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.

  • Original Article
  • Published:

Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media

Gene Therapy volume 14, pages 1705–1711 (2007)Cite this article

Abstract

Several patients with severe combined immunodeficiency-X1 disease and adenosine deaminase deficiency have been cured by retroviral-mediated gene therapy. Despite the earlier success, the production of retroviral vectors for clinical gene therapy is cumbersome, costly and lacks safety features because of the adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum. The aim of this study was to generate a retrovirus packaging cell line that could be used for the production of large clinical batch vectors. Bicistronic vectors containing an internal ribosomal entry site followed by a selection gene were used to express Moloney murine leukemia gag-pol and amphotropic envelope viral proteins in HEK293 cells. The candidate clone (293GP-A2) that was selected as the packaging cell line could release recombinant green fluorescent protein retroviruses at 4 × 107 infectious viral particles per ml. Similar titers were achieved after these cells were adapted to grow in suspension and serum-free media. Furthermore, using the same culture conditions viral titers proved to be stable for a 3-month culture period. The 293GP-A2 packaging cell line has the potential to be cultured in bioreactors, opening the possibility for large-scale use of retroviral vectors in late stage clinical trials.

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

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Cavazzana-Calvo M, Hacein-Bey S, de Saint Basile G, Gross F, Yvon E, Nusbaum P et al. Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease. Science 2000; 288: 669–672.

    Article  CAS  PubMed  Google Scholar 

  2. Aiuti A, Slavin S, Aker M, Ficara F, Deola S, Mortellaro A et al. Correction of ADA-SCID by stem cell gene therapy combined with nonmyeloablative conditioning. Science 2002; 296: 2410–2413.

    Article  CAS  PubMed  Google Scholar 

  3. Gaspar HB, Parsley KL, Howe S, King D, Gilmour KC, Sinclair J et al. Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector. Lancet 2004; 364: 2181–2187.

    Article  CAS  PubMed  Google Scholar 

  4. Sadelain M, Riviere I, Brentjens R . Targeting tumours with genetically enhanced T lymphocytes. Nat Rev Cancer 2003; 3: 35–45.

    Article  CAS  PubMed  Google Scholar 

  5. Morgan RA, Dudley ME, Wunderlich JR, Hughes MS, Yang JC, Sherry RM et al. Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 2006; 314: 126–129.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  6. Mann R, Mulligan RC, Baltimore D . Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 1983; 33: 153–159.

    Article  CAS  PubMed  Google Scholar 

  7. Miller AD, Buttimore C . Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 1986; 6: 2895–2902.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  8. Markowitz D, Goff S, Bank A . A safe packaging line for gene transfer: separating viral genes on two different plasmids. J Virol 1988; 62: 1120–1124.

    CAS  PubMed Central  PubMed  Google Scholar 

  9. Markowitz D, Goff S, Bank A . Construction and use of a safe and efficient amphotropic packaging cell line. Virology 1988; 167: 400–406.

    Article  CAS  PubMed  Google Scholar 

  10. Danos O, Mulligan RC . Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc Natl Acad Sci USA 1988; 85: 6460–6464.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Rigg RJ, Chen J, Dando JS, Forestell SP, Plavec I, Bohnlein E . A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety. Virology 1996; 218: 290–295.

    Article  CAS  PubMed  Google Scholar 

  12. Otto E, Jones-Trower A, Vanin EF, Stambaugh K, Mueller SN, Anderson WF et al. Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum Gene Ther 1994; 5: 567–575.

    Article  CAS  PubMed  Google Scholar 

  13. Chong H, Vile RG . Replication-competent retrovirus produced by a ‘split-function’ third generation amphotropic packaging cell line. Gene Therapy 1996; 3: 624–629.

    CAS  PubMed  Google Scholar 

  14. Cosset FL, Takeuchi Y, Battini JL, Weiss RA, Collins MK . High-titer packaging cells producing recombinant retroviruses resistant to human serum. J Virol 1995; 69: 7430–7436.

    CAS  PubMed Central  PubMed  Google Scholar 

  15. Forestell SP, Dando JS, Chen J, de Vries P, Bohnlein E, Rigg RJ . Novel retroviral packaging cell lines: complementary tropisms and improved vector production for efficient gene transfer. Gene Therapy 1997; 4: 600–610.

