Packaging systems for human recombinant adenovirus to be used in gene therapy

Abstract

Improved methods and products based on adenoviral materials which can advantageously be used in, for instance, gene therapy. In one aspect, an adenoviral vector is provided which has no overlap with a suitable packaging cell line, the packaging cell line comprising another aspect of the invention. This combination excludes the possibility of homologous recombination, thereby excluding the possibility of the formation of replication competent adenovirus. In another aspect, an adenovirus based helper construct which by its size is incapable of being encapsulated is disclosed. This helper virus can be transferred into any suitable host cell making it a packaging cell. Additionally, a number of useful mutations to adenoviral based materials and combinations of such mutations are disclosed. Furthermore, a method of intracellular amplification is provided.

What is claimed is:  
     
         1 . A recombinant nucleic acid comprising an adenoviral clone, said adenoviral clone selected from the group consisting of pBr/Ad.Bam-rITR (ECACC P97082122), pBr/Ad.Sal-rITR (ECACC P97082119), pBr/Ad.Cla-Bam (ECACC P97082117), uBr/Ad.Afl II-Bam (ECACC P97082114), pBr/Ad.Bam-rITRpac#2 (ECACC P97082120), pBr/Ad.Bam-rITR#8 (ECACC P97082121), pWE/Ad.AflII-rITR (ECACC P97082116), pBr/Ad.1ITR-Sal(9.4) (ECACC P97082115), and pBr/Ad. 1ITR-Sal(16.7)(ECACC P97082118).  
     
     
         2 . The recombinant nucleic acid of  claim 1 , further comprising a deletion in an E3 region.  
     
     
         3 . The recombinant nucleic acid of  claim 2 , wherein said deletion in the E3 region is replaced with a transgene.  
     
     
         4 . The recombinant nucleic acid of  claim 1 , further comprising a deletion in a gp19K region.  
     
     
         5 . The recombinant nucleic acid of  claim 4 , wherein the deletion in the gp19K region is replaced with a transgene.  
     
     
         6 . A recombinant nucleic acid comprising an adapter plasmid, said adapter plasmid selected from the group consisting of pWE/Ad.AflIII-EcoRI, pAd5/CLIP, pAd5/L420-HSA, pMV/L420-H, pMV/CMV-LacZ, and pWE/Ad-H.  
     
     
         7 . The recombinant nucleic acid of  claim 6 , wher ein said adapter plasmid is pAd5/CLIP or pAd5/L420-HSA, and said recombinant nucleic acid comprises adenovirus derived nucleotides 1-454; and adenovirus nucleotides 3511-6095.  
     
     
         8 . A recombinant nucleic acid comprising a recombinant adenovirus, wherein said recombinant adenovirus is pBS.Eco-Eco/ad5ΔHIIIΔgp19KΔXbaI.  
     
     
         9 . The recombinant nucleic acid of  claim 8 , wherein said nucleic acid further comprises a transgene.  
     
     
         10 . The recombinant nucleic acid of  claim 9 , wherein said transgene is operatively linked to an E3 promoter.  
     
     
         11 . The recombinant nucleic acid of  claim 9 , wherein said transgene is selected from the group consisting of a suicide gene, a cytokine gene, and a reporter gene.  
     
     
         12 . The recombinant nucleic acid of  claim 11 , wherein said transgene is a suicide gene comprising an HSV-TK gene.  
     
     
         13 . The recombinant nucleic acid of  claim 11 , wherein said transgene comprises a coding sequence selected from the group consisting of hIL-1α, rat IL-3, and human IL-3.  
     
     
         14 . The recombinant nucleic acid of  claim 11 , wherein said transgene comprises a coding sequence from a luciferase gene or a hacZ gene.  
     
     
         15 . The recombinant nucleic acid of  claim 14 , further comprising a heterologous nucleotide sequence.  
     
     
         16 . The recombinant nucleic acid of  claim 9 , wherein said transgene comprises a coding sequence from a human ceNOS gene.  
     
     
         17 . A recombinant nucleic acid comprising: 
 an adenoviral nucleotide sequence, said adenoviral nucleotide sequence comprising a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof acting as Inverted Terminal Repeats,    said recombinant nucleic acid lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.    
     
