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Design and Testing of Novel Oncolytic Vaccinia Strains

Part of the Methods in Molecular Biology™ book series (MIMB,volume 542)


Oncolytic or replication-selective viruses have been used as powerful tools for the delivery of therapeutic genes to tumors. Because these vectors are capable of replicating within the tumor, the therapeutic gene is amplified within the target tissue itself, resulting in the spread of the virus both within the tumor, and sometimes also between tumors. Vaccinia virus holds many advantages when serving as the backbone for oncolytic viral strains, including a large cloning capacity (at least 25 kbp) (1); a short life-cycle (2, 3); extensive previous use in humans, with contraindications and adverse reactions well described and antivirals available (4); the potential for systemic (intravenous) delivery to distant tumors; and vaccinia strains have previously demonstrated antitumor benefits in clinical trials (5).

Because vaccinia has no known receptor and is capable of infecting almost any cell type, tumor selectivity has to be engineered into vaccinia at steps after infection. We will therefore discuss potential viral virulence genes and metabolic targets that result in tumor-selective vaccinia strains. Because the virus has limited natural requirements for host cell proteins, and, instead, contains a large genome and multiple genes involved in virulence, a large number of possible attenuating gene deletions can result in the production of viral strains reliant on inherent properties of the host cell for replication.

The protocols for producing viral gene deletions and constructing viral gene expression vectors have been well established for vaccinia and are summarized briefly in this chapter. Basic assays for testing the tumor selectivity and therapeutic index of new oncolytic constructs in vitro will be covered. In addition, we describe how bioluminescence imaging can be incorporated into preclinical testing of vaccinia gene expression strains to examine the timing, biodistribution, and kinetics of viral gene expression noninvasively after delivery of the viral agents to tumor-bearing mice via different routes.


  • Oncolytic virus
  • replication-selective
  • tumor-targeting
  • vaccinia
  • virotherapy

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  • DOI: 10.1007/978-1-59745-561-9_32
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  1. Smith, G. L., and Moss, B. (1983) Infectious poxvirus vectors have capacity for at least 25 000 base pairs of foreign DNA. Gene 25, 21–8.

    PubMed  CrossRef  CAS  Google Scholar 

  2. Moss, B. (2001) (D.M., K., Fields, B. N., and Howley, P. M., Eds.) in "Field's Virology", pp. Ch.84, Lippincott-Raven, Philadelphia.

    Google Scholar 

  3. Buller, R. M., and Palumbo, G. J. (1991) Poxvirus pathogenesis. Microbiol Rev 55, 80–122.

    PubMed  CAS  Google Scholar 

  4. Moss, B. (1991) Vaccinia virus: a tool for research and vaccine development. Science 252, 1662–7.

    PubMed  CrossRef  CAS  Google Scholar 

  5. Thorne, S. H., and Kirn, D. H. (2004) Future directions for the field of oncolytic virotherapy: a perspective on the use of vaccinia virus. Expert Opin Biol Ther 4, 1307–21.

    PubMed  CrossRef  CAS  Google Scholar 

  6. Smith, G. L., Symons, J. A., Khanna, A., Vanderplasschen, A., and Alcami, A. (1997) Vaccinia virus immune evasion. Immunol Rev 159, 137–54.

    PubMed  CrossRef  CAS  Google Scholar 

  7. Thorne, S. H., Hwang, T. H., and Kirn, D. H. (2005) Vaccinia virus and oncolytic virotherapy of cancer. Curr Opin Mol Ther 7, 359–65.

    PubMed  Google Scholar 

  8. McCart, J. A., Ward, J. M., Lee, J., Hu, Y., Alexander, H. R., Libutti, S. K., Moss, B., and Bartlett, D. L. (2001) Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res 61, 8751–7.

    PubMed  CAS  Google Scholar 

  9. Chakrabarti, S., Sisler, J. R., and Moss, B. (1997) Compact, synthetic, vaccinia virus early/late promoter for protein expression. Biotechniques 23, 1094–7.

    PubMed  CAS  Google Scholar 

  10. Coupar, B. E., Oke, P. G., and Andrew, M. E. (2000) Insertion sites for recombinant vaccinia virus construction: effects on expression of a foreign protein. J Gen Virol 81, 431–9.

    PubMed  CAS  Google Scholar 

  11. Zhao, H., Doyle, T. C., Coquoz, O., Kalish, F., Rice, B. W., and Contag, C. H. (2005) Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo. J Biomed Opt 10, 41210.

    PubMed  CrossRef  Google Scholar 

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Correspondence to Steve H. Thorne .

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Thorne, S. (2009). Design and Testing of Novel Oncolytic Vaccinia Strains. In: Walther, W., Stein, U. (eds) Gene Therapy of Cancer. Methods in Molecular Biology™, vol 542. Humana Press.

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  • Print ISBN: 978-1-934115-85-5

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