Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells

Cancer Gene Therapy volume 12, pages 464–474 (2005)Cite this article

Abstract

The successful application of cancer gene therapy has been hampered by the low efficiency of in vivo gene delivery by currently used replication-defective vectors. Accordingly, considerable efforts are now being directed toward development and use of vectors capable of replicating in cancer cells. However, for replicating retroviruses, insertion of additional reading frames into the viral genome often resulted in the generation of unstable viruses. Here, we report a novel concept for the generation of replication-competent murine leukemia virus (MLV) vectors capable of mediating the secretion of soluble therapeutic proteins from infected cells. As a proof of principle, we inserted transgene regions encoding either a single-chain variable region fragment (scFv), here, the laminin-specific L36-scFv, or the T-cell-specific 7A5-scFv, or the cytokine GM-CSF into the MLV envelope (env) gene after +1 codon of the envelope (Env) protein, followed by a sequence specifying a furin protease cleavage site. The resulting viruses, termed L36-furin-A, 7A5-furin-A and GMCSF-furin-Mo, respectively, infected a variety of human cell lines, including HMEC-1 (endothelial), A301 (lymphoid), MDA-MB231 and MDA-MB468 (breast cancer) and HT1080 (fibrosarcoma) cells. Western blot analysis of conditioned culture medium from HT1080 cells infected by replicating L36-furin A, as an example, revealed that more than 90% of the Env fusion protein molecules were indeed intracellularly cleaved. After 5 days of infection, up to 3–4 μg/ml of soluble L36-scFv accumulated in the supernatant of HT1080 cells. The eukaryotically produced L36-scFv and 7A5-scFv were able to recognize their native antigens with high avidity, as assessed by ELISA and flow cytometry. Furthermore, the replicating viruses were genetically stable for more than 12 cell passages. In conclusion, a new generation of replication-competent retroviral vectors capable of mediating long-term and efficient secretion of therapeutic proteins suitable for cancer therapy was generated.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  1. Roth JA, Cristiano RJ . Gene therapy for cancer: what have we done and where are we going? J Natl Cancer Inst. 1997;89:21–39.

    Article  CAS  PubMed  Google Scholar 

  2. Vile RG, Russell SJ, Lemoine NR . Cancer gene therapy: hard lessons and new courses. Gene Therapy. 2000;7:2–8.

    Article  CAS  PubMed  Google Scholar 

  3. Ram Z, Culver KW, Oshiro EM, et al. Therapy of malignant brain tumors by intratumoral implantation of retroviral vector-producing cells. Nat Med. 1997;3:1354–1361.

    Article  CAS  PubMed  Google Scholar 

  4. Kruse CA, Roper MD, Kleinschmidt-DeMasters BK, et al. Purified herpes simplex thymidine kinase retrovector particles. I. In vitro characterization, in situ transduction efficiency, and histopathological analyses of gene therapy-treated brain tumors. Cancer Gene Ther. 1997;4:118–128.

    CAS  PubMed  Google Scholar 

  5. Russell SJ . Replicating vectors for gene therapy of cancer: risks, limitations and prospects. Eur J Cancer. 1994;30A:1165–1171.

    Article  CAS  PubMed  Google Scholar 

  6. Yoon TK, Shichinohe T, Laquerre S, Kasahara N . Selectively replicating adenoviruses for oncolytic therapy. Curr Cancer Drug Targets. 2001;1:85–107.

    Article  CAS  PubMed  Google Scholar 

  7. Mullen JT, Tanabe KK . Viral oncolysis. The Oncologist. 2002;7:106–119.

    Article  CAS  PubMed  Google Scholar 

  8. Liu BL, Robinson M, Han ZQ, et al. ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Therapy. 2003;10:292–303.

    Article  CAS  PubMed  Google Scholar 

  9. Vile RG, Sunassee K, Diaz RM . Strategies for achieving multiple layers of selectivity in gene therapy. Mol Med Today. 1998;4:84–92.

    Article  CAS  PubMed  Google Scholar 

  10. Peng KW, Russell SJ . Viral vector targeting. Curr Opin Biotechnol. 1999;10:454–457.

    Article  CAS  PubMed  Google Scholar 

  11. Spencer DM . Developments in suicide genes for preclinical and clinical applications. Curr Opin Mol Ther. 2000;2:433–440.

