The persistence of recombinant adenoviral vectors

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.