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Directed evolution of retroviruses activatable by tumour-associated matrix metalloproteases

Gene Therapy volume 10, pages 1370–1380 (2003)Cite this article

Abstract

Protease-activatable retroviral vectors offer the possibility of targeted gene transfer into cancer cells expressing a unique set of proteases as, for example, the matrix metalloproteases (MMPs). However, it is difficult to predict which substrate sequence will be optimally cleaved by a given tumour cell type. Therefore, we developed a novel approach that allows the selection of MMP-activatable retroviruses from libraries of viruses displaying combinatorially diversified protease substrates. Starting from a virus harbouring a standard MMP-2 substrate motif, after only two consecutive cycles of diversification and in vivo selection, MMP-activatable viruses were recovered. Biochemical characterization of the selected viruses revealed that their linker peptides showed a considerably increased sensitivity for MMP-2 cleavage, and interestingly also improved the particle incorporation rate of the Env protein. Owing to the optimized linker peptide, the selected viruses exhibited a greatly enhanced spreading efficiency through human fibrosarcoma cells, while having retained the dependency on MMP activation. Moreover, cell entry efficiency and virus titres were considerably improved as compared to the parental virus displaying the standard MMP-2 substrate. The results presented imply that retroviral protease substrate libraries allow the definition of MMP substrate specificities under in vivo conditions as well as the generation of optimally adapted tumour-specific viruses.

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References

  1. Kirn D, Niculescu-Duvaz I, Hallden G, Springer CJ . The emerging fields of suicide gene therapy and virotherapy. Trends Mol Med 2002; 8: S68–S73

    Article  CAS  PubMed  Google Scholar 

  2. Buchholz CJ, Stitz J, Cichutek K . Retroviral cell targeting vectors. Curr Opin Mol Ther 1999; 1: 613–621.

    CAS  PubMed  Google Scholar 

  3. Lavillette D, Russell SJ, Cosset FL . Retargeting gene delivery using surface-engineered retroviral vector particles. Curr Opin Biotechnol 2001; 12: 461–466.

    Article  CAS  PubMed  Google Scholar 

  4. Cosset FL et al. Retroviral retargeting by envelopes expressing an N-terminal binding domain. J Virol 1995; 69: 6314–6322.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Martin F et al. Retrovirus targeting by tropism restriction to melanoma cells. J Virol 1999; 73: 6923–6929.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Benedict CA et al. Targeting retroviral vectors to CD34-expressing cells: binding to CD34 does not catalyze virus-cell fusion. Hum Gene Ther 1999; 10: 545–557.

    Article  CAS  PubMed  Google Scholar 

  7. Hall FL et al. Targeting retroviral vectors to vascular lesions by genetic engineering of the MoMLV gp70 envelope protein. Hum Gene Ther 1997; 8: 2183–2192.

    Article  CAS  PubMed  Google Scholar 

  8. Boerger AL, Snitkovsky S, Young JA . Retroviral vectors preloaded with a viral receptor-ligand bridge protein are targeted to specific cell types. Proc Natl Acad Sci USA 1999; 96: 9867–9872.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Peng KW, Vile R, Cosset FL, Russell S . Selective transduction of protease-rich tumors by matrix-metalloproteinase-targeted retroviral vectors. Gene Therapy 1999; 6: 1552–1557.

    Article  CAS  PubMed  Google Scholar 

  10. Buchholz CJ et al. In vivo selection of protease cleavage sites from retrovirus display libraries. Nat Biotechnol 1998; 16: 951–954.

    Article  CAS  PubMed  Google Scholar 

  11. Peng KW et al. A gene delivery system activatable by disease-associated matrix metalloproteinases. Hum Gene Ther 1997; 8: 729–738.

    Article  CAS  PubMed  Google Scholar 

  12. Russell SJ, Cosset FL . Modifying the host range properties of retroviral vectors. J Gene Med 1999; 1: 300–311.

    Article  CAS  PubMed  Google Scholar 

  13. Nilson BHK, Morling FJ, Russell SJ . Targeting of retroviral vectors through protease–substrate interactions. Gene Therapy 1996; 3: 280–286.

