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Characterization of an immuno ‘stealth’ derivative of the herpes simplex virus thymidine-kinase gene

Cancer Gene Therapy volume 13, pages 584–591 (2006)Cite this article

Abstract

The cellular immune response against transgene-encoded neoantigens is a potential hurdle in gene therapy applications where long-term expression of transgenes is desired. Here a new optimized derivative of the herpes simplex virus 1-thymidine-kinase (HSV1-TK) gene is described. The HSV-TK gene is frequently used in experimental studies on gene-directed enzyme prodrug therapy. In the optimized gene, the HSV-TK coding region is fused with the codons for the Gly-Ala repeat of the Epstein–Barr virus nuclear-antigen 1 to prevent proteasomal degradation of the HSV-TK. To measure the protective effect in vitro, a model cytotoxic T lymphocyte epitope derived from the ovalbumin was inserted in the TK. Cells expressing the GAr-modified TK do not present TK-derived peptides in the major histocompatibility complex. Furthermore, conservative nucleotide substitutions were introduced, which prevent splicing, as well as mutations that render the TK-expressing cells more sensitive to ganciclovir (GCV). The GAr HSV-TK fusion protein is fully functional in vitro. This HSV-TK gene may be especially useful in those gene therapy applications where an immune response against the transgene-encoded product would frustrate the treatment.

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References

  1. Jooss K, Yang Y, Fisher KJ, Wilson JM . Transduction of dendritic cells by DNA viral vectors directs the immune response to transgene products in muscle fibers. J Virol 1998; 72: 4212–4223.

    CAS  PubMed  PubMed Central  Google Scholar 

  2. Abina MA, Lee MG, Descamps V, Cordier L, Lopez M, Perricaudet M et al. LacZ gene transfer into tumor cells abrogates tumorigenicity and protects mice against the development of further tumors. Gene Therapy 1996; 3: 212–216.

    CAS  PubMed  Google Scholar 

  3. Cordier L, Gao GP, Hack AA, McNally EM, Wilson JM, Chirmule N et al. Muscle-specific promoters may be necessary for adeno-associated virus-mediated gene transfer in the treatment of muscular dystrophies. Hum Gene Ther 2001; 12: 205–215.

    Article  CAS  PubMed  Google Scholar 

  4. Yang Y, Ertl HC, Wilson JM . MHC class I-restricted cytotoxic T lymphocytes to viral antigens destroy hepatocytes in mice infected with E1-deleted recombinant adenoviruses. Immunity 1994; 1: 433–442.

    Article  CAS  PubMed  Google Scholar 

  5. Yang Y, Su Q, Wilson JM . Role of viral antigens in destructive cellular immune responses to adenovirus vector-transduced cells in mouse lungs. J Virol 1996; 70: 7209–7212.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Yang Y, Wilson JM . Clearance of adenovirus-infected hepatocytes by MHC class I-restricted CD4+ CTLs in vivo. J Immunol 1995; 155: 2564–2570.

    CAS  PubMed  Google Scholar 

  7. Juillard V, Villefroy P, Godfrin D, Pavirani A, Venet A, Guillet JG . Long-term humoral and cellular immunity induced by a single immunization with replication-defective adenovirus recombinant vector. Eur J Immunol 1995; 25: 3467–3473.

    Article  CAS  PubMed  Google Scholar 

  8. Latta-Mahieu M, Rolland M, Caillet C, Wang M, Kennel P, Mahfouz I et al. Gene transfer of a chimeric trans-activator is immunogenic and results in short-lived transgene expression. Hum Gene Ther 2002; 13: 1611–1620.

    Article  CAS  PubMed  Google Scholar 

  9. Thomis DC, Marktel S, Bonini C, Traversari C, Gilman M, Bordignon C et al. A Fas-based suicide switch in human T cells for the treatment of graft-versus-host disease. Blood 2001; 97: 1249–1257.

    Article  CAS  PubMed  Google Scholar 

  10. van der Eb MM, de Leeuw B, van der Eb AJ, Hoeben RC . Side effects of suicide gene therapy. Methods Mol Med 2004; 90: 479–490.

    CAS  PubMed  Google Scholar 

  11. Morris JC, Wildner O . Therapy of head and neck squamous cell carcinoma with an oncolytic adenovirus expressing HSV-tk. Mol Ther 2000; 1: 56–62.

    Article  CAS  PubMed  Google Scholar 

  12. Fareed MU, Moolten FL . Suicide gene transduction sensitizes murine embryonic and human mesenchymal stem cells to ablation on demand – a fail-safe protection against cellular misbehavior. Gene Therapy 2002; 9: 955–962.

