System for production of helper dependent adenovirus vectors based on use of endonucleases

A system for producing a helper dependent adenovirus vector comprising:

• (a) a helper adenovirus nucleic acid sequence comprising a packaging signal, an inserted endonuclease recognition site, a first ITR located at one end of, and a second ITR located at the other end of, the nucleic acid sequence, and a third ITR located between the first and second ITRs, wherein:
• (i) said endonuclease recognition site is for an endonuclease that does not cleave anywhere else in the helper adenovirus genome, and wherein said endonuclease recognition she is disposed at a location in said helper adenovirus nucleic acid sequence 3′ to said packaging signal;
• and (ii) said third ITR is disposed at a location in said helper adenovirus nucleic acid sequence 3′ to said endonuclease recognition site;
• and (b) a helper dependent adenovirus vector comprising a left adenoviral ITR, a right adenoviral ITR, an adenoviral packaging signal and additional nucleic acid sequences, such that upon co-introduction of said helper dependent adenovirus vector into a cell with said helper adenovirus nucleic acid sequence, said helper dependent adenovirus vector is packaged.

A method for making a helper dependent adenovirus vector preparation which comprises:

• (A) making a helper adenovirus having a nucleic acid sequence comprising an endonuclease recognition site inserted 3′ to an adenoviral packaging signal, a first ITR located at one end of, and a second ITR located at the other end of, the helper adenovirus nucleic acid sequence, and a third ITR located between the first and second ITRs and disposed 3′ to said endonuclease recognition site;
• and (B) propagating said helper dependent adenovirus vector in the presence of said helper adenovirus of (A) in a cell permissive for replication of said helper dependent adenovirus vector, wherein during said propagating said helper adenovirus nucleic acid sequence is rendered incapable of being packaged into an infectious adenoviral virion due to deletion of the adenoviral packaging signal by endonuclease-mediated cleavage, alone or in combination with:
• (a) site-directed recombinatorial excision of said packaging signal:
• or (b) a deletion or mutation of adenoviral pIX encoding sequences, whereby a size restricted limitation of genome packaging prevents packaging of a genome which exceeds approximately 35 kb;
• or both (a) and (b).