Applied Immunesciences, Inc.

Rhone-poulenc Rorer Pharmaceuticals Inc.

Maureen A McNally

Jane S Lebkowski

Thomas B Okarma

Simplified methods to produce recombinant adeno-associated virus (rAAV) vectors are described. The methods involve the use of chimeric plasmids which incorporate the Epstein Barr nuclear antigen (EBNA) gene, the latent origin of replication of Epstein Barr virus (oriP), and a rAAV genome. The chimeric plasmids themselves are also a part of the present invention. These plasmids are maintained as multicopy extra-chromosomal elements in cells, such as human 293 cells. Permanent cell lines carrying these EBV/AAV plasmids are induced to produce large amounts of rAAV upon addition of wild-type, adeno-associated virus helper functions. Vectors produced in this manner are capable of transducing exogenous genes into other human cell lines and exhibit the attributes of viral elements produced by conventional methods.

Recombinant adeno-associated virus vectors

Recombinant adeno-associated virus vector packaging cells and methods

Method for producing recombinant adeno-associated virus vectors

Simplified methods to produce recombinant adeno-associated virus (rAAV) vectors are described. The AAV rep and cap genes are combined in a single recombinant adenovirus vector, i.e., a rep/cap adenovirus vector which combines in a single vector all complementing functions required for rAAV production. The rep/cap vectors of the present invention comprise: genes encoding helper functions, preferably from adenovirus, sufficient to permit AAV replication; and AAV cap and rep genes. In adenovirus rep/cap vectors the rep and cap genes may, for example, replace the adenovirus E3 gene or the adenovirus E1a and E1b genes.

Simplified methods to produce recombinant adeno-associated virus (rAAV) vectors are described. The methods involve the use of chimeric plasmids which incorporate the Epstein Barr nuclear antigen (EBNA) gene, the latent origin of replication of Epstein Barr virus (oriP), and a rAAV genome. The chimeric plasmids themselves are also a part of the present invention. These plasmids are maintained as multicopy extra-chromosomal elements in cells, such as human 293 cells. Permanent cell lines carrying these EBV/AAV plasmids are induced to produce large amounts of rAAV upon addition of wild-type, adeno-associated virus helper functions. Vectors produced in this manner are capable of transducing exogenous genes into other human cell lines and exhibit the attributes of vital elements produced by conventional methods.

Production of recombinant adeno-associated virus vectors

A simplified method to produce recombinant adeno-associated virus (AAV) vectors is described. The procedure involves the use of chimeric plasmids which incorporate the Epstein Barr nuclear antigen (EBNA) gene, the latent origin of replication of Epstein Barr Virus (oriP), and a recombinant AAV genome. The chimeric plasmids themselves are also a part of the present invention. These EBV/AAV plasmids are maintained as multicopy extra-chromosomal elements in cells, such as human 293 cells. Permanent cell lines carrying these EBV/AAV plasmids are induced to produce large amounts of recombinant AAV virus upon addition of wild-type, adeno-associated virus helper functions. Recombinant AAV vectors produced in this manner are capable of transducing exogenous genes into other human cell lines and exhibit all of the attributes of viral elements produced by conventional methods.

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Palo Alto, CA, United States of America

Maureen A McNally