High efficiency helper system for AAV vector production

Details High efficiency helper system for AAV vector production High efficiency helper system for AAV vector production

1995-08-03

1997-04-22

Guzo, David

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

4353201, 435366, 435367, 435369, 435348, 435 691, 536 2372, C12N 510, C12N 1563

Patent

active

056228562

ABSTRACT:
Novel nucleic acid molecules are provided having adeno-associated virus (AAV) coding regions that are capable of expressing necessary AAV functions to complement an AAV vector in the production of recombinant AAV (rAAV) virions. The molecules feature a nucleotide sequence that is substantially homologous to an AAV p5 promoter region, wherein the p5 promoter region is situated in the molecules in a site that is other than its natural position relative to the AAV rep coding region in the wild-type AAV genome. AAV helper function constructs are also provided, comprising the instant nucleic acid molecules embodied in a replicon that is capable of being transcribed and translated to express complementing AAV helper functions in a suitable host cell. Novel AAV packaging cells and AAV producer cells are provided, which contain the AAV helper constructs of the invention, and methods of producing enhanced levels of rAAV virions using the AAV helper constructs of the invention are also provided. Methods are also provided for producing rAAV virions without the concomitant production of significant levels of wild-type AAV.

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Jayaram, "Two-micrometer Circle Site-specific Recombination: The Minimal Substrate and the Possible Role of Flanking Sequences," Proc. Natl. Acad. Sci. USA (1985) 82:5875-5879.
McCarty et al., "Sequences Required for Coordinate Induction of Adeno-Associated Virus p19 and p40 Promoters by Rep Protein," J. Virol. (1991) 65(6):2936-2945.
O'Gorman et al., "Recombinase-mediated Gene Activation and Site-specific Integration in Mammalian Cells," Science (1991) 251:1351-1355.
Samulski et al., "Helper-free Stocks of Recombinant Adeno-associated Viruses: Normal Integration Does Not Require Viral Gene Expression," J. Virol. (1989) 63(9):3822-3828.

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