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.

  • Article
  • Published:

Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein–Barr virus

Nature Medicine volume 1, pages 1303–1308 (1995)Cite this article

Abstract

The B-lymphotrophic human herpes Epstein–Barr virus (EBV) is a 160-kilobase double-stranded DNA episomal virus carried in a persistent asymptomatic state by more than 90% of the worldwide adult population. We engineered a helper-dependent mini-EBV, With the minimal cis-EBV elements for episomal replication, viral amplification and packaging, for use as a gene delivery system. The therapeutic potential of this system was established by stably transducing B-lymphoblastoid cells from a Fanconi anaemia group C (FA-C) patient with a mini-EBV constitutively expressing the normal FACC cDNA and showing in vitro correction of the FA phenotype. In the absence of selective pressure, episomal expression persisted with a half-life of 30 days in actively growing transduced cells, indicating a retention rate of 98% expression per cell doubling. This work demonstrates the generation of an infectious non-transforming viral vector that can potentially deliver large therapeutic genes efficiently and selectively into human B cells.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Similar content being viewed by others

References

  1. Krauss, J.C. Hematopoietic stem cell gene replacement therapy. Biochem. biophys. Acta 1114, 193–207 (1992).

    CAS  PubMed  Google Scholar 

  2. Morgan, R.A. & Anderson, W.F. Human gene therapy. Annu. Rev. Biochem. 62, 191–217 (1994).

    Article  Google Scholar 

  3. Dunbar, C.E. & Emmons, R.V.B. Gene transfer into hematopoietic progenitor and stem cells: Progress and problems. Stem Cells 12, 563–576 (1994)

    Article  CAS  PubMed  Google Scholar 

  4. Culver, K.W., Anderson, W.F. & Blaese, R.M. Lymphocyte gene therapy. Hum. Gene Ther. 2, 107–109 (1991).

    Article  CAS  PubMed  Google Scholar 

  5. Blaese, R.M. & Anderson, W.F. The ADA human gene therapy: Clinical protocol. Hum. Gene Ther. 1, 327–362 (1990).

    Article  Google Scholar 

  6. Kincade, P.W. & Gibble, J.M. B lymphocytes. in Fundamental Immunology (ed.Paul, W.) 41–67 (Raven, New York, 1992).

    Google Scholar 

  7. Sprent, J. T and B memory cells. Cell 76, 315–322 (1994).

    Article  CAS  PubMed  Google Scholar 

  8. Vos, J.-M. Herpesviruses as genetic vectors. in Viruses in Human Gene Therapy (ed. Vos, J.-M.H.) 108–140 (Carolina Academic Press, Durham, North Carolina, 1994).

    Google Scholar 

  9. Kieff, E. & Liebowitz, D., Epstein Barr virus and its replication. in Virology (eds Fields, B.N. & Knipe, D.M.) 1889–1920 (Raven, New York, 1990).

    Google Scholar 

  10. Miller, G., Barr virus. in Virology (eds Fields, B.N. & Knipe, D.M.) 1921–1958 (Raven, New York, 1990).

    Google Scholar 

  11. Miyashita, E.M., Yang, B., Lam, K.M.C., Crawford, D.H. & Thorley-Lawson, D.A. A novel form of Epstein Barr virus latency in normal B-cells in vivo. Cell 80, 593–601 (1995).

    Article  CAS  PubMed  Google Scholar 

  12. Chen, F. et al. A Subpopulation of normal B-cells latently infected with Epstein Barr virus resembles Burkitt lymphoma cells in expressing EBNA-1 but not EBNA-2 or LMP-1. J. Virol. 69, 3752–3758 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Farell, P.J., Barr viral genome. Adv. Viral Oncol. 8, 103–132 (1989).

    Google Scholar 

  14. Cohen, -J.I., Picchio, G.R. & Mosier, D.E. Barr virus nuclear protein 2 is a critical determinant for tumor growth in SCID mice and for transformation in vitro. J. Virol. 66, 7555–7559 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Sun, T.-Q., Fenstermacher, D. & Vos, J.-M. Human artificial episomal chromosomes for cloning large DNA in human cells. Nature Genet. 8, 33–41 (1994).

    Article  CAS  PubMed  Google Scholar 

  16. Sun, T.-Q. & Vos, J.-M. Packaging of 200kb engineered DNA as infectious Epstein Barr virus. Int. J. Genome Res. 1, 45–57 (1992).

