Adenovirus-based methods, and cells, useful for the expression of nucleic acid sequences

Abstract

This invention provides helper-dependent adenovirus cloning vectors and helper adenoviruses, and methods for making and using such preparations, wherein the helper adenoviruses contain recombinase target sites that are useful in reducing the level of contamination of helper virus in helper-dependent adenovirus vector preparations.

What is claimed is:  
     
         1 . A method for expressing nucleic acid sequences which comprises introducing into a host a helper-dependent adenovirus vector comprising: 
 (i) a deletion of up to approximately 35,000 bp of the adenovirus genome but retaining sufficient left and right sequences including left and right ITRs and a packaging signal sufficient to support viral DNA replication and packaging; and    (ii) a fragment or fragments of foreign DNA sequences of up to about 35,000 bp.    
     
     
         2 . The method according to    claim 1    wherein said helper-dependent adenovirus vector is produced by co-infecting an isolated cell with: 
 (a) a helper adenovirus comprising a packaging signal flanked on both sides by 1ox P recombinase target sites; and  
 (b) a helper-dependent adenovirus vector comprising: 
 (i) a deletion of up to approximately 35,000 bp of the adenovirus genome but retaining an adenoviral packaging signal and sufficient left and right ITR sequences to support viral replication and packaging; and  
 (ii) a fragment or fragments of foreign DNA sequences of up to about 35,000 bp;  
 wherein said isolated cell supports replication of the helper adenovirus, and wherein said isolated cell additionally expresses Cre recombinase such that said Cre recombinase catalyzes the removal of the packaging signals from the helper adenovirus such that it replicates but does not package, and wherein the helper virus supports replication of the helper-dependent adenovirus vector such that the helper-dependent adenovirus vector is packaged into adenovirus virions which are recovered for introduction into a host.  
 
 
     
     
         3 . A cell expressing Cre recombinase and adenovirus E1.  
     
     
         4 . A human cell expressing Cre recombinase.