Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo

Hiroyuki Nakai, Sally Fuess, Theresa A. Storm, Leonard A. Meuse, Mark A. Kay

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in vivo. In hepatocyte nuclei, the incoming single-stranded (ss) vector genomes are converted into various forms of double-stranded (ds) genomes including extrachromosomal linear and circular monomers and concatemers, and a small portion of the vector genomes integrate into chromosomes. The mechanism of genome conversion is not well understood. In the present study, we analyzed the role of inverted terminal repeat (ITR) sequences of ds circular or linear rAAV vector intermediates in concatemerization. We synthesized supercoiled ds circular monomers with a double-D ITR (DDITR) (C+), and ds linear monomers with an ITR at each end (L+), and their control molecules, C- and L- , which lack the ITR-derived sequences, and transfected mouse hepatocytes with these molecules in vivo to assess their capacity for concatemerization. The transfected L+ or L-, but not C+ or C- molecules, concatemerized in vivo irrespective of the presence or absence of the ITRs. In addition, our results suggested that transfected C+ or C- species were not efficient substrates for integration. Based on these observations, we propose a model whereby ds linear molecules with free DNA ends, but not circular molecules, play an important role in rAAV vector genome concatemerization.

Original languageEnglish (US)
Pages (from-to)112-121
Number of pages10
JournalMolecular Therapy
Volume7
Issue number1
DOIs
StatePublished - Jan 1 2003

Keywords

  • Adeno-associated virus
  • Double-stranded DNA
  • Factor IX
  • Gene therapy
  • Integration
  • Inverted terminal repeat
  • Liver
  • Mouse
  • Naked DNA
  • Recombination

ASJC Scopus subject areas

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

Fingerprint Dive into the research topics of 'Free DNA ends are essential for concatemerization of synthetic double-stranded adeno-associated virus vector genomes transfected into mouse hepatocytes in vivo'. Together they form a unique fingerprint.

  • Cite this