Method for producing recombinant virus

Abstract

The invention concerns a method for producing recombinant virus. This method is based on the use of baculovirus for providing the complementary functions. It also concerns constructs used for implementing this method, the producing cells, and the resulting virus.

1 . Process for the production of defective recombinant viruses according to which the genome of the defective recombinant virus and a baculovirus comprising all or some of the functions necessary for the transcomplementation of the defective recombinant genome are introduced into a population of competent cells.  
     
     
         2 . Process according to  claim 1 , 
 characterized in that the baculovirus comprises all the functions necessary for the transcomplementation of the defective recombinant genome.    
     
     
         3 . Process according to  claim 1 , 
 characterized in that the baculovirus comprises some of the functions necessary for the transcomplementation of the defective recombinant genome, the rest being provided by the competent cell.    
     
     
         4 . Process according to  claim 1 , 
 characterized in that the functions necessary for the transcomplementation of the defective recombinant genome are provided by several baculoviruses.    
     
     
         5 . Process according to  claim 1 , 
 characterized in that the defective recombinant virus is a defective recombinant adenovirus.    
     
     
         6 . Process according to  claim 5 , 
 characterized in that the genome of the recombinant adenovirus is defective for one or more functions chosen from E1, E2, E3, E4, L1-L5, pIX and IVa2 and the baculovirus comprises all the functions necessary for the transcomplementation of the defective recombinant genome.    
     
     
         7 . Process according to  claim 5 , 
 characterized in that the genome of the recombinant adenovirus is defective for one or more functions chosen from E1, E2, E3, E4, L1-L5, pIX and IVa2, the baculovirus comprises some of the functions necessary for the transcomplementation of the defective recombinant genome, the rest of the functions being provided by one or more other baculoviruses and/or by the competent cell.    
     
     
         8 . Process according to  claim 5 , 
 characterized in that the helper baculovirus comprises all or part of the E1 region of the adenovirus, allowing the complementation of a recombinant adenovirus genome defective for the E1 region.    
     
     
         9 . Process according to  claim 5 , 
 characterized in that the helper baculovirus comprises all or part of the E2 region of the adenovirus, allowing the complementation of a recombinant adenovirus genome defective for the E2 region.    
     
     
         10 . Process according to  claim 5 , 
 characterized in that the helper baculovirus comprises all or part of the E4 region of the adenovirus, allowing the complementation of a recombinant adenovirus genome defective for the E4 region.    
     
     
         11 . Process according to  claim 5 , 
 characterized in that the helper baculovirus comprises all or part of the E1 and E4 regions of the adenovirus, allowing the complementation of a recombinant adenovirus genome defective for the E1 and E4 regions.    
     
     
         12 . Process according to  claim 5 , 
 characterized in that the recombinant adenovirus genome lacks any coding viral region and the helper baculovirus comprises all the functions allowing its complementation.    
     
     
         13 . Process according to  claim 12 , 
 characterized in that the baculovirus comprises the whole of an adenoviral genome, with the exception of the encapsidation region and possibly of the ITRs.    
     
     
         14 . Process according to  claim 1 , 
 characterized in that the complementation functions present in the baculovirus and the genome of the defective recombinant virus do not comprise a zone of homology capable of giving rise to recombination.    
     
     
         15 . Process according to  claim 14 , 
 characterized in that a recombinant adenovirus genome defective for the E1 and possibly E3 region is introduced into the competent cells, these cells are infected, simultaneously or otherwise, with a baculovirus comprising the E1 region, the adenovirus E1 region present in the baculovirus and the genome of the defective recombinant adenovirus comprising no zone of homology capable of giving rise to recombination.    
     
     
         16 . Process according to  claim 15 , 
 characterized in that the baculovirus comprises a fragment 391-3511 of the Ad5 adenovirus and in that the genome of the recombinant adenovirus defective for the E1 region carries a larger deletion.    
     
     
         17 . Process according to  claim 16 , 
 characterized in that the baculovirus comprises a fragment 391-3511 of the Ad5 adenovirus and in that the genome of the recombinant adenovirus defective for the E1 region carries a deletion covering nucleotides 383-3512 inclusive.    
     
     
         18 . Recombinant baculovirus comprising, 
 inserted into its genome, a nucleic acid encoding a complementation function of a defective virus placed under the control of a heterologous promoter.    
     
     
         19 . Baculovirus according to  claim 18 , 
 characterized in that the complementation function is chosen from all or some of the functions encoded by the E1, E2, E4, L1-L5, pIX and IVa2 regions of the adenovirus, alone or in combinations.    
     
