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Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency

Gene Therapy volume 19, pages 872–876 (2012)Cite this article

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Abstract

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68–70% normal human CD34+ cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdcscid/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34+ cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

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Acknowledgements

We thank the Translational Trials Development and Support Laboratory (TTDSL), Viral Vector Core and the Cell Processing and Manipulation Laboratory’s Normal Donor Repository of the Translational Core Laboratory (TCL) at Cincinnati Children’s Hospital Medical Center for qPCR analysis, vector titration and for providing human CD34+ cells, respectively. The Vector Production Facility is supported by Institutional funds from the Cincinnati Children’s Research Foundation. AS thanks the German Academic Exchange Service (DAAD), the European Union (FP7 program PERSIST) and the German Ministry for Research (BMBF project PIDNET.IFB-Tx) for their support. AJT acknowledges the Medical Research Council, The Wellcome Trust and Great Ormond Street Children’s Charity for their support. We thank Christopher Baum for his assistance with vector design and helpful discussions on scale-up.

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Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA

    J C M van der Loo, E Grassman, A Terwilliger, T Higashimoto, D L Nordling & P Malik

  2. Siteman Cancer Center, Barnes Jewish Hospital, Washington University St. Louis School of Medicine, St Louis, MO, USA

    W P Swaney

  3. Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany

    A Schambach

  4. Department of Biotherapy, Hospital Necker Enfants-Malades, Groupe Hospitalier Universitaire Ouest, Paris, France

    S Hacein-Bey-Abina & M Cavazzana-Calvo

  5. Clinical Investigation Center in Biotherapy, Groupe Hospitalier Universitaire Ouest, Paris, France

    S Hacein-Bey-Abina & M Cavazzana-Calvo

  6. INSERM U-768, Hopital Necker Enfants-Malades, Paris, France

    S Hacein-Bey-Abina & M Cavazzana-Calvo

  7. Université Paris Descartes, Paris, France

    S Hacein-Bey-Abina & M Cavazzana-Calvo

  8. Centre Immunodeficiency, UCL Institute of Child Health, London, UK

    A J Thrasher

  9. Department of Immunology, Great Ormond Street Hospital NHS Trust, London, UK

    A J Thrasher

  10. Division of Hematology/Oncology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA

    D A Williams

  11. Indiana University School of Medicine, Indianapolis, IN, USA

    L Reeves

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  1. J C M van der Loo
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Correspondence to J C M van der Loo.

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van der Loo, J., Swaney, W., Grassman, E. et al. Critical Variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency. Gene Ther 19, 872–876 (2012). https://doi.org/10.1038/gt.2012.37

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