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.

  • Viral Transfer Technology
  • Published:

Evaluation of recombinant alphaviruses as vectors in gene therapy

Gene Therapy volume 7, pages 472–480 (2000)Cite this article

Abstract

Alphavirus vectors based on Sindbis virus and Semliki Forest virus (SFV) were characterized as potential gene transfer vectors. Initial studies were performed using vectors engineered to transfer either lacZ or green fluorescent protein (GFP). High levels of gene transfer were achieved in human primary fibroblasts, BHK and 293T cells, with low levels of transduction observed in more than 20 other target cells. Alphavirus-based expression was generally very high, but transient in every cell type. Replication-competent alphavirus was never detected in SFV preparations but could be produced by Sindbis-based vectors at a frequency of up to 3 × 10−3 infectious units per ml. We constructed a human clotting factor IX (hFIX) cDNA-containing Sindbis virus and compared it with hFIX cDNA-harboring adenoviral and retroviral vectors. In most cases, hFIX levels obtained with Sindbis vector were initially at least an order of magnitude higher than those obtained with other viral vectors. These data demonstrate that alphavirus vectors compare favorably with adenovirus vectors as systems to promote high-level transient gene expression and should be considered as an alternative vector for gene transfer and potential gene therapy studies.

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

Access options

Figure 1
Figure 2
Figure 3
Figure 4

Similar content being viewed by others

References

  1. Johnston RE, Peters CJ . Alphaviruses. In: Fields BN, Knipe DM, Howley PM (eds) Virology Lippincott-Raven: Philadephia 1996 pp 842–898

    Google Scholar 

  2. Byrnes AP, Griffin DE . Binding of Sindbis virus to cell surface heparan sulfate J Virol 1998 72: 7349–7356

    CAS  PubMed  PubMed Central  Google Scholar 

  3. Pushko P et al. Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo Virol 1997 39: 389–401

    Article  Google Scholar 

  4. Berglund PM et al. Semliki Forest virus expression system: production of conditionally infectious recombinant particles Biotechnol 1993 11: 916–920

    CAS  Google Scholar 

  5. Liljestrom P, Garoff H . A new generation of animal cell expression vectors based on the Semliki Forest virus replicon Biotechnology 1991 9: 1356–1361

    Article  CAS  PubMed  Google Scholar 

  6. Schlesinger S . Alphaviruses – vectors for the expression of heterologous genes Trends Biotechnol 1993 11: 18–22

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schlesinger S . RNA viruses as vectors for the expression of heterologous proteins Mol Biotech 1995 3: 155–165

    Article  CAS  Google Scholar 

  8. Strauss JH, Strauss EG . The alphaviruses: gene expression, replication, and evolution Microbiol Rev 1994 58: 491–562

    CAS  PubMed  PubMed Central  Google Scholar 

  9. Lundstrom K et al. High-level expression of the human neurokinin-1 receptor in mammalian cell lines using the Semliki Forest virus expression system Eur J Biochem 1994 224: 917–921

    Article  CAS  PubMed  Google Scholar 

  10. Roks AJ et al. Vectors based on Semliki Forest virus for rapid and efficient gene transfer into non-endothelial cardiovascular cells: comparison to adenovirus Cardiovasc Res 1997 35: 498–504

    Article  CAS  PubMed  Google Scholar 

  11. Zhang J et al. Cloning of human IL-12 p40 and p35 DNA into the Semliki Forest virus vector: expression of IL-12 in human tumor cells Gene Therapy 1997 4: 367–374

    Article  CAS  PubMed  Google Scholar 

  12. Mossman SP et al. Protection against lethal simian immunodeficiency virus SIVsmmPBj14 disease by a recombinant Semliki Forest virus gp160 vaccine and by a gp120 subunit vaccine J Virol 1996 70: 1953–1960

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Corsini J et al. Efficiency of transduction by recombinant Sindbis replicon virus varies among cell lines including mosquito cells and rat sensory neurons Biotechniques 1996 21: 492–497

    Article  CAS  PubMed  Google Scholar 

  14. Altman-Hamamdzic S et al. Expression of β-galactosidase in mouse brain: utilization of a novel nonreplicative Sindbis virus vector as a neuronal gene delivery system Gene Therapy 1997 4: 815–822

    Article  CAS  PubMed  Google Scholar 

  15. Ohno K et al. Cell-specific targeting of Sindbis virus vectors displaying IgG-binding domains of protein A Nat Biotechnol 1997 15: 763–767

    Article  CAS  PubMed  Google Scholar 

  16. Sawai K, Meruelo D . Cell-specific transfection of choriocarcinoma cells by using Sindbis virus hCG expressing chimeric vector Biochem Biophys Res Commun 1998 248: 315–323

    Article  CAS  PubMed  Google Scholar 

  17. Berglund P et al. Outcome of immunization of cynomolgus monkeys with recombinant Semliki Forest virus encoding human immunodeficiency virus type 1 envelope protein and challenge with a high dose of SHIV-4 virus AIDS Res Hum Retrovir 1997 13: 1487–1495

