Methods using cre-lox for production of recombinant adeno-associated viruses
AssigneeThe Trustees of the University of Pennsylvania
InventorsJames M. Wilson, Daniel Phaneuf
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Abstract
Methods for efficient production of recombinant AAV are described. In one aspect, three vectors are introduced into a host cell. A first vector directs expression of cre recombinase, a second vector contains a promoter, a spacer sequence flanked by loxP sites and rep/cap, and a third vector contains a minigene containing a transgene and regulatory sequences flanked by AAV ITRs. In another aspect, the host cell stably or inducibly expresses cre recombinase and two vectors carrying the other elements of the system are introduced into the host cell.
Claims9
A method for production of recombinant adeno-associated virus (AAV) comprising culturing a host cell comprising and capable of expressing
- (a) a cre gene;
- (b) a nucleic acid molecule comprising a spacer sequence flanked by lox sites, and AAV rep and cap genes, wherein the spacer sequence is upstream of the AAV genes;
- and
- (c) a minigene comprising a transgene flanked by AAV inverted terminal repeats (ITRs);
- in the presence of helper virus functions which permit packaging of the minigene into an AAV capsid, whereby a recombinant AAV capable of expressing said transgene is produced.
A method for production of recombinant adeno-associated virus (AAV) comprising:
- (a) providing a host cell expressing cre;
- (b) introducing into said host cell
- a first vector comprising from 5′ to 3′, a selected promoter, a spacer sequence flanked by loxP sites, and AAV rep and cap genes;
- and
- a second vector comprising from 5′ to 3′, a minigene consisting essentially of 5′ AAV inverted terminal repeat (ITR), a selected promoter, a selected transgene, and a 3′ AAV ITR;
- (c) culturing the host cell under conditions which permit expression of the cre recombinase and replication and packaging of a recombinant AAV;
- and
- (d) recovering the recombinant AAV capable of expressing the product of the transgene.
Dependencies are inferred from the claim text.