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Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors

Gene Therapy volume 18, pages 531–538 (2011)Cite this article

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Abstract

Lentivirus can be engineered to be a highly potent vector for gene therapy applications. However, generation of clinical grade vectors in enough quantities for therapeutic use is still troublesome and limits the preclinical and clinical experiments. As a first step to solve this unmet need we recently introduced a baculovirus-based production system for lentiviral vector (LV) production using adherent cells. Herein, we have adapted and optimized the production of these vectors to a suspension cell culture system using recombinant baculoviruses delivering all elements required for a safe latest generation LV preparation. High-titer LV stocks were achieved in 293T cells grown in suspension. Produced viruses were accurately characterized and the functionality was also tested in vivo. Produced viruses were compared with viruses produced by calcium phosphate transfection method in adherent cells and polyethylenimine transfection method in suspension cells. Furthermore, a scalable and cost-effective capture purification step was developed based on a diethylaminoethyl monolithic column capable of removing most of the baculoviruses from the LV pool with 65% recovery.

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Acknowledgements

We thank Joonas Malinen, Tarja Taskinen, Jaana Siponen, Siiri Väistö, Juha Ruuskanen, Anne Martikainen and Anneli Miettinen for excellent technical assistance. This work was supported by grants from Finnish Academy, the European Union (Baculogenes, LSH-2005-1.4.4.6 and Clinigene Network of Excellence, LSHB-CT-2006-018933), Portuguese Fundação para a Ciência e Tecnologia (PTDC/EQU-EQU/71645/2006 and SFRH/BD/31257/2006) and Ark Therapeutics Group plc.

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Author notes

  1. H P Lesch and A Laitinen: These authors contributed equally to this work.

  2. C Peixoto and T Vicente: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biotechnology and Molecular Medicine, A.I. Virtanen Institute, University of Eastern Finland, Kuopio, Finland

    H P Lesch, A Laitinen, K-E Makkonen, J T Pikkarainen, H Samaranayake, S Ylä-Herttuala & K J Airenne

  2. Ark Therapeutics Oyj, Kuopio, Finland

    H P Lesch, L Laitinen, J T Pikkarainen & H Samaranayake

  3. IBET, Apartado 12, Oeiras, Portugal

    C Peixoto, T Vicente, P M Alves & M J T Carrondo

  4. ITQB-UNL, EAN, Oeiras, Portugal

    T Vicente

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

    S Ylä-Herttuala

  6. Kuopio University Hospital, Kuopio, Finland

    S Ylä-Herttuala

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  1. H P Lesch
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Correspondence to H P Lesch.

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Competing interests

HPL, JTP, HS and LL are employees of Ark Therapeutics Ltd. CP, TV, PMA and MJTC are employees of IBET.

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Lesch, H., Laitinen, A., Peixoto, C. et al. Production and purification of lentiviral vectors generated in 293T suspension cells with baculoviral vectors. Gene Ther 18, 531–538 (2011). https://doi.org/10.1038/gt.2010.162

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  • DOI: https://doi.org/10.1038/gt.2010.162

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