Production of viral vectors

Abstract

The present invention relates to methods and compositions for the production of viral vectors. In particular, the present invention provides methods and compositions for faster, higher titer and higher purity production of viral vectors (e.g. adenoviral vectors). In some embodiments, the present invention provides gutted and helper viruses with identical or similar termini. In other embodiments, the present invention provides terminal protein linked adenoviral DNA. In certain embodiments, the present invention provides template extended adenoviral DNA.

We claim: 
     
         1 . A method for producing helper-dependent viral vectors comprising:
 a) providing;
 helper-dependent viral DNA comprising a first origin of replication, 
 helper viral DNA comprising a second origin of replication, wherein said second origin of replication has similar activity level in a replication assay as said first origin of replication, and 
 target cells; and 
   b) transfecting said target cells with said helper-dependent viral DNA and said helper viral DNA under conditions such that helper-dependent viral vectors are produced.   
     
     
         2 . The method of  claim 1 , wherein said helper-dependent viral DNA comprises adenoviral DNA. 
     
     
         3 . The method of  claim 1 , wherein said helper-dependent viral DNA comprises a heterologous gene sequence. 
     
     
         4 . The method of  claim 1 , wherein said first origin of replication and second origin of replication have nucleic acid sequences that are substantially similar. 
     
     
         5 . The method of  claim 1 , wherein said helper viral DNA is adenoviral helper viral DNA. 
     
     
         6 . The method of  claim 1 , wherein said first origin of replication and said second origin of replication are not linked to terminal protein or any terminal protein remnant. 
     
     
         7 . The method of  claim 1 , wherein said helper viral DNA comprises a crippling sequence. 
     
     
         8 . The method of  claim 1 , wherein said helper viral DNA comprises recognition sites for site-specific recombinases. 
     
     
         9 . The method of  claim 1 , wherein said target cells express adenoviral DNA polymerase and preterminal protein. 
     
     
         10 . The method of  claim 8 , further comprising; providing iv) a vector encoding a site-specific recombinase, and step c) transfecting said target cells with said vector. 
     
     
         11 . The method of  claim 10 , further comprising recovering said helper-dependent vectors. 
     
     
         12 . The method of  claim 10 , wherein said recovering yields a helper-dependent titer of at least 20 fold increase compared to transfection/infection protocols in cells expressing adenoviral DNA polymerase and preterminal protein. 
     
     
         13 . A composition comprising said helper-dependent viral vectors produced by the method of  claim 1 . 
     
     
         14 . A host cell comprising;
 a) helper-dependent viral DNA comprising a first origin of replication, and b) helper viral DNA comprising a second origin of replication, wherein said second origin of replication has a similar activity level in a replication assay as said first origin of replication.   
     
     
         15 . A method for producing helper-dependent viral vectors comprising:
 a) providing;
 helper-dependent viral DNA comprising an origin of replication linked to a replication-promoting agent, and 
 target cells; and 
   b) transfecting said target cells with said helper-dependent viral DNA under conditions such that helper-dependent viral vectors are produced.   
     
     
         16 . The method of  claim 15 , further comprising; providing iii) helper viral DNA, and step c) transfecting said target cells with said helper viral DNA. 
     
     
         17 . The method of  claim 15 , wherein said replication-promoting agent is selected from Ad2 preterminal protein, Ad2 terminal protein, Ad5 preterminal protein, and Ad5 terminal protein. 
     
     
         18 . The method of  claim 15 , wherein said helper-dependent viral DNA comprises adenoviral DNA. 
     
     
         19 . The method of  claim 15 , wherein said helper-dependent viral DNA comprises a heterologous gene sequence. 
     
     
         20 . The method of  claim 15 , wherein said helper viral DNA is linked to adenoviral terminal protein.