    Article  CAS  PubMed  Google Scholar 

  16. Sheridan PL, Bodner M, Lynn A, Phuong TK, DePolo NJ, de la Vega Jr DJ et al. Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer. Mol Ther 2000; 2: 262–275.

    Article  CAS  PubMed  Google Scholar 

  17. Zhao Y, Low W, Collins MK . Improved safety and titre of murine leukaemia virus (MLV)-based retroviral vectors. Gene Therapy 2000; 7: 300–305.

    Article  CAS  PubMed  Google Scholar 

  18. Yu SS, Kim JM, Kim S . High efficiency retroviral vectors that contain no viral coding sequences. Gene Therapy 2000; 7: 797–804.

    Article  CAS  PubMed  Google Scholar 

  19. Tuschong L, Soenen SL, Blaese RM, Candotti F, Muul LM . Immune response to fetal calf serum by two adenosine deaminase-deficient patients after T cell gene therapy. Hum Gene Ther 2002; 13: 1605–1610.

    Article  CAS  PubMed  Google Scholar 

  20. Gerin PA, Gilligan MG, Searle PF, Al-Rubeai M . Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium. Hum Gene Ther 1999; 10: 1965–1974.

    Article  CAS  PubMed  Google Scholar 

  21. Gerin PA, Searle PF, Al-Rubeai M . Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18. Biotechnol Prog 1999; 15: 941–948.

    Article  CAS  PubMed  Google Scholar 

  22. Glimm H, Flugge K, Mobest D, Hofmann VM, Postmus J, Henschler R et al. Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium. Hum Gene Ther 1998; 9: 771–778.

    Article  CAS  PubMed  Google Scholar 

  23. McTaggart S, Al-Rubeai M . Effects of culture parameters on the production of retroviral vectors by a human packaging cell line. Biotechnol Prog 2000; 16: 859–865.

    Article  CAS  PubMed  Google Scholar 

  24. Chan LM, Coutelle C, Themis M . A novel human suspension culture packaging cell line for production of high-titre retroviral vectors. Gene Therapy 2001; 8: 697–703.

    Article  CAS  PubMed  Google Scholar 

  25. Schilz AJ, Kuhlcke K, Fauser AA, Eckert HG . Optimization of retroviral vector generation for clinical application. J Gene Med 2001; 3: 427–436.

    Article  CAS  PubMed  Google Scholar 

  26. Budak-Alpdogan T, Przybylowski M, Gonen M, Sadelain M, Bertino J, Riviere I . Functional assessment of the engraftment potential of gammaretrovirus-modified CD34+ cells, using a short serum-free transduction protocol. Hum Gene Ther 2006; 17: 780–794.

    Article  CAS  PubMed  Google Scholar 

  27. Kotani H, Newton PBR, Zhang S, Chiang YL, Otto E, Weaver L et al. Improved methods of retroviral vector transduction and production for gene therapy. Hum Gene Ther 1994; 5: 19–28.

    Article  CAS  PubMed  Google Scholar 

  28. Pan D, Whitley CB . Closed hollow-fiber bioreactor: a new approach to retroviral vector production. J Gene Med 1999; 1: 433–440.

    Article  CAS  PubMed  Google Scholar 

  29. Merten OW . State-of-the-art of the production of retroviral vectors. J Gene Med 2004; 6 (Suppl 1): S105–S124.

    Article  CAS  PubMed  Google Scholar 

  30. Przybylowski M, Hakakha A, Stefanski J, Hodges J, Sadelain M, Riviere I . Production scale-up and validation of packaging cell clearance of clinical-grade retroviral vector stocks produced in cell factories. Gene Therapy 2006; 13: 95–100.

    Article  CAS  PubMed  Google Scholar 

  31. Merten OW, Cruz PE, Rochette C, Geny-Fiamma C, Bouquet C, Goncalves D et al. Comparison of different bioreactor systems for the production of high titer retroviral vectors. Biotechnol Prog 2001; 17: 326–335.

