Detalle Publicación

AAV integration in the liver in a model of induced genotoxicity
Título de la revista: JOURNAL OF HEPATOLOGY (ONLINE)
ISSN: 0168-8278
Volumen: 64
Número: 2, supl.
Páginas: S303
Fecha de publicación: 2016
Lugar: WOS
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.