title: Frequency and spectrum of genomic integration of recombinant adeno-associated virus serotype 8 vector in neonatal mouse liver.
abstract: Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 x 10(11) vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a beta-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least approximately 0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was approximately 0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10(3) hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, W1244 BSTWR, 200 Lothrop Street, Pittsburgh, PA 15261, USA. nakaih@pitt.edu
authors.forename: Katsuya
authors.initials: K
authors.lastname: Inagaki
authors.forename: Chuncheng
authors.initials: C
authors.lastname: Piao
authors.forename: Nicole M
authors.initials: NM
authors.lastname: Kotchey
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Nakai
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D045744:Cell Line, Tumor; D000229:Dependovirus / Q000235:genetics* / Q000378:metabolism*; D016679:Genome, Viral; D006321:Heart / Q000821:virology; D006505:Hepatitis / Q000473:pathology / Q000821:virology; D022781:Hepatocytes / Q000821:virology; D006801:Humans; D008099:Liver / Q000821:virology*; D008115:Liver Regeneration; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000821:virology