title: Receptor-mediated delivery of engineered nucleases for genome modification.
abstract: 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.
authors: Department of Anesthesiology and Perioperative Medicine, Georgia Regents University, 1120 15th Street, Augusta, GA 30912, USA, Department of Radiation Oncology, Emory University School of Medicine, 4121 Rollins Research Center, 1510 Clifton Rd. NE, Atlanta, GA 30322, USA, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York 11724, USA, Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30322, USA, Department of Pediatrics-Divisions of Hematology/Oncology and Human Gene Therapy, Stanford University School of Medicine, Stanford, CA 94305, USA and Department of Biochemistry, Emory University School of Medicine, 4121 Rollins Research Center, 1510 Clifton Rd. NE, Atlanta, GA 30322, USA.
authors: Zhong
authors: Z
authors: Chen
authors: Lahcen
authors: L
authors: Jaafar
authors: Davies G
authors: DG
authors: Agyekum
authors: Haiyan
authors: H
authors: Xiao
authors: Marlene F
authors: MF
authors: Wade
authors: R Ileng
authors: RI
authors: Kumaran
authors: David L
authors: DL
authors: Spector
authors: Gang
authors: G
authors: Bao
authors: Matthew H
authors: MH
authors: Porteus
authors: William S
authors: WS
authors: Dynan
authors: Steffen E
authors: SE
authors: Meiler
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D023281:Genomics; D006801:Humans; D008024:Ligands; D051379:Mice; D015202:Protein Engineering; D011990:Receptors, Transferrin / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D052416:Targeted Gene Repair / Q000379:methods*; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D008024:Ligands; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D003851:Deoxyribonucleases
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