    CAS  PubMed  Google Scholar 

  12. Bansal K, Engelhard HH . Gene therapy for brain tumors. Curr Oncol Rep. 2000;2:463–472.

    Article  CAS  PubMed  Google Scholar 

  13. O'Reilly MS, Boehm T, Shing Y, et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell. 1997;88:277–285.

    Article  CAS  PubMed  Google Scholar 

  14. Regulier E, Paul S, Marigliano M, et al. Adenovirus-mediated delivery of antiangiogenic genes as an antitumor approach. Cancer Gene Ther. 2001;8:45–54.

    Article  CAS  PubMed  Google Scholar 

  15. Scappaticci FA, Smith R, Pathak A, et al. Combination angiostatin and endostatin gene transfer induces synergistic antiangiogenic activity in vitro and antitumor efficacy in leukemia and solid tumors in mice. Mol Ther. 2001;3:186–196.

    Article  CAS  PubMed  Google Scholar 

  16. Li H, Lindenmeyer F, Grenet C, et al. AdTIMP-2 inhibits tumor growth, angiogenesis, and metastasis, and prolongs survival in mice. Hum Gene Ther. 2001;12:515–526.

    Article  CAS  PubMed  Google Scholar 

  17. Sanz L, Kristensen P, Blanco B, et al. Single-chain antibody-based gene therapy: inhibition of tumor growth by in situ production of phage-derived human antibody fragments blocking functionally active sites of cell-associated matrices. Gene Therapy. 2002;9:1049–1053.

    Article  CAS  PubMed  Google Scholar 

  18. Dillon PJ, Lenz J, Rosen CA . Construction of a replication-competent murine retrovirus vector expressing the human immunodeficiency virus type 1 tat transactivator protein. J Virol. 1991;65:4490–4493.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Reik W, Weiher H, Jaenisch R . Replication-competent Moloney murine leukemia virus carrying a bacterial suppressor tRNA gene: selective cloning of proviral and flanking host sequences. Proc Natl Acad Sci USA. 1985;82:1141–1145.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Stuhlmann H, Jaenisch R, Mulligan RC . Construction and properties of replication-competent murine retroviral vectors encoding methotrexate resistance. Mol Cell Biol. 1989;9:100–108.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Buchholz CJ, Peng KW, Morling FJ, et al. In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol. 1998;16:951–954.

    Article  CAS  PubMed  Google Scholar 

  22. Nakayama K . Furin: a mammalian subtilisin/Kex2p-like endoprotease involved in processing of a wide variety of precursor proteins. Biochem J. 1997;327:625–635.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Sanz L, Kristensen P, Russell SJ, Ramirez Garcia JR, Alvarez-Vallina L . Generation and characterization of recombinant human antibodies specific for native laminin epitopes: potential application in cancer therapy. Cancer Immunol Immunother. 2001;50:557–565.

    Article  CAS  PubMed  Google Scholar 

  24. Engelstadter M, Bobkova M, Baier M, et al. Targeting human T cells by retroviral vectors displaying antibody domains selected from a phage display library. Hum Gene Ther. 2000;11:293–303.

    Article  CAS  PubMed  Google Scholar 

  25. Schneider RM, Medvedovska Y, Hartl I, et al. Directed evolution of retroviruses activatable by tumor-associated matrix metalloproteases. Gene Therapy. 2003;10:1370–1380.

    Article  CAS  PubMed  Google Scholar 

  26. Sorokin L, Girg W, Gopfert T, Hallmann R, Deutzmann R . Expression of novel 400-kDa laminin chains by mouse and bovine endothelial cells. Eur J Biochem. 1994;223:603–610.

    Article  CAS  PubMed  Google Scholar 

  27. Ekblom M, Falk M, Salmivirta K, Durbeej M, Ekblom P . Laminin isoforms and epithelial development. Ann N Y Acad Sci. 1998;857:194–211.

    Article  CAS  PubMed  Google Scholar 

  28. Miosge N . The ultrastructural composition of basement membranes in vivo. Histol Histopathol. 2001;16:1239–1248.

    CAS  PubMed  Google Scholar 

  29. Jespersen T, Duch M, Carrasco ML, Warming S, Pedersen FS . Expression of heterologous genes from an IRES translational cassette in replication competent murine leukemia virus vectors. Gene. 1999;239:227–235.

    Article  CAS  PubMed  Google Scholar 

  30. Miller AD . Development and applications of retroviral vectors. In: Coffin JM, Hughes HH, Varmus HE, eds. Retroviruses. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1997: 437–474.

