Novel adenoviral vector and methods for making and using the same

A method of preparing a recombinant adenoviral genome having an insertion nucleic acid located in an E gene region of said genome, said method comprising:

• (a) providing a first plasmid comprising an adenoviral genome having an E gene deletion region characterized by a deletion of at least one of an E1, E3 and E4 gene, where said first plasmid is further characterized by having a second restriction endonuclease site flanked by first and third restriction endonuclease sites, wherein each of said first, second and third restriction endoncuclease sites are:
• (i) different, (ii) are selected from the group consisting of I-CeuI, PI-SceI and SwaI and (iii) are located in said E gene deletion region;
• (b) providing a second plasmid comprising an insertion nucleic acid flanked by said first and third restriction endonuclease sites;
• (c) contacting each of said first and second vectors with said first and third restriction endonucleases to produce first and second cleavage products, wherein said first cleavage product that includes said adenoviral genome and said second cleavage product comprises said insertion nucleic acid;
• (d) combining said first and second cleavage products under ligation conditions sufficient to produce a ligation product composition comprising plasmid comprising said recombinant adenoviral genome having an insertion nucleic acid located in an E gene region of said genome;
• and
• (e) contacting said ligation product composition with said second restriction endonuclease;
• whereby said recombinant adenoviral genome having an insertion nucleic acid located in an E gene region of said genome is produced.

An adenoviral genome comprising:

• (a) an E gene deletion region;
• (b) first and second restriction endonuclease sites, wherein said first and second restriction endoncuclease sites are:
• (i) different, (ii) do not occur in the corresponding wild type adenoviral genome and (iii) are located in said E gene deletion region;
• and
• (c) an insertion nucleic acid positioned between said first and second restriction endonuclease sites.

A method of producing a recombinant adenovirus having a genome having an insertion nucleic acid located in an E gene region of said genome, said method comprising:

• (a) providing a first vector comprising an adenoviral genome having an E gene deletion, where said first vector is further characterized by having a second restriction endonuclease site flanked by first and third restriction endonuclease sites, wherein each of said first, second and third restriction endoncuclease sites are:
• (i) different, (ii) do not occur in the corresponding wild type adenoviral genome and (iii) are located in said E gene deletion region;
• (b) providing a second vector comprising an insertion nucleic acid flanked by said first and third restriction endonuclease sites;
• (c) contacting each of said first and second vectors with said first and third restriction endonucleases to produce first and second cleavage products, wherein said first cleavage product comprises said adenoviral genome and said second cleavage product comprises said insertion nucleic acid;
• (d) combining said first and second cleavage products under ligation conditions sufficient to produce a ligation product composition comprising a ligation product that includes said recombinant adenoviral genome having an insertion nucleic acid located in an E gene region of said genome;
• (e) transforming a cell that is transcomplementing for any inactivated E genes of said E gene region with said ligation product;
• and
• (f) maintaining said transformed cell under conditions sufficient for said virus to be produced;
• whereby said recombinant adenovirus having a genome having an insertion nucleic acid located in an E gene region of said genome is produced.

A kit for use in preparing a recombinant adenovirus, said kit comprising:

• (a) a first vector comprising an adenoviral genome comprising an E gene deletion region and first, second and third restriction endonuclease sites, wherein each of said first, second and third restriction endoncuclease sites are:
• (i) different, (ii) do not occur in the corresponding wild type adenoviral genome and (iii) are located in said E gene deletion region;
• and
• (b) first, second and third restriction endonucleases that correspond to said first, second and third restriction endonuclease sites.