    CAS  PubMed  Google Scholar 

  14. Curran S, Murray GI . Matrix metalloproteinases in tumour invasion and metastasis. J Pathol 1999; 189: 300–308.

    Article  CAS  PubMed  Google Scholar 

  15. Nagase H, Woessner Jr JF . Matrix metalloproteinases. J Biol Chem 1999; 274: 21491–21494.

    Article  CAS  PubMed  Google Scholar 

  16. Vu TH, Werb Z . Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev 2000; 14: 2123–2133.

    Article  CAS  PubMed  Google Scholar 

  17. Foda HD, Zucker S . Matrix metalloproteinases in cancer invasion, metastasis and angiogenesis. Drug Discov Today 2001; 6: 478–482.

    Article  CAS  PubMed  Google Scholar 

  18. Stack MS, Gray RD . Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide. J Biol Chem 1989; 264: 4277–4281.

    CAS  PubMed  Google Scholar 

  19. Woessner JF, Nagase H . Matrix Metalloproteinases and TIMPs. Oxford: Oxford University Press, 2000.

    Google Scholar 

  20. Balint RF, Larrick JW . Antibody engineering by parsimonious mutagenesis. Gene 1993; 137: 109–118.

    Article  CAS  PubMed  Google Scholar 

  21. Miyamori H et al. Claudin promotes activation of pro-matrix metalloproteinase-2 mediated by membrane-type matrix metalloproteinases. J Biol Chem 2001; 276: 28204–28211.

    Article  CAS  PubMed  Google Scholar 

  22. Soong NW et al. Molecular breeding of viruses. Nat Genet 2000; 25: 436–439.

    Article  CAS  PubMed  Google Scholar 

  23. Bupp K, Roth MJ . Altering retroviral tropism using a random-display envelope library. Mol Ther 2002; 5: 329–335.

    Article  CAS  PubMed  Google Scholar 

  24. Neuner P, Cortese R, Monaci P . Codon-based mutagenesis using dimer-phosphoramidites. Nucleic Acids Res 1998; 26: 1223–1227.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Liu S, Bugge TH, Leppla SH . Targeting of tumor cells by cell surface urokinase plasminogen activator-dependent anthrax toxin. J Biol Chem 2000; 276: 17976–17984.

    Article  Google Scholar 

  26. Bachrach E et al. Efficient cell infection by Moloney murine leukemia virus-derived particles requires minimal amounts of envelope glycoprotein. J Virol 2000; 74: 8480–8486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Giambernardi TA et al. Overview of matrix metalloproteinase expression in cultured human cells. Matrix Biol 1998; 16: 483–496.

    Article  CAS  PubMed  Google Scholar 

  28. Chen EI et al. A unique substrate recognition profile for matrix metalloproteinase-2. J Biol Chem 2002; 277: 4485–4491.

    Article  CAS  PubMed  Google Scholar 

  29. Morling FJ, Peng KW, Cosset FL, Russell SJ . Masking of retroviral envelope functions by oligomerizing polypeptide adaptors. Virology 1997; 234: 51–61.

    Article  CAS  PubMed  Google Scholar 

  30. Logg CR et al. 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 

  31. DuBridge RB et al. Analysis of mutation in human cells by using an Epstein–Barr virus shuttle system. Mol Cell Biol 1987; 7: 379–387.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Cosset FL et al. High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 1995; 69: 7430–7436.

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Soneoka Y et al. A transient three-plasmid expression system for the production of high titer retroviral vectors. Nucleic Acid Res 1995; 23: 628–633.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Ulrike Mueller for critical reading of the manuscript. This work was supported by grants from the 5th framework European Community programme ‘The cell factory’ to CJB and MPC.

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Authors and Affiliations

  1. Medizinische Biotechnologie, Paul-Ehrlich-Institut, Langen, Germany

    R M Schneider, Y Medvedovska, I Hartl, B Voelker, K Cichutek & C J Buchholz

  2. BioFocus plc, 10 Cambridge Science Park, Cambridge, UK

    M P Chadwick

  3. Mayo Clinic, Rochester, MN, USA

    S J Russell

Authors
  1. R M Schneider
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  2. Y Medvedovska
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  3. I Hartl
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  4. B Voelker
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  6. S J Russell
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  7. K Cichutek
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  8. C J Buchholz
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Correspondence to C J Buchholz.

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Schneider, R., Medvedovska, Y., Hartl, I. et al. Directed evolution of retroviruses activatable by tumour-associated matrix metalloproteases. Gene Ther 10, 1370–1380 (2003). https://doi.org/10.1038/sj.gt.3302007

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  • DOI: https://doi.org/10.1038/sj.gt.3302007

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