    CAS  Google Scholar 

  17. Bloss, T.A. & Sugden, B. Optimal length for DNA encapsidated by Epstein Barr virus. J. Virology 68, 8217–8222 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Fanconi, G. Familial constitutional panmyelocytopathy, Fanconi's anemia (F.A.) I. Clinical aspects. Semin. Hematol. 4, 233–240 (1967).

    CAS  PubMed  Google Scholar 

  19. Strathdee, C.A. & Buchwald, M. Molecular and cellular biology of Fanconi's anaemia. Am. J. Ped. Hemat. Oncol. 5, 177–185 (1992).

    Article  Google Scholar 

  20. Strathdee, C.A., Gavish, H., Shannon, W.R. & Buchwald, M. Cloning of cDNAs for Fanconi's anemia by functional complementation. Nature 356, 763–767 (1992).

    Article  CAS  PubMed  Google Scholar 

  21. Matsumoto, A., Vos, J.-M. & Hanawalt, P.C. Repair analysis of mitomycin C-induced DNA crosslinking in ribosomal RNA genes in lymphoblastoid cells from Fanconi's anemia patient. Mutat. Res. 217, 185–192 (1989).

    Article  CAS  PubMed  Google Scholar 

  22. Yates, J.L., Warren, N. & Sugden, B. Stable replication of plasmid derived from Epstein–Barr virus in various mammalian cells. Nature 313, 812–815 (1985).

    Article  CAS  PubMed  Google Scholar 

  23. Dillon, N. & Grosveld, F. Chromatin domain as potential units of eukaryotic gene function. Curr. Opin. genet. Dev. 4, 260–264 (1994).

    Article  CAS  PubMed  Google Scholar 

  24. Kempkes, B., Dagmar, P., Zeidler, R., Sugden, B. & Hammerschmidt, W. Immortalization of human B-lymphocytes by a plasmid containing 71 kilobase pair of Epstein Barr virus DNA. J. Virol. 69, 231–238 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Robertson, E. & Kieff, E. Reducing the complexity of the transforming Epstein Barr genome to 64 kilobase pair. J. Virol. 69, 983–993 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Edvard-Smith, C.I. et al. X-linked agammaglobulinemia and other Immunoglobulin deficiencies. Immunol. Rev. 138, 159–183 (1994).

    Article  Google Scholar 

  27. Kempkes, B., Dagmar, P., Zeidler, R. & Hammerschmidt, W. Immortalization of human primary B lymphocytes in vitro with DNA. Proc. natn. Acad. Sci. U.S.A. 92, 5875–5879 (1995).

    Article  CAS  Google Scholar 

  28. Young, L. et al. Expression of Epstein Barr virus transformation associated genes in tissues of patients with EBV lymphoproliferative disease. New Engl. J. Med. 321, 1080–1085 (1989).

    Article  CAS  PubMed  Google Scholar 

  29. Yang, Y. et al. Cellular immunity to viral antigens limits E1-deleted adenovirus for gene therapy. Proc. natn. Acad. Sci. U.S.A. 91, 4407–4411 (1994).

    Article  CAS  Google Scholar 

  30. Levitskaya, J. et al. Inhibition of antigen processing by the internal repeat region of the Epstein–Barr virus nuclear antigen-1. Nature 375, 685–688 (1995).

    Article  CAS  PubMed  Google Scholar 

  31. Heston, L., Rabson, M., Brown, N. & Miller, G., Epstein–Barr variants from cellular subclones of P3J-HR-1 Burkitt lymphoma. Nature 295, 160–163 (1982).

    Article  CAS  PubMed  Google Scholar 

  32. Kelleher, Z.T. & Vos, J.-M. Long term episomal gene delivery in human lymphoid cells using human and avian adenoviral assisted transfection. BioTechniques 17, 1110–1117 (1994).

    CAS  PubMed  Google Scholar 

  33. Reisman, D., Yates, J. & Sugden, B. A putative origin of replication of plasmids derived from Epstein Barr virus is composed of two cis-acting components. Molec. Cell. Biol. 5, 1822–1832 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7295, USA

    Subrata Banerjee, Elizabeth Livanos & Jean-Michel H. Vos

  2. Department of Biochemistry & Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 27599-7295, USA

    Jean-Michel H. Vos

Authors
  1. Subrata Banerjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Elizabeth Livanos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Michel H. Vos
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Banerjee, S., Livanos, E. & Vos, JM. Therapeutic gene delivery in human B-lymphoblastoid cells by engineered non-transforming infectious Epstein–Barr virus. Nat Med 1, 1303–1308 (1995). https://doi.org/10.1038/nm1295-1303

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm1295-1303

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing