US 6,855,534 B2 · Enhanced system for construction of adenovirus vectors

Abstract

In the present invention, viruses, plasmids or both are constructed which contain viral DNA, at least one head-to-head ITR junction, and recombinase recognition sites positioned such that site-specific recombination between recombinase recognition sites in separate plasmids results in generation of infectious viral DNA at high-efficiency in cotransfected host cells that have been engineered to express a site-specific recombinase. Because of the high-efficiency and specificity of the Cre enzyme, suitably engineered plasmids can be readily recombined to produce infectious virus at high-efficiency in cotransfected 293 cells, without, at the same time, producing wild-type adenovirus, with the attendant problems for removal thereof. Use of recombinases besides Cre and recombinase recognition sites besides lox sites, and use of cells other than 293 cells are also disclosed and enabled, as are kits incorporating the site-specific vector system, as well as compositions and methods for using such compositions as vaccines or in gene therapeutic applications. Enhancement in the efficiency of site-specific recombination is provided by inclusion of a head-to-head ITR junction in each virus, plasmid, or other nucleic acid construct.

Claims20

Dependent claims

A method for making an infectious adenovirus having enhanced efficiency which comprises contacting a cell with or introducing into a cell:

(a) a first nucleic acid sequence encoding adenovirus sequences which, in the absence of intermolecular recombination, are incapable to encode an infectious, replicable or packageable adenovirus;
and
(b) a second nucleic acid sequence encoding adenovirus sequences which, in the absence of adenoviral replication factors provided in trans or intermolecular recombination with said first nucleic acid sequence, are incapable to encode an infectious, replicable or packageable adenovirus;
provided that said first and said second nucleic acid sequences each comprise a head-to-head ITR junction and said first nucleic acid and said second nucleic acid comprise recombinase recognition sites and wherein said first and said second nucleic acids are contacted with a recombinase which recognizes said first nucleic acid and said second nucleic acid recombinase recognition sites;
whereby said first and said second nucleic acids recombine to form said infectious adenovirus.

2 more elements

A recombinant adenovirus vector system comprising:

(a) a first nucleic acid sequence encoding adenovirus sequences which, in the absence of intermolecular recombination, are incapable to encode an infectious, replicable or packageable adenovirus, said first nucleic acid sequence comprising a head-to-head ITR junction and at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase;
and,
(b) a second nucleic acid sequence encoding adenovirus sequences which, in the absence of adenoviral replication factors provided in trans or intermolecular recombination with said first nucleic acid sequence, are incapable to encode an infectious, replicable or packageable adenovirus, said second nucleic acid sequence comprising a head-to-head ITR junction and a site-specific recombinase recognition target site sufficiently identical with said recombinase recognition target site in said first nucleic acid as to be recognized by the same site-specific recombinase which recognizes said site-specific recombinase recognition target site in said first nucleic acid;
wherein said first and said second nucleic acid sequences, in combination and following site-specific intermolecular recombination, result in production of an infectious adenovirus, and wherein a site-specific recombinase which recognizes said site-specific recombinase recognition target sites either (i) is expressed by a cell into which said first and said second nucleic acids are introduced, (ii) is operatively encoded by said first nucleic acid, said second nucleic acid or both, or (iii) is provided in trans through expression from a third nucleic acid, or (iv) is provided in trans as an active protein.

1 more element

Dependencies are inferred from the claim text.

Enhanced system for construction of adenovirus vectors

Life of this patent

Enhanced system for construction of adenovirus vectors

Life of this patent

Abstract

Claims20