Skip to main content
SpringerLink
Log in

Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production with Improved Safety and Titer

  • Protocol
Viral Vectors for Gene Therapy

Part of the book series: Methods in Molecular Medicine™ ((MIMM,volume 76))

  • 888 Accesses

Abstract

Retroviral vectors based on Moloney Murine Leukemia Virus (MLV), described more than 15 years ago (1), first entered clinical trials in 1990 (2). Since then, a greater understanding of the basic retrovirus biology and how it relates to the production of recombinant retroviral vectors has helped overcome some of the initial hurdles, including low titer, outbreak of replication-competent retrovirus (RCR), and complement inactivation of vector (3–6). Subsequently, retroviral vectors have become a very safe and powerful tool for efficient in vitro, ex vivo, and in vivo gene transfer for proliferating cells. Several refinements in recombinant MLV vector technology were necessary, particularly for clinical trials using systemic administration of very high vector doses (>1010 transducing units), thus necessitating efficient large-scale manufacture of safe and high-titer retroviral vector preparations.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Mann, R., Mulligan, R. C., and Baltimore, D. (1983) Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell 33, 153–159.

    Article  PubMed  CAS  Google Scholar 

  2. Blaese, R. M., Culver, K. W., Miller, A. D., Carter, C. S., Fleisher, T., Clerici, M., et al. (1995) T lymphocyte-directed gene therapy for ADA-SCID: initial trial results after 4 years. Science 270, 475–480.

    Article  PubMed  CAS  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  4. Miller, A. D. (1990) Retrovirus packaging cells. Hum. Gene Ther. 1, 5–14.

    Article  PubMed  CAS  Google Scholar 

  5. Cosset, F. L., Takeuchi, Y., Battini, J. L., Weiss, R. A., and Collins, M. K. (1995) High-titer packaging cells producing recombinant retroviruses resistant to human serum. J. Virol. 69, 7430–7436.

    PubMed  CAS  Google Scholar 

  6. Rigg, R. J., Chen, J., Dando, J. S., Forestell, S. P., Plavec, I., and Bohnlein, E. (1996) A novel human amphotropic packaging cell line: high titer, complement resistance, and improved safety. Virology 218, 290–295.

    Article  PubMed  CAS  Google Scholar 

  7. Watanabe, S. and Temin, H. M. (1983) Construction of a helper cell line for avian reticuloendotheliosis virus cloning vectors. Mol. Cell Biol. 3, 2241–2249.

    PubMed  CAS  Google Scholar 

  8. Dornburg, R. (1995) Reticuloendotheliosis viruses and derived vectors. Gene Ther. 2, 301–310.

    PubMed  CAS  Google Scholar 

  9. Miller, A. D., Garcia, J. V., von Suhr, N., Lynch, C. M., Wilson, C., and Eiden, M. V. (1991) Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus. J. Virol. 65, 2220–2224.

    PubMed  CAS  Google Scholar 

  10. Miller, A. D. and Rosman, G. J. (1989) Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–982, 984-986, 989-990.

    PubMed  CAS  Google Scholar 

  11. Danos, O. and Mulligan, R. C. (1988) Safe and efficient generation of recombinant retroviruses with amphotropic and ecotropic host ranges. Proc. Natl. Acad. Sci. USA 85, 6460–6464.

    Article  PubMed  CAS  Google Scholar 

  12. Chong, H., Starkey, W., and Vile, R. G. (1998) A replication-competent retrovirus arising from a split-function packaging cell line was generated by recombination events between the vector, one of the packaging constructs, and endogenous retroviral sequences. J. Virol. 72, 2663–2670.

    PubMed  CAS  Google Scholar 

  13. Sorge, J., Wright, D., Erdman, V. D., and Cutting, A. E. (1984) Amphotropic retrovirus vector system for human cell gene transfer. Mol. CellBiol. 4, 1730–1737.

    CAS  Google Scholar 

  14. Sheridan, P. L., Bodner, M., Lynn, A., Phuong, T. K., DePolo, N. J., de la Vega, D. J., et al. (2000) Generation of retroviral packaging and producer cell lines for large-scale vector production and clinical application: improved safety and high titer. Mol. Ther. 2, 262–275.

    Article  PubMed  CAS  Google Scholar 

  15. (2000) Human gene marker/therapy clinical protocols (complete updated listings). Hum. Gene Ther. 11, 2543–2617.

    Google Scholar 

  16. Purcell, D. F., Broscius, C. M., Vanin, E. F., Buckler, C. E., Nienhuis, A. W., and Martin, M. A. (1996) An array of murine leukemia virus-related elements is transmitted and expressed in a primate recipient of retroviral gene transfer. J. Virol. 70, 887–897.

