Journal Article

Non-toxic and highly efficient targeting of a human chromosomal locus with a lentiviral vector-associated meganuclease

He, Chenxia, Gouble, Agnes, Duchateau, Philippe, Danos, Olivier, Paques, Frederic, Poirot, Laurent, Bourdel, Alix, Edelman, Aleksander

2013

Overview

Site specific endonucleases -- Zinc Finger Nucleases, TALE Nucleases or naturally occurring Meganucleases (MN) -- can be engineered for custom recognition of any genetic locus and used for gene targeting. Because the prolonged expression of these nucleases into cells may be toxic, due to the accumulation of DNA double strand breaks, efficient methods for their transient delivery are needed. Meganucleases (MN) can be produced as single chain proteins and are advantageous for developing approaches of protein transduction. Here, we further explore the possibilities of associating I-CreI-derived single chain MNs to lentiviral vectors. The CLS4076 is a I-CreI-derived single chain MN with a unique cutting site on human chromosome 14. Whilst a control NILV encoding CLS4076 was able to mediate up to 5% HR, up to 20-30% HR was observed when the MN protein was associated to the NILV particle. The results of gene targeting studies using this protein delivery method in human primary T or CD34+ hematopoietic cells will be presented.