    Article  CAS  PubMed  Google Scholar 

  13. Wiertz EJ, Mukherjee S, Ploegh HL . Viruses use stealth technology to escape from the host immune system. Mol Med Today 1997; 3: 116–123.

    Article  CAS  PubMed  Google Scholar 

  14. Ploegh HL . Viral strategies of immune evasion. Science 1998; 280: 248–253.

    Article  CAS  PubMed  Google Scholar 

  15. Yewdell JW, Hill AB . Viral interference with antigen presentation. Nat Immunol 2002; 3: 1019–1025.

    Article  CAS  PubMed  Google Scholar 

  16. Khanna R, Burrows SR, Kurilla MG, Jacob CA, Misko IS, Sculley TB et al. Localization of Epstein–Barr virus cytotoxic T cell epitopes using recombinant vaccinia: implications for vaccine development. J Exp Med 1992; 176: 169–176.

    Article  CAS  PubMed  Google Scholar 

  17. Khanna R, Burrows SR, Steigerwald-Mullen PM, Thomson SA, Kurilla MG, Moss DJ . Isolation of cytotoxic T lymphocytes from healthy seropositive individuals specific for peptide epitopes from Epstein–Barr virus nuclear antigen 1: implications for viral persistence and tumor surveillance. Virology 1995; 214: 633–637.

    Article  CAS  PubMed  Google Scholar 

  18. Munz C, Bickham KL, Subklewe M, Tsang ML, Chahroudi A, Kurilla MG et al. Human CD4(+) T lymphocytes consistently respond to the latent Epstein–Barr virus nuclear antigen EBNA1. J Exp Med 2000; 191: 1649–1660.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Blake N, Lee S, Redchenko I, Thomas W, Steven N, Leese A et al. Human CD8+ T cell responses to EBV EBNA1: HLA class I presentation of the (Gly-Ala)-containing protein requires exogenous processing. Immunity 1997; 7: 791–802.

    Article  CAS  PubMed  Google Scholar 

  20. Blake N, Haigh T, Shaka'a G, Croom-Carter D, Rickinson A . The importance of exogenous antigen in priming the human CD8+ T cell response: lessons from the EBV nuclear antigen EBNA1. J Immunol 2000; 165: 7078–7087.

    Article  CAS  PubMed  Google Scholar 

  21. Rickinson AB, Moss DJ . Human cytotoxic T lymphocyte responses to Epstein–Barr virus infection. Annu Rev Immunol 1997; 15: 405–431.

    Article  CAS  PubMed  Google Scholar 

  22. Levitskaya J, Coram M, Levitsky V, Imreh S, Steigerwald-Mullen PM, Klein G et al. Inhibition of antigen processing by the internal repeat region of the Epstein–Barr virus nuclear antigen-1. Nature 1995; 375: 685–688.

    Article  CAS  PubMed  Google Scholar 

  23. Levitskaya J, Sharipo A, Leonchiks A, Ciechanover A, Masucci MG . Inhibition of ubiquitin/proteasome-dependent protein degradation by the Gly-Ala repeat domain of the Epstein–Barr virus nuclear antigen 1. Proc Natl Acad Sci USA 1997; 94: 12616–12621.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Ossevoort M, Visser BM, van den Wollenberg DJ, van der Voort EI, Offringa R, Melief CJ et al. Creation of immune ‘stealth’ genes for gene therapy through fusion with the Gly-Ala repeat of EBNA-1. Gene Therapy 2003; 10: 2020–2028.

    Article  CAS  PubMed  Google Scholar 

  25. Barry SC, Harder B, Brzezinski M, Flint LY, Seppen J, Osborne WR . Lentivirus vectors encoding both central polypurine tract and posttranscriptional regulatory element provide enhanced transduction and transgene expression. Hum Gene Ther 2001; 12: 1103–1108.

    Article  CAS  PubMed  Google Scholar 

  26. DuBridge RB, Tang P, Hsia HC, Leong PM, Miller JH, Calos MP . 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 

  27. Fidler IJ . Selection of successive tumour lines for metastasis. Nat New Biol 1973; 242: 148–149.

    Article  CAS  PubMed  Google Scholar 

  28. Topp WC . Normal rat cell lines deficient in nuclear thymidine kinase. Virology 1981; 113: 408–411.

    Article  CAS  PubMed  Google Scholar 

  29. Karttunen J, Sanderson S, Shastri N . Detection of rare antigen-presenting cells by the lacZ T-cell activation assay suggests an expression cloning strategy for T-cell antigens. Proc Natl Acad Sci USA 1992; 89: 6020–6024.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Graham FL, van der Eb AJ . A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology 1973; 52: 456–467.