     
         20 . Baculovirus according to  claim 18 , 
 characterized in that the complementation function is chosen from all or some of the functions encoded by the Rep and Cap regions of the AAV, alone or in combinations.    
     
     
         21 . Baculovirus according to  claim 18 , 
 characterized in that the complementation function is chosen from all or some of the functions encoded by the gag, pol and env regions of a retrovirus, alone or in combinations.    
     
     
         22 . Baculovirus according to  claim 18 , 
 characterized in that the nucleic acid encoding the complementation function consists of a DNA corresponding to a fragment of a genome of the virus comprising the corresponding region.    
     
     
         23 . Baculovirus according to  claim 22 , 
 characterized in that the nucleic acid encoding the complementation function consists of a DNA corresponding to a fragment of a genome of serotype Ad2 or Ad5 adenovirus.    
     
     
         24 . Baculovirus according to  claim 18 , 
 characterized in that the promoter consists of the promoter region naturally responsible for the expression of the complementation functions.    
     
     
         25 . Baculovirus according to  claim 18 , 
 characterized in that the promoter is a strong cellular or viral promoter, regulated or otherwise.    
     
     
         26 . Baculovirus according to  claim 19 , 
 characterized in that the complementation function comprises the E1 region of an adenoviral genome or only a part thereof comprising at least the E1a region.    
     
     
         27 . Baculovirus according to  claim 19 , 
 characterized in that the complementation function comprises the E4 region of an adenoviral genome or only a part thereof comprising at least the frame ORF3 or ORF6.    
     
     
         28 . Baculovirus according to  claim 19 , 
 characterized in that it comprises all the coding regions of an adenoviral genome.    
     
     
         29 . Baculovirus according to  claim 28 , 
 characterized in that it comprises a complete adenoviral genome, lacking the encapsidation region.    
     
     
         30 . Baculovirus according to  claim 18 , 
 characterized in that it is an AcNPV strain.    
     
     
         31 . Baculovirus according to  claim 18 , 
 characterized in that the nucleic acid is introduced at the level of the polyhedrin locus or of the p10 locus.    
     
     
         32 . Baculovirus according to  claim 18 , 
 characterized in that the nucleic acid is introduced in the form of a cassette which is capable of being excised in the competent cell.    
     
     
         33 . Recombinant baculovirus comprising, 
 inserted into its genome, at least one DNA region flanked by two sequences allowing a site-specific recombination and positioned in direct orientation, the said DNA region comprising at least one replications origin functional in competent cells and a nucleic acid encoding a complementation function of a virus.    
     
     
         34 . Baculovirus according to  claim 33 , 
 characterized in that the sequences allowing a site-specific recombination are LoxP sequences of the P1 bacteriophage, and the recombination is obtained in the presence of the Cre protein.    
     
     
         35 . Process according to  claim 5 , 
 characterized in that the defective recombinant genome is introduced into the cell by infection with an adenovirus comprising the said genome.    
     
     
         36 . Process according to  claim 5 , 
 characterized in that the defective recombinant genome is introduced into the cell by transfection.    
     
     
         37 . Process according to  claim 1 , 
 characterized in that the defective recombinant genome is introduced into the cell with a recombinant baculovirus, distinct from the baculovirus carrying the complementation functions.    
     
     
         38 . Recombinant baculovirus comprising, 
 inserted into its genome, at least one DNA region flanked by two sequences allowing a site-specific recombination and positioned in direct orientation, the said DNA region comprising at least one replication origin functional in competent cells and a defective adenovirus genome.    
     
     
         39 . Baculovirus according to  claim 38 , 
 characterized in that the defective recombinant adenovirus genome comprises essentially the ITR regions, the encapsidation sequence and a nucleic acid of interest.    
     
     
         40 . Process for the production of defective recombinant adenoviruses, characterized in that a population of competent cells is infected with a baculovirus according to  claim 33  and with a baculovirus according to  claim 38 , the cells are put in the presence of the recombinase allowing the site-specific recombination, and then the adenoviruses produced are recovered.  
     
     
         41 . Process according to  claim 1 , 
 characterized in that the population of competent cells is a population of hepatic, muscle, fibroblast, embryonic, epithelial (particularly pulmonary), ocular (particularly retinal) or nerve cells.    
     
     
         42 . Process according to  claim 41 , 
 characterized in that the population of competent cells is chosen from the cells 293 or any derived cell comprising an additional complementation function, A549, HuH7, Hep3B, HepG2, HER, 911, HeLa or KB.    
     
     
         43 . Purified viral preparation obtained using the process according to  claim 1.