Helper-independent and AAV-ITR-independent chromosomal integration of double-stranded linear DNA vectors in mice

Hiroyuki Nakai, Eugenio Montini, Sally Fuess, Theresa A. Storm, Leonard Meuse, Milton Finegold, Markus Grompe, Mark A. Kay

Research output: Contribution to journalArticlepeer-review

46 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)101-111
Number of pages11
JournalMolecular Therapy
Issue number1
StatePublished - Jan 1 2003


  • Adeno-associated virus
  • Factor IX
  • Gene therapy
  • Hereditary tyrosinemia
  • Integration
  • Inversted terminal repeat
  • Linear DNA
  • Mouse liver
  • Naked DNA

ASJC Scopus subject areas

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery


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