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Jäger, Lorenz (2009): The persistence of recombinant adenoviral vectors. Dissertation, LMU München: Faculty of Biology
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Abstract

Recombinant adenoviral vectors (rAdV) are commonly used as gene transfer vehicles and in gene therapy. Recombinant first generation vectors lack the early genes E1 and E3 (E1/E3) which makes way for insertion of up to 8.2 kb of foreign DNA. Gene-deleted adenoviral vectors (GD AdV) lacking all viral coding sequences contain only the inverted terminal repeats (ITR) and a packaging signal at its genomic termini. Hence, there is space for the insertion of coding sequences of up to 36 kb of therapeutic transgenes and additional sequences that could stabilise the vector DNA in mitotic cells. Multiple studies for liver-based gene transfer demonstrated that non-integrative rAdV result in long-term phenotopic correction and are maintained life-long in mice and for up to 2 years in rats, dogs and non-human primates. In concordance with these reports, transgene expression in mice was also seen for several months in the present study. Therefore, it is likely that rAdV provide mechanism(s) leading to persistence in mitotic host cells. However, the mechanisms responsible for vector genome maintenance are unknown. Thus, some of the mechanisms that might mediate the persistence of rAdV genomes in vitro and in vivo were analysed. In the present study, episomal replication of GD AdV genomes was tested by a methylase/restriction endonuclease-based system. High-titre preparations of methylated GD AdV were produced and tropism and persistence of transgene expression in murine liver were analysed. It was found that the originally transduced GD AdV genomes are the persistent DNA molecules in vitro and in murine liver. Therefore, replication does not influence the persistence of GD AdV genomes. Furthermore, concatemer as well as circle formation of E1/E3 and GD AdV genomes were analysed in cell culture experiments and in the liver of mice. For these investigations, pulsed field gel electrophoresis and polymerase chain reaction assays were employed. These experiments showed that - unlike E4 mutant adenovirus that is characterised by concatemerisation - rAdV genomes are exclusively present as linear monomers. Hence, the persistence of rAdV genomes is independent of concatemeric and circular conformations. Notably, the molecular analysis of E4 mutant concatemers showed the diversity of concatemeric junctions. All possibilities of end-junctions were found having various deletions at the connected genomic termini of E4 virus. Taken together, the present study provided evidence that GD AdV genomes persist as replication-inactive linear monomers.