Helper dependent adenovirus vectors based on integrase family site-specific recombinases
AssigneeAdVec, Inc.
InventorsFrank Graham, Michael A. Rudnicki, Martina Anton
Life of this patent
Estimating the term…
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.
Claims17
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;
- (b) a recombinase 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 left and right ITR sequences to support viral replication and packaging;
- and
- (ii) a fragment or fragments of foreign nucleic acid sequences of up to about 35,000 bp.
A plasmid for making 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, said plasmid comprising a modified circular adenovirus genome wherein sequences from region E1 of the viral genome are replaced by packaging sequences from the viral genome flanked on either side by site-specific recombinase target recognition sites.
A method for making a non-packageable helper adenovirus genome comprising infecting a cell with an adenovirus comprising an E1 region wherein packaging signals contained within said E1 region are flanked on both sides by site-specific recombinase recognition sites, wherein said cell expresses a site-specific recombinase which catalyzes excision of nucleic acid sequences flanked by said site-specific recombinase recognition sites such that packaging signals contained within said E1 region are excised, whereby the resulting helper adenovirus genome does not package.
A method for making a packaged helper-dependent adenovirus vector comprising co-infecting a cell with:
- (a) a helper adenovirus comprising an E1 region wherein packaging signals contained within said E1 region are flanked on both sides by site-specific recombinase recognition sites;
- and
- (b) a helper-dependent adenovirus vector;
- wherein said cell supports replication of said helper adenovirus and wherein said cell expresses a site-specific recombinase, such that upon incubation of the co-infected cell, said recombinase catalyzes the removal of the packaging signals from the helper adenovirus such that it replicates but does not package, and wherein said helper adenovirus supports replication of said helper-dependent adenovirus vector, and wherein said 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 with recombinase recognition sites, and contacting said helper adenovirus with said recombinase to induce excision of said specific nucleic acid sequences.
A method for expressing nucleic acid sequences which comprises:
- introducing into a host a helper-dependent adenovirus Vector Comprising *1 deletion up to approximately 35,000 bp of the adenovirus genome but retaining sufficient left and right sequences including left and right ITRs and a packaging signal sufficient to support viral DNA replication and packaging, and a fragment or fragments of foreign DNA sequences of up to about 35,000 bp;
- wherein said helper-dependent adenovirus vector is produced by co-infecting an isolated cell with (a) a helper adenovirus comprising a packaging signal flanked on both sides by recombinase target sites and (b) said helper-dependent adenovirus vector wherein said isolated cell supports replication of said helper adenovirus, and wherein said isolated cell additionally expresses a recombinase such that said recombinase catalyzes the removal of the packaging signals from the helper adenovirus such that it replicates but does not package, and wherein the helper adenovirus supports replication of the helper-dependent adenovirus vector such that the helper-dependent adenovirus vector is packaged into adenovirus virions which are recovered for introduction into a host, whereby said method of expressing nucleic acid sequences results.
Dependencies are inferred from the claim text.