Production system for helper-dependent adenovirus

A method to produce helper dependent adenovirus, comprising:

• contacting an isolated mammalian cell that stably expresses a recombinase having at least 80% amino acid sequence identity to SEQ ID NO:1, a recombinant helper adenovirus vector comprising two ITRs and a packaging signal flanked by a pair of site-specific recombinase recognition sites having at least 80% nucleic acid sequence identity to SEQ ID NO:4, and a helper dependent adenovirus vector comprising a genome having two ITRs, a packaging signal, an open reading frame for a gene product of interest and a transcriptional control sequence, wherein if the open reading frame is in reverse orientation relative to the transcriptional control sequence, the open reading frame is flanked by a pair of site-specific recombinase recognition sites other than sites having at least 80% nucleic acid sequence identity to SEQ ID NO:4 or other than sites recognized by a polypeptide having at least 80°/o amino acid sequence identity to SEQ ID NO:1 or if the transcriptional control sequence is in reverse orientation relative to the open reading frame, the transcriptional control sequence is flanked by a pair of site-specific recombinase recognition sites other than sites having at least 80% nucleic acid sequence identity to SEQ ID NO:4 or other than sites recognized by a polypeptide having at least 80% amino acid sequence identity to SEQ ID NO:1;
• and
• collecting helper dependent adenovirus.

A kit comprising two or more of:

• a) an isolated mammalian cell that stably expresses a recombinase having at least 80% amino acid sequence identity to SEQ ID NO:1;
• b) a recombinant helper adenovirus vector comprising at least a portion of an adenovirus genome comprising two adenovirus ITRs and an adenovirus packaging signal flanked by a pair of site-specific recombinase recognition sites having at least 80% nucleic acid sequence identity to SEQ ID NO:4;
• and
• c) a helper dependent adenovirus vector comprising two ITRs, a packaging signal helper dependent adenovirus vector comprising a genome having two adenovirus ITRs, an adenovirus packaging signal flanked by a pair of site-specific recombinase recognition sites other than sites having at least 80% nucleic acid sequence identity to SEQ ID NO:4 or sites other than ones recognized by a polypeptide having at least 80% amino acid sequence identity to SEQ ID NO:1, and one or more restriction enzyme sites for insertion of a desired DNA fragment.