Claims
- 1. A recombinant host cell containing
(a) a first nucleic acid molecule comprising a parvovirus P5 promoter, an AAV rep gene sequence, an AAV cap gene sequence, and a spacer of sufficient size to reduce expression of the rep78 and rep68 gene products relative to other rep gene products, wherein said spacer is heterologous to said promoter and is interposed between said promoter and the start site of the rep gene, and (b) a second nucleic acid molecule comprising a minigene comprising a transgene flanked by AAV inverted terminal repeats (ITRs) and under the control of regulatory sequences directing expression thereof in a host cell; and (c) helper functions essential to the replication and packaging of rAAV.
- 2. The cell according to claim 1 wherein said spacer sequence is between about 10 bp to 10 kb in length.
- 3. The cell according to claim 1 wherein said spacer sequence is between 100 bp to 3.8 kb in length.
- 4. The cell according to claim 1 wherein said spacer sequence is about 500 bp in length.
- 5. The cell according to claim 1 wherein said spacer sequence is a random sequence of nucleotides.
- 6. The cell according to claim 1 wherein said spacer sequence encodes a gene product.
- 7. The cell according to claim 1 wherein said first nucleic acid molecule is selected from the group consisting of a plasmid, a recombinant virus, and an episome.
- 8. The cell according to claim 1 wherein said first nucleic acid molecule is integrated into the chromosome of said cell.
- 9. The cell according to claim 1 wherein said parvovirus P5 promoter is from a strain of AAV selected from the group consisting of AAV type 1, AAV type 3, AAV type HI.
- 10. The cell according to claim 1 wherein said rep gene sequences are from AAV type 1 or AAV type 5
- 11. The cell according to claim 1 wherein said second nucleic acid molecule is selected from the group consisting of a plasmid, a recombinant virus, and an episome.
- 12. The cell according to claim 1 wherein said second nucleic acid molecule is integrated into the chromosome of said cell.
- 13. The cell according to claim 1 wherein said helper functions are provided by a third nucleic acid molecule.
- 14. The cell according to claim 13 wherein said third nucleic acid molecule is selected from the group consisting of a plasmid, a recombinant or wild-type virus, and an episome.
- 15. The cell according to claim 13 wherein said third nucleic acid molecule is integrated into the chromosome of said cell.
- 16. The cell according to claim 1 which is derived from a HEK 293 cell.
- 17. A method for producing recombinant adeno-associated virus (AAV), said method comprising the steps of
(a) culturing a recombinant host cell containing
(i) a first nucleic acid molecule comprising a parvovirus P5 promoter, an AAV rep gene sequence, an AAV cap gene sequence, and a spacer of sufficient size to reduce expression of the rep78 and rep68 gene products relative to other rep gene products, wherein said spacer is heterologous to said promoter and is interposed between said promoter and the start site of the rep gene, and (ii) a second nucleic acid molecule comprising a minigene comprising a transgene flanked by AAV inverted terminal repeats (ITRs) and under the control of regulatory sequences directing expression thereof in a host cell, and (iii) helper functions essential to the replication and packaging of rAAV; and (b) isolating from said cell or cell culture, a recombinant AAV capable of expressing said transgene.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. patent application Ser. No. 09/528,470, filed Mar. 17, 2000, which is a continuation of International Patent Application No. PCT/US98/19479, filed on Sep. 18, 1998, which claims the benefit of the priority of U.S. Patent Application No. 60/059,330, filed on Sep. 19, 1997.
Government Interests
[0002] This invention was made with financial assistance from the National Institutes of Health Grant No. NIAMS P01AR/MS43648. The United States government has certain rights in this invention.
Provisional Applications (1)
|
Number |
Date |
Country |
|
60059330 |
Sep 1997 |
US |
Continuations (2)
|
Number |
Date |
Country |
Parent |
09528470 |
Mar 2000 |
US |
Child |
10255527 |
Sep 2002 |
US |
Parent |
PCT/US98/19479 |
Sep 1998 |
US |
Child |
09528470 |
Mar 2000 |
US |