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.

1 .- 37 . (canceled) 
     
     
         38 . An adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said adenoviral packaging 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 adenoviral packaging cell line and are operably linked to identical promoters. 
     
     
         39 . The adenoviral packaging cell line of  claim 38 , wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated at different sites in said adenoviral packaging cell line. 
     
     
         40 . The adenoviral packaging cell of  claim 38 , wherein said promoter that lacks substantial sequence identity with a native adenovirus E1A or E1B promoter is a retrovirus promoter. 
     
     
         41 . The adenoviral packaging cell of  claim 38 , wherein said adenovirus E1A coding sequence comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         42 . The adenoviral packaging cell of  claim 38 , wherein said adenovirus E1B coding sequence comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         43 . The adenoviral packaging cell line of  claim 39 , wherein said packaging cell line is of human origin. 
     
     
         44 . The adenoviral packaging cell line of  claim 43 , wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         45 . An adenoviral packaging cell line comprising a first expression vector and a second expression vector stably integrated into the genome of said packaging cell line, wherein said first expression vector comprises adenovirus E1A coding sequences, operably linked to a non-adenoviral heterologous promoter, and said second expression vector comprises adenovirus E1B coding sequences operably linked to a non-adenoviral heterologous promoter. 
     
     
         46 . A method of producing an adenovirus packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, 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. 
     
     
         47 . The method according to  claim 46 , wherein one of the separate vectors comprises a retroviral nucleotide sequence. 
     
     
         48 . The method according to  claim 46 , wherein each of the separate vectors comprises a retroviral nucleotide sequence. 
     
     
         49 . An adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, wherein said adenoviral packaging cell line comprises an adenovirus E1A coding sequence, each and an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter, and wherein said adenovirus EA coding sequence and said adenovirus E1B coding sequence are stably integrated into the adenoviral packaging cell line and are operable linked to different heterologous promoters. 
     
     
         50 . The adenoviral packaging cell line of  claim 49 , wherein said adenovirus E1A coding sequence and said adenovirus E1B sequence are stable integrated at different sites in said adenoviral packaging cell line. 
     
     
         51 . The adenoviral packaging cell line of  claim 49 , wherein said heterologous promoters that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter are retrovirus promoters. 
     
     
         52 . The adenoviral packaging cell of  claim 49 , wherein said adenovirus EA coding sequence comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         53 . The adenoviral packaging cell of  claim 49 , wherein said adenovirus E1B coding sequence comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         54 . The adenoviral packaging cell line of  claim 50 , wherein said adenoviral packaging cell line is of human origin. 
     
     
         55 . The adenoviral packaging cell line of  claim 50 , wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         56 . A method of producing an adenoviral packaging cell line permissive for replication of an E1A/E1B deficient adenovirus vector, 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, 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 and the promoters operably linked to the E1A and E1B coding sequences are different. 
     
     
         57 . The method according to  claim 56 , wherein one of the separate vectors comprises a retroviral nucleotide sequence. 
     
     
         58 . The method according to  claim 56 , wherein each of the separate vectors comprises a retroviral nucleotide sequence. 
     
     
         59 . An adenoviral vector substantially free of replication competent adenovirus, produced by a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to different heterologous promoters. 
     
     
         60 . The adenoviral vector of  claim 59 , wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line. 
     
     
         61 . The adenoviral vector of  claim 59 , wherein said packaging cell line is of human origin. 
     
     
         62 . The adenoviral vector of  claim 61 , wherein said adenoviral packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         63 . The adenoviral vector of  claim 59 , wherein the promoter in said adenoviral packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter. 
     
     
         64 . The adenoviral vector of  claim 59 , wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         65 . The adenoviral vector of  claim 59 , wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         66 . The adenoviral vector according to  claim 59 , 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. 
     
     
         67 . The adenoviral vector according to  claim 66 , wherein said first expression vector comprises a retroviral nucleotide sequence. 
     
     
         68 . The adenoviral vector according to  claim 66 , wherein said first and second expression vectors each comprise a retroviral nucleotide sequence. 
     
     
         69 . The adenoviral vector according to  claim 59 , wherein said adenoviral vector is replication defective. 
     
     
         70 . A composition comprising the adenoviral vector of  claim 59 , together with a pharmaceutically acceptable excipient. 
     
