ADENOASSOCIATED VIRUS VECTORS CAN BE EFFICIENTLY PRODUCED WITHOUT HELPER VIRUS

The purpose of this work was to develop an efficient method for the pr oduction of adeno-associated virus (AAV) vectors in the absence of hel per virus. The adenovirus regions that mediate AAV vector replication were identified and assembled into a helper plasmid. These included th e VA, E2A and E4 regions. When this helper plasmid was cotransfected i nto 293 cells, along with plasmids encoding the AAV vector, and rep an d cap genes, AAV vector was produced as efficiently as when using aden ovirus infection as a source of help. CMV-driven constructs expressing the E4orf6 and the 72-M-r, E2A proteins were able to functionally rep lace the E4 and E2A regions, respectively. Therefore the minimum set o f genes required to produce AAV helper activity equivalent to that pro vided by adenovirus infection consists of, or is a subset of, the foll owing genes: the E4orf6 gene, the 72-M-r, E2A protein gene, the VA RNA genes and the E1 region. AAV vector preparations made with adenovirus and by the helper virus-free method were essentially indistinguishabl e with respect to particle density, particle to infectivity ratio, cap simer ratio and efficiency of muscle transduction in vivo. Only AAV ve ctor preparations made by the helper virus-free method were not reacti ve with anti-adenovirus sera.