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Gene Ther. 2014 Aug;21(8):759-66. doi: 10.1038/gt.2014.51. Epub 2014 Jun 12.

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

Author information

1
INSERM U845, Université Paris Descartes, Faculté de Médecine Paris Descartes-Site Necker, Paris, France.
2
Cellectis Therapeutics, Paris, France.
3
Cellectis SA, Paris, France.
4
1] INSERM U845, Université Paris Descartes, Faculté de Médecine Paris Descartes-Site Necker, Paris, France [2] Cancer Institute, University College London, London, UK.

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.

PMID:
24919419
DOI:
10.1038/gt.2014.51
[Indexed for MEDLINE]

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