Homologous and heterologous recombination between adenovirus vector DNA and chromosomal DNA

• Published in: The journal of gene medicine 2008-11, Vol.10 (11), p.1176-1189
• Main Authors: Stephen, Sam Laurel, Sivanandam, Vijayshankar Ganesh, Kochanek, Stefan
• Format: Article
• Language: English
• Subjects: Adenoviridae - genetics; adenovirus vector; Base Sequence; Cells, Cultured; Chromosomes - genetics; Chromosomes - metabolism; DNA - chemistry; DNA - metabolism; DNA, Viral - chemistry; Gene therapy; Genetic Vectors - chemistry; genotoxicity; Humans; integration; Models, Genetic; Molecular Sequence Data; Pentosyltransferases - genetics; Pentosyltransferases - metabolism; recombination; Recombination, Genetic - genetics
• ISSN: 1099-498X; 1521-2254
• DOI: 10.1002/jgm.1246

Background Adenovirus vector DNA is perceived to remain as episome following gene transfer. We quantitatively and qualitatively analysed recombination between high capacity adenoviral vector (HC‐AdV) and chromosomal DNA following gene transfer in vitro. Methods We studied homologous and heterologous recombination with a single HC‐AdV carrying (i) a large genomic HPRT fragment with the HPRT CHICAGO mutation causing translational stop upon homologous recombination with the HPRT locus and (ii) a selection marker to allow for clonal selection in the event of heterologous recombination. We analysed the sequences at the junctions between vector and chromosomal DNA. Results In primary cells and in cell lines, the frequency of homologous recombination ranged from 2 × 10−5 to 1.6 × 10−6. Heterologous recombination occurred at rates between 5.5 × 10−3 and 1.1 × 10−4. HC‐AdV DNA integrated via the termini mostly as intact molecules. Analysis of the junction sequences indicated vector integration in a relatively random manner without an obvious preference for particular chromosomal regions, but with a preference for integration into genes. Integration into protooncogenes or tumor suppressor genes was not observed. Patchy homologies between vector termini and chromosomal DNA were found at the site of integration. Although the majority of integrations had occurred without causing mutations in the chromosomal DNA, cases of nucleotide substitutions and insertions were observed. In several cases, deletions of even relative large chromosomal regions were likely. Conclusions These results extend previous information on the integration patterns of adenovirus vector DNA and contribute to a risk–benefit assessment of adenovirus‐mediated gene transfer. Copyright © 2008 John Wiley & Sons, Ltd.