Receptor-mediated delivery of engineered nucleases for genome modification

Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucl...

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Bibliographic Details
Published in:Nucleic acids research 2013-10, Vol.41 (19), p.e182-e182
Main Authors: Chen, Zhong, Jaafar, Lahcen, Agyekum, Davies G, Xiao, Haiyan, Wade, Marlene F, Kumaran, R Ileng, Spector, David L, Bao, Gang, Porteus, Matthew H, Dynan, William S, Meiler, Steffen E
Format: Article
Language:English
Subjects:
Animals
Cell Line
Cells, Cultured
Deoxyribonucleases - genetics
Deoxyribonucleases - metabolism
DNA Cleavage
Endocytosis
Genomics
Humans
Ligands
Methods Online
Mice
Protein Engineering
Receptors, Transferrin - metabolism
Recombinant Fusion Proteins - metabolism
Targeted Gene Repair - methods
Transferrin - genetics
Transferrin - metabolism
Zinc Fingers
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Summary:Engineered nucleases, which incise the genome at predetermined sites, have a number of laboratory and clinical applications. There is, however, a need for better methods for controlled intracellular delivery of nucleases. Here, we demonstrate a method for ligand-mediated delivery of zinc finger nucleases (ZFN) proteins using transferrin receptor-mediated endocytosis. Uptake is rapid and efficient in established mammalian cell lines and in primary cells, including mouse and human hematopoietic stem-progenitor cell populations. In contrast to cDNA expression, ZFN protein levels decline rapidly following internalization, affording better temporal control of nuclease activity. We show that transferrin-mediated ZFN uptake leads to site-specific in situ cleavage of the target locus. Additionally, despite the much shorter duration of ZFN activity, the efficiency of gene correction approaches that seen with cDNA-mediated expression. The approach is flexible and general, with the potential for extension to other targeting ligands and nuclease architectures.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/gkt710