     
         71 . An adenoviral vector substantially free of replication competent adenovirus, produced by a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to identical promoters. 
     
     
         72 . The adenoviral vector of  claim 71 , wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line. 
     
     
         73 . The adenoviral vector of  claim 71 , wherein said packaging cell line is of human origin. 
     
     
         74 . The adenoviral vector of  claim 73 , wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         75 . The adenoviral vector of  claim 71 , wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter. 
     
     
         76 . The adenoviral vector of  claim 71 , wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         77 . The adenoviral vector of  claim 71 , wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         78 . The adenoviral vector according to  claim 71 , wherein said adenoviral vector is replication defective. 
     
     
         79 . A composition comprising the adenoviral vector of  claim 71 , together with a pharmaceutically acceptable excipient. 
     
     
         80 . 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 and are operably linked to identical promoters. 
     
     
         81 . A method of producing a modified cell line, the method comprising: introducing into a first 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. 
     
     
         82 . A cell line comprising an adenovirus E1A coding sequence, each and an adenovirus E1B coding sequence operably linked to a promoter that lacks substantial sequence identity to a native adenovirus E1A or E1B promoter, and wherein said adenovirus E1A coding sequence and said adenovirus E1B coding sequence are stably integrated into the cell line and are operable linked to different heterologous promoters. 
     
     
         83 . A method of producing a modified cell line, the method comprising:
 introducing into a first 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, 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 and the promoters operably linked to the E1A and E1B coding sequences are different.   
     
     
         84 . A system comprising an adenoviral vector substantially free of wild-type replication competent adenovirus, and a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to different heterologous promoters. 
     
     
         85 . The system of  claim 84 , wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line. 
     
     
         86 . The system of  claim 84 , wherein said packaging cell line is of human origin. 
     
     
         87 . The system of  claim 86 , wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         88 . The system of  claim 84 , wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter. 
     
     
         89 . The system of  claim 84 , wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         90 . The system of  claim 84 , wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         91 . The system according to  claim 84 , 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. 
     
     
         92 . The system according to  claim 91 , wherein said first expression vector comprises a retroviral nucleotide sequence. 
     
     
         93 . The system according to  claim 91 , wherein said first and second expression vectors each comprise a retroviral nucleotide sequence. 
     
     
         94 . The system according to  claim 84 , wherein said adenoviral vector is replication defective. 
     
     
         95 . The system according to  claim 84 , wherein no wild type replication competent adenovirus is detected following 18 cycles of infection. 
     
     
         96 . A system comprising an adenoviral vector substantially free of wild type replication competent adenovirus, and a packaging cell line permissive for replication of an E1A/E1B deficient adenoviral vector, wherein said packaging cell line comprises an adenoviral E1A coding sequence and an adenoviral E1B coding sequence operably linked to a promoter that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter, and wherein said adenoviral E1A coding sequence and said adenoviral E1B coding sequence are stably integrated into said packaging cell line and are operably linked to identical promoters. 
     
     
         97 . The system of  claim 96 , wherein said adenoviral E1A coding sequence and said adenoviral E1B sequence are stable integrated at different sites in said packaging cell line. 
     
     
         98 . The system of  claim 96 , wherein said packaging cell line is of human origin. 
     
     
         99 . The system of  claim 98 , wherein said packaging cell line originates from a cell line selected from the group consisting of A549 cells permissive for adenovirus replication, PC-3 cells, and primary cells permissive for adenovirus production. 
     
     
         100 . The system of  claim 96 , wherein the promoter in said packaging cell line that lacks substantial sequence identity with a native adenoviral E1A or E1B promoter is a retrovirus promoter. 
     
     
         101 . The system of  claim 96 , wherein said adenoviral E1A coding sequence in said packaging cell line comprises the E1A coding sequence of GenBank Accession Number M73260. 
     
     
         102 . The system of  claim 96 , wherein said adenoviral E1B coding sequence in said packaging cell line comprises the E1B coding sequence of GenBank Accession Number M73260. 
     
     
         103 . The system according to  claim 96 , wherein said adenoviral vector is replication defective. 
     
     
         104 . The system according to  claim 96 , wherein no wild type replication competent adenovirus is detected following 18 cycles of infection.