Vectors, cells and methods for the production of deleterious viral eukaryotic gene transfer vectors

Abstract

The present invention provides a method of in vitro propagation of a viral eukaryotic gene transfer vector comprising a deleterious, i.e., a cytostatic, cytotoxic, or apoptotic, gene in a eukaryotic, e.g., a mammalian, host-production cell, comprising a blocking gene. The blocking gene inhibits the adverse effects of the deleterious gene on the eukaryotic host-production cell. Vectors and cells useful in the context of the present inventive method are also provided.

What is claimed is:  
     
         1 . A eukaryotic cell comprising a blocking gene and a viral eukaryotic gene transfer vector, which is derived from a deleterious virus or comprises a deleterious gene.  
     
     
         2 . The eukaryotic cell of  claim 1 , wherein said blocking gene is integrated in the nuclear genome of the eukaryotic cell.  
     
     
         3  The eukaryotic cell of  claim 1 , wherein said blocking gene is on a plasmid or a vector, wherein said vector is other than the viral eukaryotic gene transfer vector.  
     
     
         4 . The eukaryotic cell of  claim 1 , wherein said blocking gene encodes a caspase inhibitor, crmA, baculoviral p35 protein, an IAP gene product or a FLIP gene product.  
     
     
         5 . The eukaryotic cell of  claim 1 , wherein said viral eukaryotic gene transfer vector is an adenoviral vector.  
     
     
         6 . The eukaryotic cell of  claim 1 , wherein said eukaryotic cell is a mammalian cell.  
     
     
         7 . The eukaryotic cell of  claim 1 , wherein said eukaryotic cell is HEK 293 or AE25.  
     
     
         8 . The eukaryotic cell of  claim 1 , which comprises at least one gene that complements in trans for at least one essential gene function of said viral eukaryotic gene transfer vector.  
     
     
         9 . The eukaryotic cell of  claim 4 , wherein said viral eukaryotic gene transfer vector is an adenoviral vector.  
     
     
         10 . A mammalian cell for the production of a eukaryotic gene transfer vector that comprises a gene encoding and expressing an inducible nitric oxide synthase, wherein said cell comprises a dominant negative calmodulin variant and is permissive for the replication of said eukaryotic gene transfer vector.  
     
     
         11 . A mammalian cell for the production of a eukaryotic gene transfer vector that comprises a gene encoding and expressing an inducible nitric oxide synthase, wherein said mammalian cell comprises a gene that encodes and overexpresses a polypeptide that binds to biopterin and calmodulin, and wherein said polypeptide prevents the activation of human inducible nitric oxide synthase.  
     
     
         12 . The mammalian cell of  claim 11 , wherein said polypeptide is a dominant negative inducible nitric oxide synthase.  
     
     
         13 . The mammalian cell of  claim 11 , wherein said eukaryotic gene transfer vector is an adenoviral vector that is deficient in at least one essential gene function of the E1 region of an adenovirus.