Adeno-associated viruses (AAVs) are a group of naturally defective parvoviruses that are being developed as gene therapy vectors for the treatment of diabetes, obesity, diabetes-associated erectile dysfunction, cystic fibrosis, hemophilia B, and glycogen storage diseases. AAVs require co-infection with a helper virus, usually an adenovirus or herpesvirus, for efficient productive infection. In the absence of helper virus, AAV type 2 (AAV2) DNA can integrate into the host genome with a strong preference (70%-90% of integration events) for a 4 kilobase region of human chromosome 19, designated AAVS1 (the only example of site-specific integration in a mammalian virus system). This ability to preferentially integrate also contributes to AAV2's attractiveness as a vector for gene therapy, since this could potentially limit the dangers associated with insertional mutagenesis. The Rep68 and Rep78 proteins (Rep68/78), encoded by AAV2 have binding and nicking sites within AAV2 and AAVS1 DNA. Rep68/78 are required for preferential integration, as well as virus replication. ? ? We have amplified and sequenced over 60 integration junctions between AAV2 and AAVS1 DNA. Several patterns have emerged. First, all integration junctions are on the same side of the Rep68/78 nicking site of AAVS1, consistent with the hypothesis that unidirectional DNA replication, initiated at the nick is somehow involved. The integration junction sequences show the three classical types of non-homologous end joining joints; microhomology at junctions, insertion of sequences at the junction and direct joining. This suggests that cellular DNA repair proteins may be involved in the integration process. We also detected a cluster of integration junctions within AAVS1, near the region of homology with AAV2 that contains the Rep68/78 binding site. In addition, multiple junctions were found in regions enriched for polypyrimidine tracts on the nicked strand of AAVS1. Recent work from other laboratories suggests that such polypyrimidine tracts may stimulate displacement loop formation (strand invasion) and thus stimulate recombination. We have also identified several junctions consistent with homeologous recombination (recombination between slightly mismatched sequences). For example, two junctions at approximately the same site (just upstream of an 11 base polypyrimidine tract in AAVS1), technically defined as a 2 base microhomology junction and a direct joining, each had 8 of 10 bases in common between AAVS1 and AAV2. We hypothesize that the recombination events were initiated by a strand invasion event, followed by the use of the partially mismatched sequence as a primer for DNA synthesis. This would have been followed by DNA mismatch repair that in some cases corrected the mismatch in favor of AAV2 and in other cases corrected in favor of AAVS1. ? ? Pancreatic islets are important targets for diabetes gene therapies. Although several diabetes model systems involve rats, transduction of rat pancreatic islets by AAV vectors has not been well characterized. Since the AAV packaging signals are highly conserved, it is possible to package a recombinant AAV2 genome, containing a marker gene, in the capsids of multiple AAV types (pseudotyping). We tested the abilities of several pseudotyped AAV vectors, containing the gene encoding green fluorescent protein, to transduce rat islets. We have found vectors packaged into an AAV5 capsid to be suitable for transduction of rat pancreatic islets.
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