Recombinase-based system for combustion of adenovirus vectors

A recombinant adenovirus vector system comprising:

• (a) either (i) a first nucleic acid sequence encoding adenovirus sequences which, in the absence of intermolecular recombination, are insufficient to encode an infectious, replicable or packageable adenovirus, said first nucleic acid sequence comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase or (ii) a first nucleic acid sequence encoding adenovirus sequences which are sufficient to encode an infectious, replicable or packageable adenovirus and comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase, wherein contact of said first nucleic acid with said site-specific recombinase results in excision of sequences front said first nucleic acid sequence such that, in the absence of intermolecular recombination, said adenovirus of (ii) is rendered replication or packaging defective;
• (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 insufficient to encode an infectious, replicable or packageable adenovirus, said second nucleic acid sequence comprising at least one 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;
• whereby said first and said second nucleic acid sequences, in combination and following site-specific intermolecular recombination, result in production of an infectious adenovirus, wherein a site-specific recombinase which recognizes said site-specific recombinase recognition target sites is either (i) expressed by a cell into which said first and said second nucleic acids are introduced, (ii) 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 is provided in trans as an active protein, and with the proviso that an adenovirus encoded terminal protein is not linked to the first or to the said second nucleic acid sequence.

A kit for construction of a recombinant adenovirus vector comprising:

• (a) either (i) a first nucleic acid sequence encoding adenovirus sequences which, in the absence of intermolecular recombination, are insufficient to encode an infectious, replicable or packageable adenovirus, said first nucleic acid sequence comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase or (ii) a first nucleic acid sequence encoding adenovirus sequences which are sufficient to encode an infectious, replicable or packageable adenovirus and comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase, wherein contact of said first nucleic acid with said site-specific recombinase results in excision of sequences from said first nucleic acid sequence such that, in the absence of intermolecular recombination, said adenovirus of (ii) is rendered replication or packaging defective;
• (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 insufficient to encode an infectious, replicable or packageable adenovirus, said second nucleic acid sequence comprising at least one 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;
• and
• (c) a cell wherein, when said first nucleic acid sequence and said second nucleic acid sequence are cotransfected and recombined through the action of a recombinase which recognizes said recombinase recognition sites, said recombinant adenovirus vector, packaged and infectious, is constructed, with the proviso that an adenovirus encoded terminal protein is not linked to the first or to the said second nucleic acid sequence.

A recombinant adenovirus vector system comprising:

• (a) either (i) a first nucleic acid sequence encoding adenovirus sequences which, in the absence of intermolecular recombination, are insufficient to encode an infectious, replicable or packageable adenovirus, said first nucleic acid sequence comprising at least one site-specific recombinase recognition target site which is recognized by a site-specific recombinase or (ii) a first nucleic acid sequence encoding adenovirus sequences which are sufficient to encode an infectious, replicable or packageable adenovirus, said first nucleic acid sequence comprising (A) at least one restriction enzyme recognition site such that upon restriction of said nucleic acid with a restriction enzyme which recognizes said site, a site-specific recombinase recognition target site remains intact, but said adenovirus of (ii) is rendered replication or packaging deficient, or (B) wherein said nucleic acid comprises at least one site-specific recombinase recognition site which is recognized by a site-specific recombinase, wherein contact of said first nucleic acid with said site-specific recombinase results in excision of sequences from said first nucleic acid sequence such that, in the absence of intermolecular recombination, said adenovirus of (ii) is rendered replication or packaging defective;
• (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 insufficient to encode an infectious, replicable or packageable adenovirus, said second nucleic acid sequence comprising at least one 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;
• with the proviso that an adenovirus encoded terminal protein is not linked to the first or to the said second nucleic acid sequence, 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 is either (i) expressed by a cell into which said first and said second nucleic acids are introduced, (ii) 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 is provided in trans as an active protein.