    Article  CAS  PubMed  Google Scholar 

  18. Berglund P et al. Enhancing immune responses using suicidal DNA vaccines Nat Biotechnol 1998 16: 562–565

    Article  CAS  PubMed  Google Scholar 

  19. Tsuji M et al. Recombinant Sindbis viruses expressing a cytotoxic T-lymphocyte epitope of a malaria parasite or of influenza virus elicit protection against the corresponding pathogen in mice J Virol 1998 72: 6907–6910

    CAS  PubMed  PubMed Central  Google Scholar 

  20. Tubulekas I, Berglund P, Fleeton M, Liljestrom P . Alphavirus expression vectors and their use as recombinant vaccines: a minireview Gene 1997 190: 191–195

    Article  CAS  PubMed  Google Scholar 

  21. Frolov I et al. Alphavirus-based expression vectors: strategies and applications Proc Natl Acad Sci USA 1996 93: 11371–11377

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Bunnell BA et al. High-efficiency retroviral-mediated gene transfer into human and non-human primate peripheral blood lymphocytes Proc Natl Acad Sci USA 1995 92: 7739–7743

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Hanenberg H et al. Colocalization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells Nature Med 1996 8: 876–882

    Article  Google Scholar 

  24. Miller AD, Buttimore C . Redesign of retroviral packaging cell lines to avoid recombination leading to helper virus production Mol Cell Biol 1986 6: 2895–2903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Bredenbeek PJ, Frolov I, Rice CM, Schlesinger S . Sindbis virus expression vectors: packaging of RNA replicons by using defective helper RNAs J Virol 1993 67: 6439–6446

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Frolov I, Frolova E, Schlesinger S . Sindbis virus replicons and sindbis virus: assembly of chimeras and of particles deficient in virus RNA J Virol 1997 71: 2819–2829

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Polo JM et al. Stable alphavirus packaging cell lines for Sindbis virus- and Semliki Forest virus-derived vectors Proc Natl Acad Sci USA 1999 96: 4598–4603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mastromarino P et al. Effect of polyions on the early events of Sindbis virus infection of Vero cells Arch Virol 1991 121: 19–27

    Article  CAS  PubMed  Google Scholar 

  29. Lu YE, Cassese T, Kielian M . The cholesterol requirement for Sindbis virus entry and exit and characterization of a spike protein region involved in cholesterol dependence J Virol 1999 73: 4272–4278

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Klimstra WB, Ryman KD, Johnston RE . Adaptation of Sindbis virus to BHK cells selects for use of heparan sulfate as an attachment receptor J Virol 1998 72: 7357–7366

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Chen M et al. Transient gene expression from yeast artificial chromosome DNA in mammalian cells is enhanced by adenovirus Nucleic Acids Res 1997 25: 4416–4418

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Lozier JN et al. Gut epithelial cells as targets for gene therapy of hemophilia Hum Gene Ther 1997 8: 1481–1490

    Article  CAS  PubMed  Google Scholar 

  33. Loimas S, Wahlfors J, Janne J . Herpes simplex virus thymidine kinase-green fluorescent protein fusion gene: new tool for gene transfer studies and gene therapy Biotechniques 1998 24: 614–618

    Article  CAS  PubMed  Google Scholar 

  34. Wahlfors JJ, Xanthopoulos KG, Morgan RA . Semliki Forest virus-mediated production of retroviral vector RNA in retroviral packaging cells Hum Gene Ther 1997 8: 2031–2041

    Article  CAS  PubMed  Google Scholar 

  35. Nelson DM et al. Characterization of diverse viral vector preparations, using a simple and rapid whole-virion dot-blot method Hum Gene Ther 1998 9: 2401–2405

    Article  CAS  PubMed  Google Scholar 

  36. Caplen NJ et al. Liposome-mediated CFTR gene transfer to the nasal epithelium of patients with cystic fibrosis Nature Med 1995 1: 39–46 (addendum 1: 272)

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

SL obtained financial support from Saastamoinen Foundation and Finnish Cultural Foundation of Northern Savo. We thank Drs G Nolan (Stanford University, Stanford, CA, USA), E Oldfield (National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA), S Rosenberg (National Cancer Institute, National Institutes of Health, Bethesda, MD, USA) and N Fusening (German Cancer Research Center, Heidelberg, Germany), for the cell lines, and Drs J Lozier and L Chen for the FIX adenoviral and retroviral vectors, respectively.

Author information

Author notes

  1. D M Nelson

    Present address: Aurora Biosciences Corporation, San Diego, 92121, CA, USA

Authors and Affiliations

  1. Clinical Gene Therapy Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA

    J J Wahlfors, S A Zullo, D M Nelson & R A Morgan

  2. AI Virtanen Institute, University of Kuopio, Kuopio, Finland

    J J Wahlfors & S Loimas

  3. Graduate Genetics Program, The George Washington University, Washington, DC, USA

    S A Zullo

Authors
  1. J J Wahlfors
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S A Zullo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. S Loimas
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D M Nelson
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. R A Morgan
    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

Wahlfors, J., Zullo, S., Loimas, S. et al. Evaluation of recombinant alphaviruses as vectors in gene therapy. Gene Ther 7, 472–480 (2000). https://doi.org/10.1038/sj.gt.3301122

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3301122

Keywords

This article is cited by

Search

Advanced search

Quick links