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  • Viral Transfer Technology
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Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron

Gene Therapy volume 7, pages 1979–1985 (2000)Cite this article

Abstract

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

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References

  1. Hollingsworth SJ et al. The effect of combined expression of interleukin 2 and interleukin 4 on the tumorigenicity and treatment of B16F10 melanoma Br J Cancer 1996 74: 6–15

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Gäken JA et al. Irradiated NC adenocarcinoma cells transduced with both B7.1 and interleukin-2 induce CD4+-mediated rejection of established tumors Hum Gene Ther 1997 8: 477–488

    Article  PubMed  Google Scholar 

  3. Putzer BM et al. Interleukin 12 and B7–1 costimulatory molecule expressed by an adenovirus vector act synergistically to facilitate tumor regression Proc Natl Acad Sci USA 1997 94: 10889–10894

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Sumimoto H et al. GM-CSF and B7–1 (CD80) co-stimulatory signals co-operate in the induction of effective anti-tumor immunity in syngeneic mice Int J Cancer 1997 73: 556–561

    Article  CAS  PubMed  Google Scholar 

  5. Pizzoferrato E et al. Enhanced immunogenicity of B cell lymphoma genetically engineered to express both B7-1 and interleukin-12 Hum Gene Ther 1997 8: 2217–2228

    Article  CAS  PubMed  Google Scholar 

  6. Carroll MW et al. Construction and characterization of a triple-recombinant vaccinia virus encoding B7–1, interleukin 12, and a model tumor antigen J Natl Cancer Inst 1998 90: 1881–1887

    Article  CAS  PubMed  Google Scholar 

  7. Emtage PC et al. A double recombinant adenovirus expressing the costimulatory molecule B7–1 (murine) and human IL-2 induces complete tumor regression in a murine breast adenocarcinoma model J Immunol 1998 160: 2531–2538

    CAS  PubMed  Google Scholar 

  8. Chong H et al. Tumour cell expression of B7 costimulatory molecules and interleukin-12 or granulocyte-macrophage colony-stimulating factor induces a local antitumour response and may generate systemic protective immunity Gene Therapy 1998 5: 223–232

    Article  CAS  PubMed  Google Scholar 

  9. Yoon SJ et al. Synergistic anti-tumor effects with co-expression of GM-CSF and IFN-gamma in murine tumors Int J Cancer 1998 77: 907–912

    Article  CAS  PubMed  Google Scholar 

  10. Putzer BM et al. Combination therapy with interleukin-2 and wild-type p53 expressed by adenoviral vectors potentiates tumor regression in a murine model of breast cancer Hum Gene Ther 1998 9: 707–718

    Article  CAS  PubMed  Google Scholar 

  11. Joki T et al. Induction of effective antitumor immunity in a mouse brain tumor model using B7–1 (CD80) and intercellular adhesive molecule 1 (ICAM-1; CD54) transfection and recombinant interleukin 12 Int J Cancer 1999 82: 714–720

    Article  CAS  PubMed  Google Scholar 

  12. Emerman M, Temin HM . Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism Cell 1984 39: 449–467

    Article  CAS  PubMed  Google Scholar 

  13. Emerman M, Temin HM . Comparison of promoter suppression in avian and murine retrovirus vectors Nucleic Acids Res 1986 14: 9381–9396

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Vile R, Miller N, Chernajovsky Y, Hart IA . Comparison of the properties of different retroviral vectors containing the murine tyrosinase promoter to achieve transcriptionally targeted expression of the HSVtk or IL-2 genes Gene Therapy 1994 1: 307–316

    CAS  PubMed  Google Scholar 

  15. Morgan RA et al. Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy Nucleic Acids Res 1992 20: 1293–1299

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Zitvogel L et al. Construction and characterization of retroviral vectors expressing biologically active human interleukin-12 Hum Gene Ther 1994 5: 1493–1506

