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Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity

Gene Therapy volume 21, pages 759–766 (2014)Cite this article

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Abstract

Site-specific endonucleases can be engineered for custom recognition of any genetic locus and used for gene targeting. Yet, the prolonged expression and accumulation of these nucleases in cells lead to toxic effect. Here we describe an efficient and quantitative method for introducing nucleases into cells as proteins packaged within lentiviral vector particles. I-CreI-derived meganucleases, which can be engineered as single-chain proteins, were incorporated into lentiviral vector particles either without modification or as fusions with cyclophilin A. The small amount of nuclease delivered by the viral particles is sufficient to induce efficient targeted mutagenesis in human HEK293H and primary T cells. When a repair template sequence was packaged in the lentiviral vector, high levels of homologous gene targeting were obtained and toxicity was markedly reduced.

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Acknowledgements

We thank A Henry and C Bregnard for technical help and S Basmaciogullari and A Izmiryan for discussions. This work was supported by the French National Research Agency (ANR 2010 BIOT 007 01). Funding for open access charge: French National Research Agency.

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Authors and Affiliations

  1. INSERM U845, Université Paris Descartes, Faculté de Médecine Paris Descartes-Site Necker, Paris, France

    C He, A Bourdel, V Manchev, A Edelman & O Danos

  2. Cellectis Therapeutics, Paris, France

    A Gouble & L Poirot

  3. Cellectis SA, Paris, France

    F Paques & P Duchateau

  4. Cancer Institute, University College London, London, UK

    O Danos

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  1. C He
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Correspondence to O Danos.

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He, C., Gouble, A., Bourdel, A. et al. Lentiviral protein delivery of meganucleases in human cells mediates gene targeting and alleviates toxicity. Gene Ther 21, 759–766 (2014). https://doi.org/10.1038/gt.2014.51

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