title: DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors.
abstract: DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.
authors.affiliation: Department of Biomedicine, Aarhus University, DK-8000 Aarhus C, Denmark and Department of Immunology and Microbial Science and IAVI Neutralizing Antibody Center, The Scripps Research Institute, La Jolla, CA 92037, USA.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Cai
authors.forename: Rasmus O
authors.initials: RO
authors.lastname: Bak
authors.forename: Louise Bechmann
authors.initials: LB
authors.lastname: Krogh
authors.forename: Nicklas H
authors.initials: NH
authors.lastname: Staunstrup
authors.forename: Brian
authors.initials: B
authors.lastname: Moldt
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Corydon
authors.forename: Lisbeth Dahl
authors.initials: LD
authors.lastname: Schrøder
authors.forename: Jacob Giehm
authors.initials: JG
authors.lastname: Mikkelsen
mesh_terms: D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D015683:Gene Products, gag / Q000235:genetics*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D011498:Protein Precursors / Q000235:genetics; D011506:Proteins / Q000235:genetics / Q000378:metabolism; D019895:Transposases / Q000235:genetics / Q000378:metabolism*; D014771:Virion / Q000235:genetics / Q000378:metabolism
chemical_list: D015683:Gene Products, gag; D011498:Protein Precursors; D011506:Proteins; D019895:Transposases