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Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap

Gene Therapy volume 8, pages 190–198 (2001)Cite this article

Abstract

Retroviral vectors have become the primary tool for gene delivery into hematopoietic cells, including T lymphocytes. Lentiviral vectors offer an advantage over Moloney murine leukemia virus (MuLV) vectors because of their ability to translocate across an intact nuclear membrane and integrate into the genome of nonproliferating cells. We have recently demonstrated that a central strand displacement event, controlled by the central polypurine tract (cPPT) and the central termination sequence (CTS), results in the formation of a central DNA flap which acts as a cis-determinant of HIV-1 genome nuclear import. Here, we show that insertion of this DNA determinant in a classical lentiviral vector resulted in a significantly higher level of transduction in activated T cells (51 ± 12.7% versus 15 ± 1.4%). CD4+ and CD8+ T cells were transduced at equivalent levels. Importantly, freshly isolated T cells stimulated only during the 12-h transduction period could be efficiently transduced with this new flap-containing lentiviral vector, but not with the parental lentiviral vector nor an MuLV vector. Transgene expression in the flap-containing lentiviral vector, under the control of either an internal cytomegalovirus or the elongation factor-1 alpha (EF1α) promoter, was significant and expression remained elevated in resting T cells. Thus, this system allows stable expression of transgenes in T lymphocytes following a short ex vivo transduction protocol.

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Acknowledgements

We are grateful to M Sitbon for critical insights during the course of this work. Dr Ikunoshin Kato and Setsuko Yoshimura of Takara Shuzo Co Ltd are generously acknowledged for providing the recombinant fibronectin fragment and for their continuing assistance. We are indebted to Drs C June, D Cantrell and S Nagata for generously providing reagents. Supported by grants from the ANRS and AFM (NT and PC), March of Dimes Grant No. 6.FY99-406 and ARC (NT).

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Author notes

  1. V Dardalhon and B Herpers: The first two authors contributed equally to this work

Authors and Affiliations

  1. Institut de Génétique Moléculaire de Montpellier, UMR 5535/IFR 22, Paris, France

    V Dardalhon, B Herpers, N Noraz & N Taylor

  2. INSERM U474, Maternité de Port Royal, Paris, France

    F Pflumio & A Dubart-Kupperschmitt

  3. Unité d'Oncologie Virale, Institut Pasteur, Paris, France

    D Guetard, C Leveau & P Charneau

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  1. V Dardalhon
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Dardalhon, V., Herpers, B., Noraz, N. et al. Lentivirus-mediated gene transfer in primary T cells is enhanced by a central DNA flap. Gene Ther 8, 190–198 (2001). https://doi.org/10.1038/sj.gt.3301378

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