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A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration

Gene Therapy volume 17, pages 272–280 (2010)Cite this article

Abstract

The clinical application of self-inactivating (SIN) retroviral vectors has been hampered by the lack of reliable and efficient vector production technologies. To enable production of SIN γ-retroviral vectors from stable producer clones, a new PG13-based packaging cell, known as PG368, was developed. Viral vector expression constructs can be reliably inserted at a predefined genomic locus of PG368 packaging cells by an Flp-recombinase-mediated targeted cassette exchange (RMCE) reaction. A new, carefully designed vector-targeting construct, pEMTAR-1, eliminated the co-packaging of the selectable marker gene used for the identification of successful recombination at the predefined genomic locus and thus, improved the safety of the production system. Selected clones produced vector supernatants at consistent titers. The targeted insertion of therapeutically relevant SIN vectors for chronic granulomatous disease and X-linked severe combined immunodeficiency into PG368 cells results in stable titers within the range necessary for clinical application. The production of retroviral SIN vectors from stable clinical-grade producer cells is feasible and will contribute to the safe production and application of SIN γ-retroviral vectors for clinical trials.

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Acknowledgements

We thank Dr Angelika Lehr for the critical reading of the paper. This study was supported by grants of the German Ministry for Research and Education (TreatID), the Deutsche Forschungsgemeinschaft (DFG, that is, SPP1230 and Excellence Cluster REBIRTH), the CGD Research Trust (Grant J4G/04B/GT to MG and AJT), and the European Union (CONSERT, LSHB-CT-2004–005242; Clinigene, LSHB-CT-2006–018933), and the Else-Kröner-Fresenius-Stiftung (fellowship to AS).

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Authors and Affiliations

  1. EUFETS AG, Idar-Oberstein, Germany

    R Loew, Y Meyer & K Kuehlcke

  2. Helmholtz Center for Infection Research, Braunschweig, Germany

    L Gama-Norton, D Wirth & H Hauser

  3. Georg Speyer Haus (GSH), Frankfurt/Main, Germany

    S Stein & M Grez

  4. Centre for Immunodeficiency, Molecular Immunology Unit, UCL Institute of Child Health, University College London, London, UK

    S Thornhill & A Thrasher

  5. Department of Experimental Hematology, Hannover Medical School, Hannover, Germany

    C Baum & A Schambach

  6. Instituto de Biologia Experimental e Tecnológica, Universidade Nova de Lisboa, Oeiras, Portugal

    L Gama-Norton

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Correspondence to R Loew or A Schambach.

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Loew, R., Meyer, Y., Kuehlcke, K. et al. A new PG13-based packaging cell line for stable production of clinical-grade self-inactivating γ-retroviral vectors using targeted integration. Gene Ther 17, 272–280 (2010). https://doi.org/10.1038/gt.2009.134

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  • DOI: https://doi.org/10.1038/gt.2009.134

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