    Article  CAS  PubMed  Google Scholar 

  32. Ghani K, Garnier A, Coelho H, Transfiguracion J, Trudel P, Kamen A . Retroviral vector production using suspension-adapted 293GPG cells in a 3L acoustic filter-based perfusion bioreactor. Biotechnol Bioeng 2006; 95: 653–660.

    Article  CAS  PubMed  Google Scholar 

  33. Pizzato M, Merten OW, Blair ED, Takeuchi Y . Development of a suspension packaging cell line for production of high titre, serum-resistant murine leukemia virus vectors. Gene Therapy 2001; 8: 737–745.

    Article  CAS  PubMed  Google Scholar 

  34. Miller AD, Garcia JV, von Suhr N, Lynch CM, Wilson C, Eiden MV . Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J Virol 1991; 65: 2220–2224.

    CAS  PubMed Central  PubMed  Google Scholar 

  35. Young WB, Link Jr CJ . Chimeric retroviral helper virus and picornavirus IRES sequence to eliminate DNA methylation for improved retroviral packaging cells. J Virol 2000; 74: 5242–5249.

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  36. Reeves L, Smucker P, Cornetta K . Packaging cell line characteristics and optimizing retroviral vector titer: the National Gene Vector Laboratory experience. Hum Gene Ther 2000; 11: 2093–2103.

    Article  CAS  PubMed  Google Scholar 

  37. Kelly PF, Vandergriff J, Nathwani A, Nienhuis AW, Vanin EF . Highly efficient gene transfer into cord blood nonobese diabetic/severe combined immunodeficiency repopulating cells by oncoretroviral vector particles pseudotyped with the feline endogenous retrovirus (RD114) envelope protein. Blood 2000; 96: 1206–1214.

    CAS  PubMed  Google Scholar 

  38. Relander T, Brun AC, Olsson K, Pedersen L, Richter J . Overexpression of gibbon ape leukemia virus (GALV) receptor (GLVR1) on human CD34(+) cells increases gene transfer mediated by GALV pseudotyped vectors. Mol Ther 2002; 6: 400–406.

    Article  CAS  PubMed  Google Scholar 

  39. Relander T, Johansson M, Olsson K, Ikeda Y, Takeuchi Y, Collins M et al. Gene transfer to repopulating human CD34+ cells using amphotropic-, GALV-, or RD114-pseudotyped HIV-1-based vectors from stable producer cells. Mol Ther 2005; 11: 452–459.

    Article  CAS  PubMed  Google Scholar 

  40. Cote J, Garnier A, Massie B, Kamen A . Serum-free production of recombinant proteins and adenoviral vectors by 293SF-3F6 cells. Biotechnol Bioeng 1998; 59: 567–575.

    Article  CAS  PubMed  Google Scholar 

  41. Qiao J, Roy V, Girard M-H, Caruso M . High translation efficiency is mediated by the encephalomyocarditis virus IRES if the natural sequence surrounding the 11th AUG is retained. Hum Gene Ther 2002; 13: 881–887.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We are grateful to Pedro Otavio de Campos-Lima for critical reading of the manuscript and to Stephen Goff for the MLV provirus used to generate the 3T3 chronically infected cell line. This study was initiated with a grant from the Canadian Institute of Health Research (CIHR) (IC074582). MC is a Senior Research Scholar of the Fonds de la Recherche en Santé du Québec (FRSQ).

Author information

Authors and Affiliations

  1. Le Centre de Recherche en Cancérologie de l'Université Laval, L'Hôtel Dieu de Québec, Centre Hospitalier Universitaire de Québec, Québec, Canada

    K Ghani, S Cottin & M Caruso

  2. Biotechnology Research Institute, National Research Council Canada, Montréal, Québec, Canada

    A Kamen

Authors
  1. K Ghani
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S Cottin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A Kamen
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. M Caruso
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to M Caruso.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ghani, K., Cottin, S., Kamen, A. et al. Generation of a high-titer packaging cell line for the production of retroviral vectors in suspension and serum-free media. Gene Ther 14, 1705–1711 (2007). https://doi.org/10.1038/sj.gt.3303039

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.gt.3303039

Keywords

This article is cited by

Search

Advanced search

Quick links