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Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS

Gene Therapy volume 16, pages 605–619 (2009)Cite this article

Abstract

The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) is widely used in retroviral gene transfer vectors. However, this element contains an open-reading frame (ORF) encoding a truncated peptide of the woodchuck hepatitis virus X protein (WHX). Because we are developing a lentiviral vector for the gene therapy of Wiskott–Aldrich syndrome (WAS), we evaluated whether the WPRE was needed in the gene transfer cassette and tested the possibility of replacing it with a mutated derivative. The transcriptional activity of the WPRE was undetectable in the context of the lentiviral vector but the element was capable of translating a polypeptide. This capability was abrogated by mutating the WHX ORF translation start. The WPRE was required to express high levels of the transgene and for that, the native form or mutated derivatives functioned equivalently. The vector using a WAS gene promoter and the mut6 WPRE induced long-term expression of the WAS transgene in vivo, correcting cytoskeletal defects, thymocyte and B-cell numbers and improved the colitis of WAS-null mice. By providing additional evidence of efficacy of this WAS lentiviral vector with improved safety features, our results validate a mutated WPRE, which should be useful in future gene therapy applications.

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Acknowledgements

We are grateful for excellent technical help from Genethon colleagues, notably Khalil Seye for the qPCR assays, Johanna Blondeau for western blots, Anne Samba for virus stock production, Bernard Gjata for histology, Séverine Charles for bioexperimentation and the zootechnicians for animal care. We also thank MG Roncarolo and Dr L Naldini (Tiget, Milan) for providing the initial lentiviral plasmids. The work was supported in part from funds from the FP6 CONSERT European project (FP6-2003-LIFESCIHEALTH-I-005242) and from AFM (French Muscular Dystrophy Association). AJT was supported by the Wellcome Trust.

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Author notes

  1. M A Zanta-Boussif and S Charrier: These authors contributed equally to this work.

Authors and Affiliations

  1. Translational Research and Development, Genethon, Evry, France

    M A Zanta-Boussif, A Brice-Ouzet & S Martin

  2. INSERM U790, Genethon, Evry, France

    S Charrier & A Galy

  3. Université Paris-Sud, Genethon, Orsay, France

    S Charrier & A Galy

  4. UMR 8121, Institut Gustave Roussy, Villejuif, France

    P Opolon

  5. Centre for Immunodeficiency, Institute of Child Health, University College London, London, UK

    A J Thrasher

  6. Great Ormond Street Hospital NHS Trust, London, UK

    A J Thrasher

  7. Department of Cell and Molecular Biology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA

    T J Hope

Authors
  1. M A Zanta-Boussif
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  2. S Charrier
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  4. S Martin
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  6. A J Thrasher
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  7. T J Hope
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  8. A Galy
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Corresponding author

Correspondence to A Galy.

Additional information

This work was performed at Généthon, 1 bis, rue de l'Internationale, 91002 Evry, France.

Supplementary Information accompanies the paper on Gene Therapy website (http://www.nature.com/gt)

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Zanta-Boussif, M., Charrier, S., Brice-Ouzet, A. et al. Validation of a mutated PRE sequence allowing high and sustained transgene expression while abrogating WHV-X protein synthesis: application to the gene therapy of WAS. Gene Ther 16, 605–619 (2009). https://doi.org/10.1038/gt.2009.3

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