Resumen:
Background and Aims: Recombinant AAV transduction of
postmitotic cells, such as hepatocytes, leads mainly to formation of
episomal monomeric and concatameric circles or linear episomes,
which assimilate into chromatin with a typical nucleosomal pattern.
However in proliferating cells, nonintegrated viral genomes are
instable and are lost soon upon proliferation of transduced cells.
Integration of AAV vectors can occur in presence of a transcriptionally
active or damaged genome. Therefore, the objective of this project
was to analyze the integration of AAV vectors in presence or absence
of genotoxic damage.
Methods: To perform this study we have compare the integration
profile of AAV genomes in the liver of mice after the administration
into adult mice of an AAV vector expressing PBGD (the therapeutic
vector for acute intermittent porphyria) or an AAV vector expressing
Thymidine Kinase (TK) followed by Gancyclovir administration,
which causes genotoxicity. Integration analysis was performed by
the method called LAM-PCR.
Results: Here we showed that when hepatocyte proliferation is
induced by a genotoxic injury, AAV genomes are efficiently integrated.
We found that while the percentage of AAV genome integration in the
liver of AAV-PBGD treated mice is lower than 1% and 99% of the viral
genomes remain episomal, in the case of the animals receiving
AAV-TK in combination with Gancyclovir the situation is completely
the opposite, with a percentage of integration higher than 99%.
We analyzed the relative sequence count (which might act as marker
for clonal outgrowth) of each exact mappable integration sites (IS)
in relation to all sequences resembling concatemeric or proviral
structures and we detected 13 IS with a high relative sequence count
of >10%. The genes which is located nearby the IS with a high relative
sequence count were determined by ingenuity pathway analyzing
tools and found that they regulate different cellular functions.
Conclusions: AAV genomes stay mainly forming episomal structures
in absence of injury, but the 99% of these genomes integrate in the
cellular DNA in presence of genotoxic damage. We are also able to
determine the exact IS of the AAV genome.
