Claims for Patent: 6,274,354

Summary for Patent: 6,274,354

Title:	Methods using cre-lox for production of recombinant adeno-associated viruses
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.
Inventor(s):	Wilson; James M. (Gladwyne, PA), Phaneuf; Daniel (Philadelphia, PA)
Assignee:	The Trustees of the University of Pennsylvania (Philadelphia, PA)
Application Number:	09/242,743
Patent Claims:	1. 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. 2. The method according to claim 1 further comprising: (a) introducing into a host cell (i) a first vector comprising a cre gene under control of sequences which permit expression of cre recombinase; (ii) a second vector comprising from 5' to 3', a selected promoter, a spacer sequence flanked by loxP sites, and AAV rep and AAV cap genes; (iii) a third vector comprising a minigene consisting essentially of, from 5' to 3', a 5' AAV ITR, a promoter, a transgene and 3' AAV ITR; (b) culturing the host cell under conditions which permit expression of the cre recombinase; and (c) recovering recombinant AAV capable of expressing the product of said transgene. 3. The method according to claim 1 wherein the spacer sequence is selected from the group consisting of: (a) a 1300 bp fragment containing translational start and stop sequences; (b) a 1600 bp fragment containing the green fluorescent protein (GFP) cDNA, an intron and a polyadenylation signal; and (c) a 1000 bp fragment containing the neomycin coding sequence and a polyadenylation signal. 4. The method according to claim 2 wherein at least one of said vectors is a recombinant adenovirus and the host cell is a 293 cell. 5. The method according to claim 2 wherein the first vector is a recombinant adenovirus and the sequences which permit expression comprise a cytomegalovirus promoter, the vector further comprising a nuclear localization signal operably linked to the cre gene. 6. The method according to claim 2 wherein the second vector is a recombinant adenovirus and comprises an AAV P5 promoter. 7. 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. 8. The method according to claim 7 wherein the first and second vectors are recombinant adenoviruses. 9. The method according to claim 8 wherein the spacer sequence is selected from the group consisting of: (a) a 1300 bp fragment containing translational start and stop sequences; (b) a 1600 bp fragment containing the GFP cDNA, an intron and a polyadenylation signal; and (c) a 1000 bp fragment containing the neomycin coding sequence and a polyadenylation signal.

Details for Patent 6,274,354

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2016-09-06
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2016-09-06
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2016-09-06