    PubMed  CAS  Google Scholar 

  17. Donahue, R. E., Kessler, S. W., Bodine, D., McDonagh, K., Dunbar, C., Goodman, S., et al. (1992) Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer. J. Exp. Med. 176, 1125–1135.

    Article  PubMed  CAS  Google Scholar 

  18. Otto, E., Jones-Trower, A., Vanin, E. F., Stambaugh, K., Mueller, S. N., Anderson, W. F., and McGarrity, G. J. (1994) Characterization of a replication-competent retrovirus resulting from recombination of packaging and vector sequences. Hum. Gene Ther. 5, 567–575.

    Article  PubMed  CAS  Google Scholar 

  19. Lower, R., Lower, J., and Kurth, R. (1996) The viruses in all of us: characteristics and biological significance of human endogenous retrovirus sequences. Proc. Natl. Acad. Sci. USA 93, 5177–5184.

    Article  PubMed  CAS  Google Scholar 

  20. Jenkins, N. A., Copeland, N. G., Taylor, B. A., and Lee, B. K. (1982) Organization, distribution, and stability of endogenous ecotropic murine leukemia virus DNA sequences in chromosomes of Mus musculus. J. Virol. 43, 26–36.

    PubMed  CAS  Google Scholar 

  21. Hatzoglou, M., Hodgson, C. P., Mularo, F., and Hanson, R. W. (1990) Efficient packaging of a specific VL30 retroelement by psi 2 cells which produce MoMLV recombinant retroviruses. Hum. Gene Ther. 1, 385–397.

    Article  PubMed  CAS  Google Scholar 

  22. Scadden, D. T., Fuller, B., and Cunningham, J. M. (1990) Human cells infected with retrovirus vectors acquire an endogenous murine provirus. J. Virol. 64, 424–427.

    PubMed  CAS  Google Scholar 

  23. Howard, B., Burrascano, M., McCallister, T., Chong, K., Gangavalli, R., Severins-son, L., et al. (1994) Retrovirus-mediated gene transfer of the human gamma-IFN gene: a therapy for cancer. Ann. NY Acad. Sci. 716, 167–187.

    Article  PubMed  CAS  Google Scholar 

  24. Farson, D., McGuinness, R., Dull, T., Limoli, K., Lazar, R., Jalali, S., et al. (1999) Large-scale manufacturing of safe and efficient retrovirus packaging lines for use in immunotherapy protocols. J. Gene Med. 1, 195–209.

    Article  PubMed  CAS  Google Scholar 

  25. Bender, M. A., Palmer, T. D., Gelinas, R. E., and Miller, A. D. (1987) Evidence that the packaging signal of Moloney murine leukemia virus extends into the gag region. J. Virol. 61, 1639–1646.

    PubMed  CAS  Google Scholar 

  26. Morgenstern, J. P. and Land, H. (1990) Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res. 18, 3587–3596.

    Article  PubMed  CAS  Google Scholar 

  27. Salmons, B. and Gunzburg, W. H. (1993) Targeting of retroviral vectors for gene therapy. Hum. Gene Ther. 4, 129–141.

    Article  PubMed  CAS  Google Scholar 

  28. Dzierzak, E. A., Papayannopoulou, T., and Mulligan, R. C. (1988) Lineagespecific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature 331, 35–41.

    Article  PubMed  CAS  Google Scholar 

  29. Yu, S. F., von Ruden, T., Kantoff, P. W., Garber, C., Seiberg, M., Ruther, U., et al. (1986) Self-inactivating retroviral vectors designed for transfer of whole genes into mammalian cells. Proc. Natl. Acad. Sci. USA 83, 3194–3198.

    Article  PubMed  CAS  Google Scholar 

  30. Hantzopoulos, P. A., Sullenger, B. A., Ungers, G., and Gilboa, E. (1989) Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector. Proc. Natl. Acad. Sci. USA 86, 3519–3523.

    Article  PubMed  CAS  Google Scholar 

  31. Hawley, R. G., Sabourin, L. A., and Hawley, T. S. (1989) An improved retroviral vector for gene transfer into undifferentiated cells. Nucleic Acids Res. 17, 4001.

    Article  PubMed  CAS  Google Scholar 

  32. VandenDriessche, T., Vanslembrouck, V., Goovaerts, I., Zwinnen, H., Vanderhaeghen, M. L., Collen, D., and Chuah, M. K. (1999) Long-term expression of human coagulation factor VIII and correction of hemophilia A after in vivo retroviral gene transfer in factor VIII-deficient mice. Proc. Natl. Acad. Sci. USA 96, 10,379–10,384.

