Production of adenovirus vectors with reduced levels of replication competent adenovirus contamination

A recombinant adenoviral nucleic acid sequence comprising:

• a. a left-end inverted terminal repeat (ITR) sequence;
• b. a packaging signal inverted with respect to its normal orientation along the DNA sequence;
• c. a deletion in early region 1 (E1) to eliminate expression of E1A and E1B genes;
• d. an insertion of foreign DNA in E1, in E3, or in both E1 and E3, wherein foreign DNA inserted into E3 comprises an expression cassette;
• and
• e. a right-end inverted terminal repeat (ITR) sequence.

A system for propagation of recombinant adenoviral vector comprising

• a. a culture vessel comprising culture media into which are added:
• b. a recombinant adenoviral vector comprising a nucleic acid sequence comprising:
• i. a left-end inverted terminal repeat (ITR) sequence;
• ii. a deletion in early region 1 (E1) to eliminate expression of E1A and E1B genes;
• iii. an insertion of an expression cassette in E1, in E3, or in both E1 and E3;
• iv. a packaging signal inverted with respect to a normal orientation along the nucleic acid sequence;
• and
• v. a right-end inverted terminal repeat (ITR) sequence, and
• c. a cell comprising an adenoviral DNA sequence expressing E1A and E1B genes.

A method for producing a recombinant adenoviral vector comprising a gene of interest, said method not producing a vector having a functional E1 region, said method comprising:

• a. providing a complementing cell harboring a first nucleic acid comprising adenoviral nucleic acid sequences encoding functional E1A protein and functional E1B protein;
• b. transferring a second nucleic acid into said complementing cell, said second nucleic acid comprising a recombinant adenoviral nucleic acid sequence comprising functional Inverted Terminal Repeats (ITRs) at or near both of its termini, and a functional packaging signal inverted with respect to a normal orientation along the adenoviral DNA sequence with respect to the nearest of the ITRs, the second nucleic acid further comprising a foreign DNA sequence comprising the gene of interest, and all sequences required for replication of said second nucleic acid which are not provided by said complementing cell;
• c. culturing said complementing cell;
• and
• d. harvesting the recombinant adenoviral vector produced from the complementing cell.