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FORGEN II VV5 Genomic Targeting of recombinant Adeno-Associated Virus

The adeno-associated virus (AAV) has promising features as a vector for somatic gene therapy, e.g. AAV displays a wide tropism, transduces terminally differentiated as well as nondividing cells, shows low immunogenicity and lacks any apparent pathologenicity. A unique characteristic of wild-type AAV is its ability to integrate site-specifically into the human chromosome 19q13.3-qter (= AAVS1). Although recombinant AAV (rAAV) can integrate into the genome, the specificity for AAVS1 appears to be lost due to the absence of the viral-encoded Rep proteins. The goal of our project is the generation of rAAV with the ability for site-specific integration in AAVS1 without limiting its transgene coding capacity of 4.7 KB. In the first round of experiments we produced rAAV, encoding the genes for Green Fluorescence Protein and Hygromycine resistance by the adenovirus dependent production method. Then, HeLa cells were co-transduced with a Rep expressing plasmid and the rAAV(GFP/Hygro). From the co-transduced and GFP expressing cells, single cell clones were generated. 70% of these single cell clones (Rep expressing plasmid and rAAV) showed site-specific integration of the transgenes in AAVS1, whereas none of the controls (only rAAV) were positive for site specific integration. Therfore, it is possible to integrate transgenes site specifically into the human chromosome 19q13.3-qter in vitro by this method. However, co-transduction is not efficient enough for an in vivo application. The next step is to establish such methods, which is done in collaboration with MediGene AG in Martinsried.


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