    Article  CAS  PubMed  Google Scholar 

  17. Schalken JA et al. Fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas J Clin Invest 1987 80: 1545–1549

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Shapiro J et al. Localization of endogenous furin in cultured cell lines J Histochem Cytochem 1997 45: 3–12

    Article  CAS  PubMed  Google Scholar 

  19. Barr PJ et al. cDNA and gene structure for a human subtilisin-like protease with cleavage specificity for paired basic amino acid residues DNA Cell Biol 1991 10: 319–328

    Article  CAS  PubMed  Google Scholar 

  20. Bresnahan PA et al. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo J Cell Biol 1990 111: 2851–2859

    Article  CAS  PubMed  Google Scholar 

  21. Hosaka M et al. Arg-X-Lys/Arg-Arg motif as a signal for precursor cleavage catalyzed by furin within the constitutive secretory pathway J Biol Chem 1991 266: 12127–12130

    CAS  PubMed  Google Scholar 

  22. Matthews DJ, Goodman LJ, Gorman CM, Wells JA . A survey of furin substrate specificity using substrate phage display Protein Sci 1994 3: 1197–1205

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Liu YC et al. Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector Proc Natl Acad Sci USA 1993 90: 8957–8961

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Groskreutz DJ, Sliwkowski MX, Gorman CM . Genetically engineered proinsulin constitutively processed and secreted as mature, active insulin J Biol Chem 1994 269: 6241–6245

    CAS  PubMed  Google Scholar 

  25. Chen L et al. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer J Immunol 1997 159: 351–359

    CAS  PubMed  Google Scholar 

  26. Mattner F et al. Treatment with homodimeric interleukin-12 (IL-12) p40 protects mice from IL-12-dependent shock but not from tumor necrosis factor alpha-dependent shock Infect Immun 1997 65: 4734–4737

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Methot D et al. Tissue targeting of angiotensin peptides J Biol Chem 1997 272: 12994–12999

    Article  CAS  PubMed  Google Scholar 

  28. Wise RJ et al. Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site Proc Natl Acad Sci USA 1990 87: 9378–9382

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Smeekens SP et al. Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3 Proc Natl Acad Sci USA 1992 89: 8822–8826

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Watanabe T et al. Sequence requirements for precursor cleavage within the constitutive secretory pathway J Biol Chem 1992 267: 8270–8274

    CAS  PubMed  Google Scholar 

  31. Hatsuzawa K et al. Structure and expression of mouse furin, a yeast Kex2-related protease – lack of processing of coexpressed prorenin in GH4C1 J Biol Chem 1990 265: 22075–22078

    CAS  PubMed  Google Scholar 

  32. Schoenhaut D et al. Cloning and expression of murine IL-12 J Immunol 1992 148: 3433–3440

    CAS  PubMed  Google Scholar 

  33. Döhring C, Angman L, Spagnoli G, Lanzavecchia A . T-helper- and accessory-cell-independent cytotoxic responses to human tumor cells transfected with a B7 retroviral vector Int J Cancer 1994 57: 754–759

    Article  PubMed  Google Scholar 

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Acknowledgements

This work was supported by grant number 9605 from the Leukaemia Research Fund.

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Authors and Affiliations

  1. Department of Molecular Medicine, Immune Gene Therapy Programme, The Rayne Institute, London, UK

    J Gäken, J Jiang, E van Berkel, C Hughes, M Kuiper, D Darling, J Galea-Lauri, K Ford & F Farzaneh

  2. Department of Immunology, Immune Gene Therapy Programme, The Rayne Institute, London, UK

    K Daniel & M Kemeny

  3. Department of Oral Oncology King's College School of Medicine and Dentistry, Immune Gene Therapy Programme, The Rayne Institute, London, UK

    M Tavassoli

  4. Mayo Clinic, Rochester, Minnesota, USA

    S Russell

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  1. J Gäken
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Gäken, J., Jiang, J., Daniel, K. et al. Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron. Gene Ther 7, 1979–1985 (2000). https://doi.org/10.1038/sj.gt.3301341

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