    Article  PubMed  CAS  Google Scholar 

  33. Nemunaitis, J., Fong, T., Burrows, F., Bruce, J., Peters, G., Ognoskie, N., et al. (1999) Phase I trial of interferon gamma retroviral vector administered intratumorally with multiple courses in patients with metastatic melanoma. Hum. Gene Ther. 10, 1289–1298.

    Article  PubMed  CAS  Google Scholar 

  34. Karavodin, L. M., Robbins, J., Chong, K., Hsu, D., Ibanez, C., Mento, S., et al. (1998) Generation of a systemic antitumor response with regional intratumoral injections of interferon gamma retroviral vector. Hum. Gene Ther. 9, 2231–2241.

    Article  PubMed  CAS  Google Scholar 

  35. Greengard, J. S. and Jolly, D. J. (1999) Animal testing of retroviral-mediated gene therapy for factor VIII deficiency. Thromb. Haemost. 82, 555–561.

    PubMed  CAS  Google Scholar 

  36. Sallberg, M., Hughes, J., Javadian, A., Ronlov, G., Hultgren, C., Townsend, K., et al. (1998) Genetic immunization of chimpanzees chronically infected with the hepatitis B virus, using a recombinant retroviral vector encoding the hepatitis B virus core antigen. Hum. Gene Ther. 9, 1719–1729.

    Article  PubMed  CAS  Google Scholar 

  37. Ghivizzani, S. C., Lechman, E. R., Tio, C., Mule, K. M., Chada, S., McCormack, J. E., et al. (1997) Direct retrovirus-mediated gene transfer to the synovium of the rabbit knee: implications for arthritis gene therapy. Gene Ther. 4, 977–982.

    Article  PubMed  CAS  Google Scholar 

  38. Wang, G., Davidson, B. L., Melchert, P., Slepushkin, V. A., van Es, H. H., Bodner, M., et al. (1998) Influence of cell polarity on retrovirus-mediated gene transfer to differentiated human airway epithelia. J. Virol. 72, 9818–9826.

    PubMed  CAS  Google Scholar 

  39. Le Doux, J. M., Morgan, J. R., Snow, R. G., and Yarmush, M. L. (1996) Proteoglycans secreted by packaging cell lines inhibit retrovirus infection. J. Virol. 70, 6468–6473.

    PubMed  Google Scholar 

  40. Forestell, S. P., Bohnlein, E., and Rigg, R. J. (1995) Retroviral end-point titer is not predictive of gene transfer efficiency: implications for vector production. Gene Ther. 2, 723–730.

    PubMed  CAS  Google Scholar 

  41. Battini, J. L., Danos, O., and Heard, J. M. (1995) Receptor-binding domain of murine leukemia virus envelope glycoproteins. J. Virol. 69, 713–719.

    PubMed  CAS  Google Scholar 

  42. DePolo, N. J., Harkleroad, C. E., Bodner, M., Watt, A. T., Anderson, C. G., Greengard, J. S., et al. (1999) The resistance of retroviral vectors produced from human cells to serum inactivation in vivo and in vitro is primate species dependent. J. Virol. 73, 6708–6714.

    PubMed  CAS  Google Scholar 

  43. Slingsby, J. H., Baban, D., Sutton, J., Esapa, M., Price, T., Kingsman, S. M., et al. (2000) Analysis of 4070A envelope levels in retroviral preparations and effect on target cell transduction efficiency. Hum. Gene Ther. 11, 1439–1451.

    Article  PubMed  CAS  Google Scholar 

  44. Yap, M. W., Kingsman, S. M., and Kingsman, A. J. (2000) Effects of stoichiometry of retroviral components on virus production. J. Gen. Virol. 81 Pt 9, 2195–2202.

    PubMed  CAS  Google Scholar 

  45. Coffin J. M., Hughes S. H., and Varmus H. E. (eds.) (1997) Retroviruses. Cold Spring Harbor Laboratory Press, New York.

    Google Scholar 

  46. Yoshimatsu, T., Tamura, M., Kuriyama, S., and Ikenaka, K. (1998) Improvement of retroviral packaging cell lines by introducing the polyomavirus early region. Hum. Gene Ther. 9, 161–172.

    Article  PubMed  CAS  Google Scholar 

  47. Muenchau, D. D., Freeman, S. M., Cornetta, K., Zwiebel, J. A., and Anderson, W. F. (1990) Analysis of retroviral packaging lines for generation of replication-competent virus. Virology 176, 262–265.

