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Generation of lentivirus vectors using recombinant baculoviruses

Gene Therapy volume 15, pages 1280–1286 (2008)Cite this article

Abstract

In spite of advances in conventional four-plasmid transient transfection methods and development of inducible stable production cell lines, production of replication-defective lentiviral vectors in clinical scale has been challenging. Baculovirus technology offers an alternative to scalable virus production as a result of fast and easy production of baculoviruses, efficient transduction of mammalian cells and safety of the baculoviruses. As a first step toward scalable lentiviral production system, we have constructed four recombinant baculoviruses: the BAC-transfer virus expresses green fluorescent protein (GFP) as a transgene and BAC-gag-pol, BAC-vesicular stomatitis virus glycoprotein G and BAC-rev express all elements required for a safe lentivirus vector generation. Following 293T cell transduction with recombinant baculoviruses functional lentiviruses were produced. Different baculovirus concentrations, mediums and transduction times were used to find optimal conditions for lentivirus production. The unconcentrated lentiviral titers in cell culture mediums were on average 2.5 × 106 TU ml−1, which are comparable to titers of the lentiviruses produced by conventional four-plasmid methods. Lentiviruses produced by baculovirus method transduced HeLa cells and showed sustained GFP expression. No evidence of the formation of replication competent lentiviruses was detected by p24 enzyme-linked immunosorbent assay. Our results show that baculoviruses are an attractive alternative for the production of lentiviruses in mammalian cells.

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Acknowledgements

We thank Miia Roschier, Tarja Taskinen, Erik Peltomaa, Riikka Eisto, Joonas Malinen, Anne Martikainen and Anneli Miettinen for technical assistance, Dr Olli Laitinen, Minna Kaikkonen and Petri Mäkinen for their invaluable discussions, and Dr Jani Räty and Dr Roseanne Girnary for reviewing the manuscript. This study was supported by Ark Therapeutics Group Plc and EU Clinigene (LSHB-CT-2006-018933) Consortium Flexibility Funds.

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Authors and Affiliations

  1. Department of Biotechnology and Molecular Medicine, AI Virtanen Institute for Molecular Sciences, University of Kuopio, Kuopio, Finland

    H P Lesch, S Turpeinen, A J Mähönen, K J Airenne & S Ylä-Herttuala

  2. Ark Therapeutics Oyj, Kuopio, Finland

    H P Lesch, S Turpeinen & A J Mähönen

  3. Department of Biological and Environmental Science, NanoScience Center, University of Jyväskylä, Finland

    E A Niskanen

  4. Department of Medicine and Gene Therapy Unit, University of Kuopio, Kuopio, Finland

    S Ylä-Herttuala

  5. Kuopio University Hospital, Kuopio, Finland

    S Ylä-Herttuala

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  1. H P Lesch
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Correspondence to S Ylä-Herttuala.

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Lesch, H., Turpeinen, S., Niskanen, E. et al. Generation of lentivirus vectors using recombinant baculoviruses. Gene Ther 15, 1280–1286 (2008). https://doi.org/10.1038/gt.2008.76

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