pp 1-23 | Cite as

High-Titer Production of HIV-Based Lentiviral Vectors in Roller Bottles for Gene and Cell Therapy

  • Hazal Banu Olgun
  • Hale M. Tasyurek
  • Ahter Dilsad Sanlioglu
  • Salih Sanlioglu
Protocol
Part of the Methods in Molecular Biology book series

Abstract

Lentiviral vectors are becoming preferred vectors of choice for clinical gene therapy trials due to their safety, efficacy, and the long-term gene expression they provide. Although the efficacy of lentiviral vectors is mainly predetermined by the therapeutic genes they carry, they must be produced at high titers to exert therapeutic benefit for in vivo applications. Thus, there is need for practical, robust, and scalable viral vector production methods applicable to any laboratory setting. Here, we describe a practical lentiviral production technique in roller bottles yielding high-titer third-generation lentiviral vectors useful for in vivo gene transfer applications. CaPO4-mediated transient transfection protocol involving the use of a transfer vector and three different packaging plasmids is employed to generate lentivectors in roller bottles. Following clearance of cellular debris via low-speed centrifugation and filtration, virus is concentrated by high-speed ultracentrifugation over sucrose cushion.

Keywords

Gene therapy Lentivirus Roller bottles 

Notes

Acknowledgments

This study is supported by grants from Akdeniz University Scientific Research Administration Division (TYL-2015-1027) and the Scientific and Technological Research Council of Turkey (TUBITAK-112S114).

