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Delivery of an EBV episome by a self-circularizing helper-dependent adenovirus: long-term transgene expression in immunocompetent mice

Gene Therapy volume 17, pages 1288–1293 (2010)Cite this article

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Abstract

Epstein–Barr virus (EBV) evolved an episomal system for maintaining life-long, latent infection of human B lymphocytes. Circular episomes engineered from EBV components required for this latent form of infection have the capacity to persist in most types of replicating mammalian cells without DNA integration and the pitfalls of insertional mutagenesis. EBV episomes are typically transduced using low-efficiency methods. Here we present a method for efficient delivery of EBV episomes to nuclei of hepatocytes in living mice using a helper-dependent adenoviral vector and Cre-mediated recombination in vivo to generate circular EBV episomes following infection. Cre is transiently expressed from a hepatocyte-specific promoter so that vector generation and transgene expression are tissue specific. We show long-term persistence of the circularized vector DNA and expression of a reporter gene in hepatocytes of immunocompetent mice.

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References

  1. Tsurumi T, Fujita M, Kudoh A . Latent and lytic Epstein–Barr virus replication strategies. Rev Med Virol 2005; 15: 3–15.

    Article  CAS  PubMed  Google Scholar 

  2. Yates J, Warren N, Sugden B . Stable replication of plasmid derived from Epstein–Barr virus in various mammalian cells. Nature 1985; 313: 812–815.

    Article  CAS  PubMed  Google Scholar 

  3. Krysan PJ, Calos MP . Epstein–Barr virus-based vectors that replicate in rodent cells. Gene 1993; 136: 137–143.

    Article  CAS  PubMed  Google Scholar 

  4. Lindner SE, Sugden B . The plasmid replicon of Epstein–Barr virus: mechanistic insights into efficient, licensed, extrachromosomal replication in human cells. Plasmid 2007; 58: 1–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Nanbo A, Sugden A, Sugden B . The coupling of synthesis and partitioning of EBV's plasmid replicon is revealed in live cells. EMBO J 2007; 26: 4252–4262.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Wohlgemuth JG, Kang SH, Bulboaca GH, Nawotka KA, Calos MP . Long-term gene expression from autonomously replicating vectors in mammalian cells. Gene Therapy 1996; 3: 503–512.

    CAS  PubMed  Google Scholar 

  7. Stoll SM, Sclimenti CR, Baba EJ, Meuse L, Kay MA, Calos MP . Epstein–Barr virus/human vector provides high-level, long-term expression of alpha1-antitrypsin in mice. Mol Ther 2001; 4: 122–129.

    Article  CAS  PubMed  Google Scholar 

  8. Segura MM, Alba R, Bosch A, Chillon M . Advances in helper-dependent adenoviral vector research. Curr Gene Ther 2008; 8: 222–235.

    Article  CAS  PubMed  Google Scholar 

  9. Parks RJ, Chen L, Anton M, Sankar U, Rudnicki MA, Graham FL . A helper-dependent adenovirus vector system: removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 1996; 93: 13565–13570.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Brunetti-Pierri N, Ng P . Progress and prospects: gene therapy for genetic diseases with helper-dependent adenoviral vectors. Gene Therapy 2008; 15: 553–560.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Waddington SN, McVey JH, Bhella D, Parker AL, Barker K, Atoda H et al. Adenovirus serotype 5 hexon mediates liver gene transfer. Cell 2008; 132: 397–409.

    Article  CAS  PubMed  Google Scholar 

  12. Lowenstein PR . With a little help from my f(X)riends!: the basis of Ad5-mediated transduction of the liver revealed. Mol Ther 2008; 16: 1004–1006.

    Article  CAS  PubMed  Google Scholar 

  13. Okuyama T, Huber RM, Bowling W, Pearline R, Kennedy SC, Flye MW et al. Liver-directed gene therapy: a retroviral vector with a complete LTR and the ApoE enhancer-alpha 1-antitrypsin promoter dramatically increases expression of human alpha 1-antitrypsin in vivo. Hum Gene Ther 1996; 7: 637–645.