    Article  CAS  PubMed  Google Scholar 

  31. Chalmers D, Ferrand C, Apperley JF, Melo JV, Ebeling S, Newton I et al. Elimination of the truncated message from the herpes simplex virus thymidine kinase suicide gene. Mol Ther 2001; 4: 146–148.

    Article  CAS  PubMed  Google Scholar 

  32. Cowsill C, Southgate TD, Morrissey G, Dewey RA, Morelli AE, Maleniak TC et al. Central nervous system toxicity of two adenoviral vectors encoding variants of the herpes simplex virus type 1 thymidine kinase: reduced cytotoxicity of a truncated HSV1-TK. Gene Therapy 2000; 7: 679–685.

    Article  CAS  PubMed  Google Scholar 

  33. Carbone FR, Bevan MJ . Induction of ovalbumin-specific cytotoxic T cells by in vivo peptide immunization. J Exp Med 1989; 169: 603–612.

    Article  CAS  PubMed  Google Scholar 

  34. Rotzschke O, Falk K, Stevanovic S, Jung G, Walden P, Rammensee HG . Exact prediction of a natural T cell epitope. Eur J Immunol 1991; 21: 2891–2894.

    Article  CAS  PubMed  Google Scholar 

  35. Black ME, Kokoris MS, Sabo P . Herpes simplex virus-1 thymidine kinase mutants created by semi-random sequence mutagenesis improve prodrug-mediated tumor cell killing. Cancer Res 2001; 61: 3022–3026.

    CAS  PubMed  Google Scholar 

  36. Kokoris MS, Black ME . Characterization of herpes simplex virus type 1 thymidine kinase mutants engineered for improved ganciclovir or acyclovir activity. Protein Sci 2002; 11: 2267–2272.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Carlotti F, Zaldumbide A, Martin P, Boulukos KE, Hoeben RC, Pognonec P . Development of an inducible suicide gene system based on human caspase 8. Cancer Gene Ther 2005; 12: 627–639.

    Article  CAS  PubMed  Google Scholar 

  38. Fan L, Freeman KW, Khan T, Pham E, Spencer DM . Improved artificial death switches based on caspases and FADD. Hum Gene Ther 1999; 10: 2273–2285.

    Article  CAS  PubMed  Google Scholar 

  39. Sharipo A, Imreh M, Leonchiks A, Imreh S, Masucci MG . A minimal glycine-alanine repeat prevents the interaction of ubiquitinated I kappaB alpha with the proteasome: a new mechanism for selective inhibition of proteolysis. Nat Med 1998; 4: 939–944.

    Article  CAS  PubMed  Google Scholar 

  40. Sharipo A, Imreh M, Leonchiks A, Branden C, Masucci MG . cis-Inhibition of proteasomal degradation by viral repeats: impact of length and amino acid composition. FEBS Lett 2001; 499: 137–142.

    Article  CAS  PubMed  Google Scholar 

  41. Tellam J, Connolly G, Green KJ, Miles JJ, Moss DJ, Burrows SR et al. Endogenous presentation of CD8+ T cell epitopes from Epstein–Barr virus-encoded nuclear antigen 1. J Exp Med 2004; 199: 1421–1431.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Lee SP, Brooks JM, Al Jarrah H, Thomas WA, Haigh TA, Taylor GS et al. CD8 T cell recognition of endogenously expressed Epstein–Barr virus nuclear antigen 1. J Exp Med 2004; 199: 1409–1420.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands

    M Ossevoort, A Zaldumbide, S J Cramer & R C Hoeben

  2. Department of Immunohematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands

    E I H van der Voort & R E M Toes

  3. Department of Rheumatology, Leiden University Medical Center, Leiden, The Netherlands

    E I H van der Voort & R E M Toes

Authors
  1. M Ossevoort
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  2. A Zaldumbide
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  3. S J Cramer
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  4. E I H van der Voort
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  5. R E M Toes
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  6. R C Hoeben
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Corresponding author

Correspondence to R C Hoeben.

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Ossevoort, M., Zaldumbide, A., Cramer, S. et al. Characterization of an immuno ‘stealth’ derivative of the herpes simplex virus thymidine-kinase gene. Cancer Gene Ther 13, 584–591 (2006). https://doi.org/10.1038/sj.cgt.7700925

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  • DOI: https://doi.org/10.1038/sj.cgt.7700925

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