title: Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice.
abstract: Nonviral plasmid DNA is a promising vector for achieving ex vivo and in vivo gene transfer. However, transgene expression is usually transient, especially in dividing target cells due to loss of vector genomes. Here we describe the use of naked double-stranded (ds) linear DNA as a way to insert exogenous DNA sequences into chromosomes of mouse hepatocytes in vivo, without helper components such as integrase or transposase. We constructed ds linear DNA vectors with or without adeno-associated virus inverted terminal repeats (AAV-ITRs), introduced them into mouse hepatocytes in vivo using a hydrodynamics-based transfection technique, and analyzed for vector genome integration in various ways. Surprisingly, these linear DNA molecules integrated in mouse hepatocytes in vivo at a level of 0.3-0.5 vector genome, or more, per diploid genomic equivalent irrespective of the AAV-ITR sequences. Our results establish a novel and simple way to engineer chromosomes in vivo and provide further insights into the mechanisms of recombinant AAV vector integration in vivo. In addition, they may provide a clue for developing new nonviral integrating gene delivery vector systems.
authors.affiliation: Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
authors.forename: Eugenio
authors.initials: E
authors.lastname: Montini
authors.forename: Sally
authors.initials: S
authors.lastname: Fuess
authors.forename: Theresa A
authors.initials: TA
authors.lastname: Storm
authors.forename: Leonard
authors.initials: L
authors.lastname: Meuse
authors.forename: Milton
authors.initials: M
authors.lastname: Finegold
authors.forename: Markus
authors.initials: M
authors.lastname: Grompe
authors.forename: Mark A
authors.initials: MA
authors.lastname: Kay
mesh_terms: D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002875:Chromosomes; D004247:DNA / Q000008:administration & dosage* / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data
chemical_list: D004247:DNA