    Article  CAS  PubMed  Google Scholar 

  14. Gallaher SD, Gil JS, Dorigo O, Berk AJ . Robust in vivo transduction of a genetically stable Epstein–Barr virus episome to hepatocytes in mice by a hybrid viral vector. J Virol 2009; 83: 3249–3257.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Tan BT, Wu L, Berk AJ . An adenovirus-Epstein-Barr virus hybrid vector that stably transforms cultured cells with high efficiency. J Virol 1999; 73: 7582–7589.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Dorigo O, Gil JS, Gallaher SD, Tan BT, Castro MG, Lowenstein PR et al. Development of a novel helper-dependent adenovirus-Epstein-Barr virus hybrid system for the stable transformation of mammalian cells. J Virol 2004; 78: 6556–6566.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Al-Dosari M, Zhang G, Knapp JE, Liu D . Evaluation of viral and mammalian promoters for driving transgene expression in mouse liver. Biochem Biophys Res Commun 2006; 339: 673–678.

    Article  CAS  PubMed  Google Scholar 

  18. Pastore L, Morral N, Zhou H, Garcia R, Parks RJ, Kochanek S et al. Use of a liver-specific promoter reduces immune response to the transgene in adenoviral vectors. Hum Gene Ther 1999; 10: 1773–1781.

    Article  CAS  PubMed  Google Scholar 

  19. Martin GR, Evans MJ . Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro. Proc Natl Acad Sci USA 1975; 72: 1441–1445.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Umana P, Gerdes CA, Stone D, Davis JR, Ward D, Castro MG et al. Efficient FLPe recombinase enables scalable production of helper-dependent adenoviral vectors with negligible helper-virus contamination. Nat Biotechnol 2001; 19: 582–585.

    Article  CAS  PubMed  Google Scholar 

  21. Tao N, Gao GP, Parr M, Johnston J, Baradet T, Wilson JM et al. Sequestration of adenoviral vector by Kupffer cells leads to a nonlinear dose response of transduction in liver. Mol Ther 2001; 3: 28–35.

    Article  CAS  PubMed  Google Scholar 

  22. Barr D, Tubb J, Ferguson D, Scaria A, Lieber A, Wilson C et al. Strain related variations in adenovirally mediated transgene expression from mouse hepatocytes in vivo: comparisons between immunocompetent and immunodeficient inbred strains. Gene Therapy 1995; 2: 151–155.

    CAS  PubMed  Google Scholar 

  23. Brooks AR, Harkins RN, Wang P, Qian HS, Liu P, Rubanyi GM . Transcriptional silencing is associated with extensive methylation of the CMV promoter following adenoviral gene delivery to muscle. J Gene Med 2004; 6: 395–404.

    Article  CAS  PubMed  Google Scholar 

  24. Loser P, Jennings GS, Strauss M, Sandig V . Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFkappaB. J Virol 1998; 72: 180–190.

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Lowenstein PR, Kroeger K, Castro MG . Immunology of neurological gene therapy: how T cells modulate viral vector-mediated therapeutic transgene expression through immunological synapses. Neurotherapeutics 2007; 4: 715–724.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by USPHS Grant R37 CA025235 and the Eli and Edythe Broad Center of JSG was partially supported by the Ruth L Kirchstein National Research Service Award GM007185, and SDG by UCLA Training Grant in Genetic Mechanisms T32-GM07104.

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Authors and Affiliations

  1. Molecular Biology Institute, University of California, Los Angeles, CA, USA

    J S Gil, S D Gallaher & A J Berk

  2. Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA, USA

    J S Gil, S D Gallaher & A J Berk

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  1. J S Gil
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  2. S D Gallaher
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  3. A J Berk
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Correspondence to A J Berk.

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Gil, J., Gallaher, S. & Berk, A. Delivery of an EBV episome by a self-circularizing helper-dependent adenovirus: long-term transgene expression in immunocompetent mice. Gene Ther 17, 1288–1293 (2010). https://doi.org/10.1038/gt.2010.75

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