    Google Scholar 

  31. Logg CR, Tai CK, Logg A, Anderson WF, Kasahara N . A uniquely stable replication-competent retrovirus vector achieves efficient gene delivery in vitro and in solid tumors. Hum Gene Ther. 2001;12:921–932.

    Article  CAS  PubMed  Google Scholar 

  32. Logg CR, Logg A, Tai CK, Cannon PM, Kasahara N . Genomic stability of murine leukemia viruses containing insertions at the Env-3′ untranslated region boundary. J Virol. 2001;75:6989–6998.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wang WJ, Tai C-K, Kasahara N, Chen TC . Highly efficient and tumor-restricted gene transfer to malignant gliomas by replication-competent retroviral vectors. Hum Gene Ther. 2003;14:117–127.

    Article  CAS  PubMed  Google Scholar 

  34. Liu YC, Kawagishi M, Mikayama T, 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 

  35. 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 

  36. Gaken J, Jiang J, Daniel K, et al. Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron. Gene Therapy. 2000;7:1979–1985.

    Article  CAS  PubMed  Google Scholar 

  37. Sandrin V, Russell SJ, Cosset FL . Targeting retroviral and lentiviral vectors. Curr Top Microbiol Immunol. 2003;281:137–178.

    CAS  PubMed  Google Scholar 

  38. Murray L, Luens K, Tushinski R, et al. Optimization of retroviral gene transduction of mobilized primitive hematopoietic progenitors by using thrombopoietin, Flt3, and Kit ligands and RetroNectin culture. Hum Gene Ther. 1999;10:1743–1752.

    Article  CAS  PubMed  Google Scholar 

  39. Donahue RE, Kessler SW, Bodine D, et al. Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J Exp Med. 1992;176:1125–1135.

    Article  CAS  PubMed  Google Scholar 

  40. Marshall E . Clinical research. Gene therapy a suspect in leukemia-like disease. Science. 2002;298:34–35.

    Article  CAS  PubMed  Google Scholar 

  41. Nilson BH, Morling FJ, Cosset FL, Russell SJ . Targeting of retroviral vectors through protease-substrate interactions. Gene Therapy. 1996;3:280–286.

    CAS  PubMed  Google Scholar 

  42. Logg CR, Logg A, Matusik RJ, Bochner BH, Kasahara N . Tissue-specific transcriptional targeting of a replication-competent retroviral vector. J Virol. 2002;76:12783–12791.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Diaz RM, Eisen T, Hart IR, et al. Exchange of viral promoter/enhancer elements with heterologous regulatory sequences generates targeted hybrid long terminal repeat vectors for gene therapy of melanoma. J Virol. 1998;72:789–795.

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Jager U, Zhao Y, Porter CD . Endothelial cell-specific transcriptional targeting from a hybrid long terminal repeat retrovirus vector containing human prepro-endothelin-1 promoter sequences. J Virol. 1999;73:9702–9709.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by grant 2002.067.1 of the Wilhelm-Sander-Stiftung to CJB and KC and from the Ministerio de Ciencia y Tecnología (grant BIO2001-0385) and the 5th framework of the European Community (Grant EC QLK3-CT-1999-00386) to L Á-V.

Author information

Authors and Affiliations

  1. Division of Medical Biotechnology, Paul-Ehrlich-Institut, 63225, Langen, Germany

    Carsten Finger, Yuansheng Sun, Christian J Buchholz & Klaus Cichutek

  2. Department of Immunology, Hospital Universitario Clinica Puerta de Hierro, 28035, Madrid, Spain

    Laura Sanz & Luis Alvarez-Vallina

Authors
  1. Carsten Finger
    View author publications

    Search author on:PubMed Google Scholar

  2. Yuansheng Sun
    View author publications

    Search author on:PubMed Google Scholar

  3. Laura Sanz
    View author publications

    Search author on:PubMed Google Scholar

  4. Luis Alvarez-Vallina
    View author publications

    Search author on:PubMed Google Scholar

  5. Christian J Buchholz
    View author publications

    Search author on:PubMed Google Scholar

  6. Klaus Cichutek
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to Klaus Cichutek.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Finger, C., Sun, Y., Sanz, L. et al. Replicating retroviral vectors mediating continuous production and secretion of therapeutic gene products from cancer cells. Cancer Gene Ther 12, 464–474 (2005). https://doi.org/10.1038/sj.cgt.7700805

Download citation

  • Received:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700805

Keywords

This article is cited by

Search

Advanced search

Quick links