US 5,780,280 A · Recombinant adeno-associated virus vectors

Abstract

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.

Claims12

Dependent claims

A method for producing a recombinant adeno-associated virus comprising the steps of:

(a) introducing into a mammalian cell an AAV/EBV plasmid vector, said AAV/EBV plasmid vector comprising an Epstein Barr nuclear antigen gene, an Epstein Barr virus latent origin of replication and an adeno-associated virus transducing vector comprising exogenous genetic material and lacking functional adeno-associated virus rep and cap genes;
thereby producing a recombinant adeno-associated virus producer cell;
(b) growing said producer cell in a cell growth medium;
(c) introducing into said producer cell an adenovirus vector comprising an adeno-associated virus cap gene and an adeno-associated virus rep gene, thereby producing a recombinant adeno-associated virus;
and
(d) isolating said recombinant adeno-associated virus.

3 more elements

Dependencies are inferred from the claim text.

Recombinant adeno-associated virus vectors

Life of this patent

Recombinant adeno-associated virus vectors

Life of this patent

Abstract

Claims12