Helper dependent adenovirus vectors based on integrase family site-specific recombinases
AssigneeAdVec, Inc.
InventorsFrank L. Graham, Michael A. Rudnicki, Martina Anton
Life of this patent
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Abstract
This invention provides helper-dependent adenovirus cloning vectors and helper adenoviruses, and methods for making and Using such preparations, wherein the helper adenoviruses contain recombinase target sites that are useful in reducing the level of contamination of helper virus in helper-dependent adenovirus vector preparations.
Claims18
An adenovirus vector system for expressing foreign DNA sequences, comprising:
- a. a helper adenovirus comprising a modified early region 1 (E1) wherein adenoviral packaging signals contained within said E1 region are flanked on both sides by site-specific recombinase target recognition sites other than lox sites;
- b. an integrase family recombinase other than Cre which catalyzes site-specific recombination between said site-specific recombinase target recognition sites;
- and,
- c. a helper-dependent adenovirus vector, comprising:
- i. a deletion of up to approximately 35,000 bp of adenoviral sequences but retaining sufficient right and left ITR sequences to support viral replication and packaging;
- and
- ii. foreign nucleic acid sequences of up to about 37,000 bp.
A plasmid for making a helper adenovirus comprising a modified circular adenoviral genome having a modified early region 1 (E1) wherein adenoviral packaging signals contained within said E1 region are flanked on both sides by first site-specific recombinase target recognition sites other than lox sites, said sites corresponding to said first site-specific recombinase target recognition sites of a integrase family recombinase other than Cre.
A method for making a packaged helper-dependent adenovirus vector comprising co-infecting a cell with:
- a. at least one helper adenovirus comprising an E1 region wherein packaging signals contained within said E1 region are flanked on both sides by site-specific integrase family recombinase target recognition sites other than lox sites;
- and
- b. at least one helper-dependent adenovirus vector;
- wherein said cell supports replication of said at least one helper adenovirus and wherein said cell expresses a site-specific integrase family recombinase other than Cre, such that upon incubation of the co-infected cell, said recombinase catalyzes the removal of the packaging signals from said at least one helper adenovirus such that said least one helper adenovirus without packaging signals does not package, wherein said at least one helper adenovirus replicates, wherein said at least one helper adenovirus supports replication of said at least one helper-dependent adenovirus vector, and wherein said at least one helper-dependent adenovirus vector is packaged into adenovirus virions.
A method for excising specific nucleic acid sequences from a helper adenovirus, which helper adenovirus supports replication of a helper-dependent adenoviral vector co-infected in a cell with said helper adenovirus, which comprises constructing said helper adenovirus such that said specific nucleic acid sequences are flanked on both sides wit site-specific integrase family recombinase target recognition sites other than lox, and contacting said helper adenovirus with a integrase family recombinase other tan Cre to induce excision of said specific nucleic acid sequences.
Dependencies are inferred from the claim text.