GENERATION OF RECOMBINANT ADENO-ASSOCIATED VIRUS TYPE 3 BASED VECTORS

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Adeno-associated viruses (AAVs) are small, non-enveloped, single-stranded DNA viruses belonging to the Porvoviridae family. AAVs have been isolated from a variety of different species, where they appear to be non-pathogenic. To date, five primate AAVs have been described, distinguished serologically by their antigenically distinct capsid proteins. Isolates of AAV-2, AAV-3 and AAV-5 have been obtained directly from human clinical specimens, and man appears to be the natural host. Until recently only AAV-2 had been characterized at the genomic level. Much of the current interest in AAVs stems from their potential use as a vector for gene therapy, with virtually all studies using AAV-2. Although AAV-2 has a broad tissue host range and can transduce a wide variety of tissue types, some cells, specifically erythroid cells have been shown to be non permissive. We have previously cloned and characterized the full-length genome of AAV-3 and produced an infectious clone. In this study, we inserted either the genes for green fluorescence protein (GFP) or beta-galactosidase into AAV-2 and AAV-3 based plasmids, and produced recombinant virus. Recombinant virus was then used to transduce hematopoietic cells, and the transduction efficiencies compared. Recombinant AAV-3 virus successfully transduced erythroid and megakaryoblastoid cells, in contrast to rAAV-2. In contrast rAAV-2 appeared better at transducing lymphocytes. These results suggest not only that there are different cellular receptors for AAV-2 and AAV-3, but that rAAV-3 vectors may be better for transduction of some hematopoietic cell types. We could also show that AAV-3 vector transduced NT2 cells in some process of differentiation, indicating that AAV-3 vectors can be used to modulate neuronal differentiation. AAV vector-mediated gene expression was persisted in the rat striatum at 7 months after injection of AAV-TH vectors.