Résumé :
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Adeno-associated virus (AAV) vectors are one of the most promising viral vectors used in gene therapy clinical trials. In the absence of an immune response directed against the transgene, they can drive long-term expression of therapeutic genes after a single intra-muscular injection. Nevertheless, the mechanism by which the genome of rAAV persists is not completely elucidated. Previous studies demonstrated that rAAV genomes persist in the skeletal muscle mainly in high-molecular weight (HMW) episomes. However, the persistence of the expression has not been correlated directly with a particular viral DNA conformation. The aim of our study was to determine if the rAAV genome adopts a chromatin-like structure in vivo providing an explanation of stable vector genome maintenance and transgene expression. We chose a model that is relevant in the clinical setting: gene transfer in skeletal muscle of the non-human primate mediated by an rAAV coding an immunosuppressive molecule. Muscular biopsies were performed more than one year after administration of AAV serotypes 1 and 8. As previously described in the literature, we have shown that the rAAV persists as HMW episomes comprised of vector genomes in head-to-head and head-to-tail orientations. In accordance with previous studies realized in the skeletal muscle, LAM-PCR (linear amplification-mediated PCR) data obtained from total DNA of transduced primate muscle indicate that integration corresponds to a very minor pathway for vector genome maintenance in this organ. Furthermore, a micrococcal nuclease digestion assay (MNase) performed on myocyte nuclei isolated from injected muscle demonstrated, for the first time, that the rAAV genome assembles and persists as chromatin with a typical nucleosome structure in vivo. These results suggest that the association of histones with rAAV genomes, and probably other proteins, leads to the stable maintenance of the vector genome, and transgene expression may be subjected to epigenetic regulation. To contact the author:: magalie.penaud-budloo@univ-nantes.fr (1) INSERM UMR 649, Nantes, FRANCE. (2) Department of Translational Oncology, National Center for Tumor Diseases, Heidelberg, GERMANY. (3) Ecole Nationale Vétérinaire, Nantes, FRANCE. (4) Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville, USA.
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