    Article  PubMed  CAS  Google Scholar 

  48. Lynch, C. M. and Miller, A. D. (1991) Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges. J. Virol. 65, 3887–3890.

    PubMed  CAS  Google Scholar 

  49. Kinsella, T. M. and Nolan, G. P. (1996) Episomal vectors rapidly and stably produce high-titer recombinant retrovirus. Hum. Gene Ther. 7, 1405–1413.

    Article  PubMed  CAS  Google Scholar 

  50. Kim, Y. S., Lim, H. K., and Kim, K. J. (1998) Production of high-titer retroviral vectors and detection of replication-competent retroviruses. Mol. Cells 8, 36–42.

    PubMed  CAS  Google Scholar 

  51. Yee, J. K., Friedmann, T., and Burns, J. C. (1994) Generation of high-titer pseudotyped retroviral vectors with very broad host range. Meth. Cell Biol. 43 Pt A, 99–112.

    Article  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  54. Paul, R. W., Morris, D., Hess, B. W., Dunn, J., and Overell, R. W. (1993) Increased viral titer through concentration of viral harvests from retroviral packaging lines. Hum. Gene Ther. 4, 609–615.

    Article  PubMed  CAS  Google Scholar 

  55. Printz, M., Reynolds, J., Mento, S. J., Jolly, D., Kowal, K., and Sajjadi, N. (1995) Recombinant retroviral vector interferes with the detection of amphotropic replication competent retrovirus in standard culture assays. Gene Ther. 2, 143–150.

    PubMed  CAS  Google Scholar 

  56. Miller, A. D. and Buttimore, C. (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol. Cell Biol. 6, 2895–2902.

    PubMed  CAS  Google Scholar 

  57. Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993) Vesicular stomatitis virus G glycoprotein pseudotyped retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc. Natl. Acad. Sci. USA 90, 8033–8037.

    Article  PubMed  CAS  Google Scholar 

  58. Hwang, L. H. and Gilboa, E. (1984) Expression of genes introduced into cells by retroviral infection is more efficient than that of genes introduced into cells by DNA transfection. J. Virol. 50, 417–424.

    PubMed  CAS  Google Scholar 

  59. Verzeletti, S., Bonini, C., Marktel, S., Nobili, N., Ciceri, F., Traversari, C., and Bordignon, C. (1998) Herpes simplex virus thymidine kinase gene transfer for controlled graft-versus-host disease and graft-versus-leukemia: clinical follow-up and improved new vectors. Hum. Gene Ther. 9, 2243–2251.

    Article  PubMed  CAS  Google Scholar 

  60. Jung, D., Jaeger, E., Cayeux, S., Blankenstein, T., Hilmes, C., Karbach, J., et al. (1998) Strong immunogenic potential of a B7 retroviral expression vector: generation of HLA-B7-restricted CTL response against selectable marker genes. Hum. Gene Ther. 9, 53–62.

    Article  PubMed  CAS  Google Scholar 

  61. DuBridge, R. B., Tang, P., Hsia, H. C., Leong, P. M., Miller, J. H., and Calos, M. P. (1987) Analysis of mutation in human cells by using an Epstein-Barr virus shuttle system. Mol. Cell Biol. 7, 379–387.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Research, Cancer Vaccines, Cerus Corporation, Concord, CA

    Thomas W. Dubensky Jr. (Vice President)

  2. GenStar Therapeutics Inc., San Diego, CA

    Sybille L. Sauter (Director for Vaccines & Immunotherapy)

Authors
  1. Thomas W. Dubensky Jr.
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sybille L. Sauter
    View author publications

    You can also search for this author in PubMed Google Scholar

Editor information

Editors and Affiliations

  1. Department of Oral Molecular Biology, School of Dentistry Oregon Health & Science University, Portland, OR

    Curtis A. Machida

Rights and permissions

Reprints and permissions

Copyright information

© 2003 Humana Press Inc.

About this protocol

Cite this protocol

Dubensky, T.W., Sauter, S.L. (2003). Generation of Retroviral Packaging and Producer Cell Lines for Large-Scale Vector Production with Improved Safety and Titer. In: Machida, C.A. (eds) Viral Vectors for Gene Therapy. Methods in Molecular Medicine™, vol 76. Humana Press. https://doi.org/10.1385/1-59259-304-6:309

Download citation

  • DOI: https://doi.org/10.1385/1-59259-304-6:309

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-019-9

  • Online ISBN: 978-1-59259-304-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation