin-dependent transactivator (ET), constructed by fusing the prokaryotic repressor E to a eukaryotic transactivation domain that binds and activates transcription from ETR-containing synthetic eukaryotic promoters (PETR). Addition of macrolide antibiotic results in repression of transgene expression. The EON system is based on E binding to artificial ETR-derived operators cloned adjacent to constitutive promoters, resulting in repression of transgene expression. In the presence of macrolides, gene expression is induced. Control of transgene expression in primary cells, cell lines, and microencapsulated human cells transplanted into mice was demonstrated using the E.REX (EOFF and EON) systems. The macrolide-responsive E.REX technology was functionally compatible with the streptogramin (PIP)–regulated and tetracycline (TET)–regulated expression systems, and therefore may be combined for multiregulated multigene therapeutic interventions in mammalian cells and tissues.

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Figure 1: Regulation of gene expression by macrolide antibiotics.
Figure 2: E.REX-based autoregulated expression technology.
Figure 3: Dose-response expression characteristics and production kinetics of the erythromycin-inducible gene control system.
Figure 4: E.REX-based multiregulated multigene expression technology.
Figure 5: Macrolide-adjustable EPO secretion from microencapsulated HT-1080 cell derivatives in mice and culture.

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References

  1. Fussenegger, M. The impact of mammalian gene regulation concepts on functional genomic research, metabolic engineering, and advanced gene therapies. Biotechnol. Prog. 17, 1–51 (2001).

    Article  CAS  Google Scholar 

  2. Chin, L. et al. Essential role for oncogenic Ras in tumour maintenance. Nature 400, 468–472 (1999).

    Article  CAS  Google Scholar 

  3. Malleret, G. et al. Inducible and reversible enhancement of learning, memory, and long-term potentiation by genetic inhibition of calcineurin. Cell 104, 675–686 (2001).

    Article  CAS  Google Scholar 

  4. Niwa, H., Miyazaki, J. & Smith, A.G. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat. Genet. 24, 372–376 (2000).

    Article  CAS  Google Scholar 

  5. Ewald, D. et al. Time-sensitive reversal of hyperplasia in transgenic mice expressing SV40 T antigen. Science 273, 1384–1386 (1996).

    Article  CAS  Google Scholar 

  6. Aubel, D. et al. Design of a novel mammalian screening system for the detection of bioavailable, non-cytotoxic streptogramin antibiotics. J. Antibiot. 54, 44–55 (2001).

    Article  CAS  Google Scholar 

  7. Rivera, V.M. et al. A humanized system for pharmacologic control of gene expression. Nat. Med. 2, 1028–1032 (1997).

    Article  Google Scholar 

  8. Bohl, D., Naffakh, N. & Heard, J.M. Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nat. Med. 3, 299–305 (1997).

    Article  CAS  Google Scholar 

  9. Serguera, C., Bohl, D., Rolland, E., Prevost, P. & Heard, J.M. Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells. Hum. Gene Ther. 19, 375–383 (1999).

    Article  Google Scholar 

  10. Wang, Y., O'Malley Jr., B.W., Tsai, S. & O'Malley, B. A regulatory system for use in gene transfer. Proc. Natl. Acad. Sci. USA 91, 8180–8184 (1994).

    Article  CAS  Google Scholar 

  11. Ye, X. et al. Regulation of therapeutic proteins after in vivo somatic cell gene transfer. Science 283, 88–91 (1999).

    Article  CAS  Google Scholar 

  12. Fussenegger, M., Bailey, J.E. & Varner, J. A mathematical model of caspase function in apoptosis. Nat. Biotechnol. 18, 768–774 (2000).

    Article  CAS  Google Scholar 

  13. Fussenegger, M., Schlatter, S., Dätwyler, D., Mazur, X. & Bailey, J.E. Controlled proliferation by multigene metabolic engineering enhances the productivity of Chinese hamster ovary cells. Nat. Biotechnol. 16, 468–472 (1998).

    Article  CAS  Google Scholar 

  14. Fux, C. et al. Streptogramin- and tetracycline-responsive dual regulated expression of p27Kip1 sense and antisense enables positive and negative growth control of Chinese hamster ovary cells. Nucleic Acids Res. 29, E19 (2001).

    Article  CAS  Google Scholar 

  15. Moutsatos, I.K. et al. Exogenously regulated stem cell-mediated gene therapy for bone regeneration. Mol. Ther. 3, 449–461 (2001).

    Article  Google Scholar 

  16. Rossi, F.M.V. et al. Tetracycline-regulated factors with distinct dimerization domains allow reversible growth inhibition by p16. Nat. Genet. 20, 389–393 (1998).

    Article  CAS  Google Scholar 

  17. Umaña, P., Jean-Mairet, J., Moudry, R., Amstutz, H. & Bailey, J.E. Engineered glycoforms of an antineuroblastoma IgG1 with optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 17, 176–180 (1999).

    Article  Google Scholar 

  18. Gossen, M. & Bujard, H. Tight control of gene expression in mammalian cells by tetracycline-responsive promoters. Proc. Natl. Acad. Sci. USA 89, 5547–5551 (1992).

    Article  CAS  Google Scholar 

  19. Gossen, M. et al. Transcriptional activation by tetracyclines in mammalian cells. Science 268, 1766–1769 (1995).

    Article  CAS  Google Scholar 

  20. Fussenegger, M. et al. Streptogramin-based gene regulation systems for mammalian cells. Nat. Biotechnol. 18, 1203–1208 (2000).

    Article  CAS  Google Scholar 

  21. Boorsma, M. et al. A novel temperature inducible replicon-based DNA expression system. Nat. Biotechnol. 18, 429–432 (2000).

    Article  CAS  Google Scholar 

  22. Moser, S. et al. Dual-regulated expression technology: a new era in adjusting heterologous gene expression in mammalian cells. J. Gen. Med. 3, 529–549 (2001).

    Article  CAS  Google Scholar 

  23. Guay, D.R.P. Macrolide antibiotics in paediatric infection diseases. Drugs 51, 515–536 (1996).

    Article  CAS  Google Scholar 

  24. Williams, J.D. & Sefton, A.M. Comparison of macrolide antibiotics. J. Antimicrob. Chemother. (suppl. C) 31, 11–26 (1993).

    Article  CAS  Google Scholar 

  25. Noguchi, N. et al. Nucleotide sequence and characterization of erythromycin resistance determinant that encodes macrolide 2'-phosphotransferase I in Escherichia coli. Antimicrob. Agents Chemother. 39, 2359–2363 (1995).

    Article  CAS  Google Scholar 

  26. Noguchi, N., Takada, K., Katayama, J., Emura, A. & Sasatsu, M. Regulation of transcription of the mph(A) gene for macrolide 2'phosphotransferase I in Escherichia coli: characterization of the regulatory gene mphR(A). J. Bacteriol. 182, 5052–5058 (2000).

    Article  CAS  Google Scholar 

  27. Triezenberg, S.J., Kingsbury, R.C. & McKnight, S.L. Functional dissection of VP16, the transactivator of herpes simplex virus immediate early gene expression. Genes Dev. 2, 718–729 (1988).

    Article  CAS  Google Scholar 

  28. Fussenegger, M., Mazur, X. & Bailey, J.E. A novel cytostatic process enhances the productivity of Chinese hamster ovary cells. Biotechnol. Bioeng. 55, 927–939 (1997).

    Article  CAS  Google Scholar 

  29. Rodvold, K.A. Clinical pharmacokinetics of clarithromycin. Clin. Pharmacokinet. 37, 385–398 (1999).

    Article  CAS  Google Scholar 

  30. Fussenegger, M., Moser, S., Mazur, X. & Bailey, J.E. Autoregulated multicistronic expression vectors provide one-step cloning of regulated product gene expression in mammalian cells. Biotechnol. Prog. 13, 733–740 (1997).

    Article  CAS  Google Scholar 

  31. Moosmann, P., Georgiev, O., Thiesen, H.J., Hagmann, M. & Schaffner, W. Silencing of RNA polymerases II and III-dependent transcription by the KRAB protein domain of Kox1, a kruppel-type zinc finger factor. Biol. Chem. 387, 669–677 (1997).

    Google Scholar 

  32. Moser, S. et al. An update of pTRIDENT multicistronic expression vectors: pTRIDENTs containing novel streptogramin-responsive promoters. Biotechnol. Prog. 16, 724–735 (2000).

    Article  CAS  Google Scholar 

  33. Cheung, W., Minton, N. & Gunawardena, K. Pharmacokinetics and pharmacodynamics of epoietin alfa once weekly and three times weekly. Eur. J. Clin. Pharmacol. 57, 411–418 (2001).

    Article  CAS  Google Scholar 

  34. Aiello, L., Guilfoyle, R., Huebner, K. & Weinmann, R. Adenovirus 5 DNA sequences present and RNA sequences transcribed in transformed human embryo kidney cells (HEK-Ad-5 or 293). Virology 94, 460–469 (1979).

    Article  CAS  Google Scholar 

  35. Schlatter, S., Rimann, M., Kelm, J. & Fussenegger, M. SAMY, a novel mammalian reporter gene derived from Bacillus stearothermophilus α-amylase. Gene 282, 19–31 (2002).

    Article  CAS  Google Scholar 

  36. Bristow, A. A collaborative study for the establishment of a biological reference preparation for erythropoietin. Pharmeuropa 2, 31–48 (1997).

    Google Scholar 

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Acknowledgements

We thank N. Noguchi for providing pTZ3509, A. Weber for bibliographic work, E. Niederer (ETH Institute of Biomedical Engineering) for FACS, and J. Zanghi for critical comments on the manuscript. This work was supported by Cistronics Cell Technology (Zurich, Switzerland) and the Swiss National Science Foundation (grant 631-065946). This paper is dedicated to James E. Bailey, an outstanding scientist, friend, and mentor who passed away during the final revision of this manuscript.

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  1. James E. Bailey: Deceased.

Authors and Affiliations

  1. Institute of Biotechnology, Swiss Federal Institute of Technology, ETH Zurich, Zurich, CH-8093, Switzerland

    Wilfried Weber, Cornelia Fux, Bettina Keller, Beat P. Kramer, James E. Bailey & Martin Fussenegger

  2. IUTA Département Génie Biologique, Institut Universitaire de Technologie, IUTA, 43 Boulevard du 11 Novembre 1918, Villeurbanne Cedex, F-69200, France

    Marie Daoud-El Baba & Dominique Aubel

  3. Institute for Biomedical Engineering, Swiss Federal Institute of Technology, ETH Zurich, Zurich, CH-8044, Switzerland

    Cornelia C. Weber

  4. Inotech Encapsulation AG, Kirchstrasse 1, Dottikon, CH-5605, Switzerland

    Christoph Heinzen

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Correspondence to Martin Fussenegger.

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Weber, W., Fux, C., Daoud-El Baba, M. et al. Macrolide-based transgene control in mammalian cells and mice. Nat Biotechnol 20, 901–907 (2002). https://doi.org/10.1038/nbt731

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