Adenovirus vectors for gene therapy
AssigneeAdVec, Inc.
InventorsFrank L. Graham, Andrew Bett, Ludvik Prevec, Wael M. Haddara
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Abstract
The invention comprises a series of adenovirus-based vectors having deletions in the E1 and/or E3 regions, and also insertions of pBR322 sequences, which can be used to deliver nucleic acid inserts into host cells, tissues or organisms that then can express the insert. The invention includes the use of the vectors in introducing genes into cells, in making vaccines and in gene therapy.
Claims27
A kit for making an adenovirus vector comprising nucleic acid sequences of a first plasmid and a second plasmid, said kit comprising a first and a second plasmid:
- (a) wherein said first plasmid comprises:
- (i) a modified adenovirus genome, wherein said genome comprises a modification within early region 1 (E1) that comprises a deletion of a packaging signal of the early region 1 which renders said first plasmid incapable of forming viable viral particles in host cells, by eliminating susceptibility of adenoviral sequences encoded by said first plasmid to being encapsidated into a viral particle, but does not affect the ability of the adenovirus genome to replicate, and
- (ii) at least one nucleic acid sequence for (A) encoding antibiotic resistance and (B) replication of said first plasmid in host cells;
- and
- (b) wherein said second plasmid is an E1 shuttle plasmid, which comprises at least one nucleic acid sequence comprising adenovirus genome E1 region sufficient to rescue said first plasmid via recombination of said first plasmid and said second plasmid to produce said adenovirus vector.
A method for introducing and expressing a foreign nucleic acid sequence in a host cell, comprising:
- (a) introducing into a mammalian host cell a first plasmid comprising a modified adenovirus genome having a modification within early region 1 (E1) sufficient to render said first plasmid unable to form viable viral particles in mammalian host cells, said modification eliminating susceptibility of said first plasmid to being encapsidated into a viral particle, said first plasmid further comprising at least one nucleic acid sequence for (A) encoding antibiotic resistance and (B) for replication of said modified adenovirus genome in bacterial host cells, and
- (b) introducing into said host cell recited in (a), an E1 shuttle plasmid that contains a sequence comprising adenovirus genome E1 region, comprising a packaging signal, and a nucleic acid sequence comprising a coding sequence having an open reading frame inserted into the E1 region and comprising sequences that regulate the expression of said open reading frame;
- (c) isolating virus particles wherein said first plasmid has recombined with said shuttle plasmid to yield a recombinant modified viral genome containing elements of said first plasmid plus the packaging signal and the inserted nucleic acid sequence of the E1 shuttle plasmid;
- (d) introducing said recombinant modified viral genome into a host cell;
- and
- (e) expressing the coding sequences contained in said modified recombinant viral genome.
A plasmid that comprises a modified Ad5 genome, selected from the group consisting of:
- pBHGE3, pBHG9, pBHG10, pBHG10B and pBHG11.
A method for making an infectious adenoviral vector by recombining two plasmids, neither of which separately comprises sufficient sequences to produce an infectious adenoviral vector which comprises:
- infecting a cell with:
- (a) a first plasmid comprising adenoviral sequences in which adenoviral packaging sequences have been deleted, such that said first plasmid encodes a viral genome which cannot be packaged;
- and (b) a second plasmid comprising sufficient adenoviral sequences, upon recombination with sequences in said first plasmid, to reconstitute said deleted adenoviral packaging sequences in said first plasmid to produce an infectious adenoviral vector.
Dependencies are inferred from the claim text.