References

  1. 1.
    Sakuma T et al (2012) Lentiviral vectors: basic to translational. Biochem J 443(3):603–618Google Scholar
  2. 2.
    Goff S (2001) Retroviridae: the retroviruses and their replication. In: Howley P et al (eds) Fields Virology. Lippincott, Williams and Wilkins, Philadelphia, PA, pp 1871–1939Google Scholar
  3. 3.
    Vogt PK (1997) Historical introduction to the general properties of retroviruses. In: Coffin JM et al (eds) Retroviruses. CSHL Press, Cold Spring Harbor, NY, pp 1–26Google Scholar
  4. 4.
    Tasyurek MH et al (2014) GLP-1-mediated gene therapy approaches for diabetes treatment. Expert Rev Mol Med 16:e7Google Scholar
  5. 5.
    Templeton NS (2009) Gene and cell therapy : therapeutic mechanisms and strategies, 3rd edn. CRC Press, Boca RatonGoogle Scholar
  6. 6.
    Powell DM et al (1997) HIV Rev-dependent binding of SF2/ASF to the Rev response element: possible role in Rev-mediated inhibition of HIV RNA splicing. Proc Natl Acad Sci U S A 94(3):973–978Google Scholar
  7. 7.
    Lever A et al (1989) Identification of a sequence required for efficient packaging of human immunodeficiency virus type 1 RNA into virions. J Virol 63(9):4085–4087Google Scholar
  8. 8.
    McBride MS, Panganiban AT (1996) The human immunodeficiency virus type 1 encapsidation site is a multipartite RNA element composed of functional hairpin structures. J Virol 70(5):2963–2973Google Scholar
  9. 9.
    Ciuffi A (2016) The benefits of integration. Clin Microbiol Infect 22(4):324–332Google Scholar
  10. 10.
    Frankel AD, Young JA (1998) HIV-1: fifteen proteins and an RNA. Annu Rev Biochem 67:1–25Google Scholar
  11. 11.
    Herrmann CH, Rice AP (1995) Lentivirus Tat proteins specifically associate with a cellular protein kinase, TAK, that hyperphosphorylates the carboxyl-terminal domain of the large subunit of RNA polymerase II: candidate for a Tat cofactor. J Virol 69(3):1612–1620Google Scholar
  12. 12.
    Kim YK et al (2002) Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation. Mol Cell Biol 22(13):4622–4637Google Scholar
  13. 13.
    Jones KA, Peterlin BM (1994) Control of RNA initiation and elongation at the HIV-1 promoter. Annu Rev Biochem 63:717–743Google Scholar
  14. 14.
    Pollard VW, Malim MH (1998) The HIV-1 Rev protein. Annu Rev Microbiol 52:491–532Google Scholar
  15. 15.
    Collins DR, Collins KL (2014) HIV-1 accessory proteins adapt cellular adaptors to facilitate immune evasion. PLoS Pathog 10(1):e1003851Google Scholar
  16. 16.
    Simon V, Ho DD (2003) HIV-1 dynamics in vivo: implications for therapy. Nat Rev Microbiol 1(3):181–190Google Scholar
  17. 17.
    Naldini L et al (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272(5259):263–267Google Scholar
  18. 18.
    Naldini L, Verma IM (2000) Lentiviral vectors. Adv Virus Res 55:599–609Google Scholar
  19. 19.
    Zufferey R et al (1997) Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol 15(9):871–875Google Scholar
  20. 20.
    Dull T et al (1998) A third-generation lentivirus vector with a conditional packaging system. J Virol 72(11):8463–8471Google Scholar
  21. 21.
    Miyoshi H et al (1998) Development of a self-inactivating lentivirus vector. J Virol 72(10):8150–8157Google Scholar
  22. 22.
    Zufferey R et al (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72(12):9873–9880Google Scholar
  23. 23.
    Zufferey R et al (1999) Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. J Virol 73(4):2886–2892Google Scholar
  24. 24.
    Stacey GN, M.O.-W. (2011) Host cells and cell banking. In: Merten OW, Al-Rubai M (eds) Viral vectors for gene therapy. Humana Press, Totowa, NJ, pp 45–88Google Scholar
  25. 25.
    Merten OW et al (2016) Production of lentiviral vectors. Mol Ther Methods Clin Dev 3:16017Google Scholar
  26. 26.
    Warnock JN et al (2006) Cell culture processes for the production of viral vectors for gene therapy purposes. Cytotechnology 50(1–3):141–162Google Scholar
  27. 27.
    Segura MM et al (2007) Production of lentiviral vectors by large-scale transient transfection of suspension cultures and affinity chromatography purification. Biotechnol Bioeng 98(4):789–799Google Scholar
  28. 28.
    Geisse S (2009) Reflections on more than 10 years of TGE approaches. Protein Expr Purif 64(2):99–107Google Scholar
  29. 29.
    Ansorge S et al (2010) Recent progress in lentiviral vector mass production. Biochem Eng J 48(3):362–377Google Scholar
  30. 30.
    Ansorge S et al (2009) Development of a scalable process for high-yield lentiviral vector production by transient transfection of HEK293 suspension cultures. J Gene Med 11(10):868–876Google Scholar
  31. 31.
    Follenzi A, Naldini L (2002) Generation of HIV-1 derived lentiviral vectors. Methods Enzymol 346:454–465Google Scholar
  32. 32.
    Toledo JR et al (2009) Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors. Appl Biochem Biotechnol 157(3):538–544Google Scholar
  33. 33.
    Pham PL et al (2006) Large-scale transfection of mammalian cells for the fast production of recombinant protein. Mol Biotechnol 34(2):225–237Google Scholar
  34. 34.
    Jordan M et al (1996) Transfecting mammalian cells: optimization of critical parameters affecting calcium-phosphate precipitate formation. Nucleic Acids Res 24(4):596–601Google Scholar
  35. 35.
    Mitta B et al (2005) Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles. Metab Eng 7(5–6):426–436Google Scholar
  36. 36.
    al Yacoub N et al (2007) Optimized production and concentration of lentiviral vectors containing large inserts. J Gene Med 9(7):579–584Google Scholar
  37. 37.
    Sena-Esteves M et al (2004) Optimized large-scale production of high titer lentivirus vector pseudotypes. J Virol Methods 122(2):131–139Google Scholar
  38. 38.
    Karolewski BA et al (2003) Comparison of transfection conditions for a lentivirus vector produced in large volumes. Hum Gene Ther 14(14):1287–1296Google Scholar
  39. 39.
    Luthman H, Magnusson G (1983) High efficiency polyoma DNA transfection of chloroquine treated cells. Nucleic Acids Res 11(5):1295–1308Google Scholar
  40. 40.
    Olgun H (2017) Optimized production and purification methods of third generation HIV-based Lentiviral Vectors for in vivo applications. Master’s thesis. Retrieved from Turkey Council of Higher Education Thesis CenterGoogle Scholar
  41. 41.
    Tasyurek HM et al (2018) Therapeutic potential of lentivirus-mediated glucagon-like peptide-1 (GLP-1) gene therapy for diabetes. Hum Gene Ther.  https://doi.org/10.1089/hum.2017.180
  42. 42.
    Tasyurek MH, Eksi YE, Sanlioglu AD, Altunbas HA, Balci MK, Griffith TS, Sanlioglu S (2018) HIV-based lentivirus-mediated vasoactive intestinal peptide gene delivery protects against DIO animal model of type 2 diabetes. Gene Ther.  https://doi.org/10.1038/s41434-018-0011-1
  43. 43.
    Backliwal G et al (2008) High-density transfection with HEK-293 cells allows doubling of transient titers and removes need for a priori DNA complex formation with PEI. Biotechnol Bioeng 99(3):721–727Google Scholar
  44. 44.
    Derouazi M et al (2004) Serum-free large-scale transient transfection of CHO cells. Biotechnol Bioeng 87(4):537–545Google Scholar
  45. 45.
    Durocher Y et al (2002) High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human 293-EBNA1 cells. Nucleic Acids Res 30(2):E9Google Scholar
  46. 46.
    Yang YW, Yang JC (1997) Calcium phosphate as a gene carrier: electron microscopy. Biomaterials 18(3):213–217Google Scholar
  47. 47.
    Jordan M (2000) Transient gene expression in mammalian cells based on the calcium phosphate transfection method. In: Al-Rubeai M (ed) Cell engineering. Springer, DordrechtGoogle Scholar
  48. 48.
    Kingston RE et al (2003) Calcium phosphate transfection. Curr Protoc Mol Biol Chapter 9:Unit 9.1Google Scholar
  49. 49.
    Tom R et al (2008) Transfection of HEK293-EBNA1 cells in suspension with 293fectin for production of recombinant proteins. CSH Protoc 2008:pdb prot4979Google Scholar

Copyright information

© Springer Science+Business Media New York 2018

Authors and Affiliations

  • Hazal Banu Olgun
    • 1
  • Hale M. Tasyurek
    • 1
  • Ahter Dilsad Sanlioglu
    • 1
  • Salih Sanlioglu
    • 1
  1. 1.Human Gene and Cell Therapy Center of Akdeniz University HospitalsAntalyaTurkey

Personalised recommendations