Complementing cell lines

Abstract

A packaging cell line that complements recombinant adenoviruses based on serotypes from subgroup B, preferably adenovirus type 35. The cell line is preferably derived from primary, diploid human cells that are transformed by adenovirus E1 sequences either operatively linked on one DNA molecule or located on two separate DNA molecules, the sequences being operatively linked to regulatory sequences enabling transcription and translation of encoded proteins. Also disclosed is a cell line derived from PER.C6 that expresses functional Ad35 E1B sequences. The Ad35-E1B sequences are driven by the E1B promoter or a heterologous promoter and terminated by a heterologous poly-adenylation signal. The cell lines are useful for producing recombinant adenoviruses designed for gene therapy and vaccination. The cell lines can also be used for producing human recombinant therapeutic proteins such as human growth factors and human antibodies. Also, the cell lines are useful for producing human viruses other than adenovirus such as influenza virus, herpes simplex virus, rotavirus, and measles virus.

What is claimed is: 
     
         1 . An adenovirus packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said cell line comprises an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said cell line. 
     
     
         2 . The adenovirus packaging cell line of  claim 1 , wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated at different sites in said cell line. 
     
     
         3 . The adenovirus packaging cell line of  claim 2 , wherein said packaging cell line is of human origin. 
     
     
         4 . An adenovirus packaging cell line comprising a first expression vector and a second expression vector stably integrated into the genome of said cell line, wherein said first expression vector comprises human adenovirus E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises human adenovirus E1B coding sequences operably linked to a non-adenoviral heterologous promoter. 
     
     
         5 . A method of producing the adenovirus packaging cell line of  claim 1 , the method comprising: introducing into a cell line permissive for adenovirus replication, nucleic acid comprising (i) an adenovirus E1A coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter and (ii) an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein the nucleic acid comprising the adenovirus E1A coding sequence and the nucleic acid comprising the adenovirus E1B coding sequence are present on separate vectors. 
     
     
         6 . The adenoviral vector according to  claim 7 , wherein said packaging cell line comprises a first expression vector and a second expression vector stably integrated into said packaging cell line's genome, wherein said first expression vector comprises adenoviral E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenoviral E1B coding sequences operably linked to a non-adenoviral heterologous promoter. 
     
     
         7 . A method of producing an adenoviral vector substantially free of replication competent adenovirus, the method comprising: producing an andenoviral vector substantially free of replication competent adenovirus with the adenovirus packaging cell line of  claim 1 . 
     
     
         8 . The method according to  claim 7 , wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line. 
     
     
         9 . The method according to  claim 7 , wherein said packaging cell line is of human origin. 
     
     
         10 . The method according to  claim 7 , wherein said adenoviral vector is replication defective. 
     
     
         11 . The method according to  claim 7 , further comprising admixing the adenoviral vector substantially free of replication competent adenovirus together with a pharmaceutically acceptable excipient. 
     
     
         12 . A cell comprising an adenovirus E1A coding sequence and an adenovirus E1B coding sequence each operably linked to a promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into said packaging cell line. 
     
     
         13 . The adenovirus packaging cell line of  claim 1  together with an adenoviral vector substantially free of wild type replication competent adenovirus. 
     
     
         14 . The adenovirus packaging cell line of  claim 13 , wherein said adenoviral vector is replication defective. 
     
     
         15 . The adenovirus packaging cell line of  claim 13 , wherein no wild type replication competent adenovirus is detected following 18 cycles of infection.