     
         18 . A recombinant nucleic acid for incorporation into a cell comprising: 
 an adenoviral nucleotide sequence comprising sufficient adenoviral sequences necessary for replication and capsid gene expression, but lacking at least the E1 region and an adenoviral encapsulating signal, wherein said adenoviral nucleotide sequence further lacks overlapping sequences allowing for homologous recombination leading to replication competent virus in the cell into which said recombinant nucleic acid is to be transferred.    
     
     
         19 . The recombinant nucleic acid of  claim 18 , further comprising a deletion in an E3 region of said recombinant nucleic acid.  
     
     
         20 . The recombinant nucleic acid of  claim 18 , further comprising a deletion in a gp19K region of said recombinant nucleic acid  
     
     
         21 . A recombinant nucleic acid comprising a construct comprising pWE/Ad.Δ5′.  
     
     
         22 . The recombinant nucleic acid of  claim 21 , further comprising a deletion in an E3 region of said recombinant nucleic acid.  
     
     
         23 . The recombinant nucleic acid of  claim 21 , further comprising a deletion in a gp19K region of said recombinant nucleic acid.  
     
     
         24 . A recombinant nucleic acid comprising: 
 an adenoviral nucleotide sequence comprising sufficient adenovirus sequences needed for replication and capsid gene expression and a complementary sequence to an upstream part of said recombinant nucleic acid, wherein said complementary sequence can base-pair with said upstream part so that it functions as a start-site for a nucleic acid polymerase, said adenoviral nucleotide sequence having a deletion of one Inverted Terminal Repeat, the E1 region, and the adenoviral encapsulating signal,    wherein said recombinant nucleic acid has no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.    
     
     
         25 . The recombinant nucleic acid according to  claim 25 , wherein said recombinant nucleic acid comprises a construct pWE/AAV.Δ5′.  
     
     
         26 . A recombinant nucleic acid comprising: 
 an adenoviral nucleotide sequence comprising a sequence for adenovirus-independent replication and sufficient adenoviral sequences necessary for replication;    wherein said adenoviral nucleotide sequence has a deletion of at least the E1 region and encapsulating signal of adenovirus, said recombinant nucleic acid comprising no overlapping sequences which allow for homologous recombination leading to replication competent virus in a cell into which said recombinant nucleic acid is transferred.    
     
     
         27 . The recombinant nucleic acid of  claim 26 , wherein said adenoviral nucleotide sequence further has a deletion of at least one of the Inverted Terminal Repeats of said adenovirus.  
     
     
         28 . The recombinant nucleic acid of  claim 26 , wherein said adenoviral nucleotide sequence for adenovirus-independent replication comprises an SV40 origin of replication.  
     
     
         29 . An adapter plasmid comprising: 
 an adenoviral nucleotide sequence comprising, in operable configuration, at least one functional Inverted Terminal Repeat and one functional encapsulating signal, said adapter plasmid having no sequences which allow for homologous recombination leading to replication competent virus in a cell into which said adapter plasmid is transferred.    
     
     
         30 . The adapter plasmid of  claim 29 , wherein said adapter plasmid has no E1 region sequences.  
     
     
         31 . The adapter plasmid of  claim 29 , further comprising a multiple cloning site.  
     
     
         32 . The adapter plasmid of  claim 31 , further comprising a nucleic acid inserted into said multiple cloning site.  
     
     
         33 . A method of generating a recombinant adenovirus having an E1 deletion and a gp19K deletion, said method comprising:  
       growing a cell comprising adenovirus complementing sequences transfected with: 
 i) an adapter plasmid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said nucleotide sequence comprises in operable configuration one functional Inverted Terminal Repeat, one functional encapsulating signal and adenoviral sequences which allow for homologous recombination leading to the generation of a replication-defective, recombinant adenovirus genome in a cell into which said adapter plasmid is transferred and having no E1 region sequences, and  
 ii) a recombinant nucleic acid comprising at least one second nucleotide sequence based on or derived from an adenovirus, wherein said at least one second nucleotide sequence comprises one Inverted Terminal Repeat and sufficient adenovirus sequences for replication and a partial overlap with said adapter plasmid, wherein said at least one second nucleotide sequence comprises a deletion of at least the E1 region, encapsulating signal and gp19K sequences;  
 wherein said complementing sequences, said first nucleotide sequence and said at least one second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus,  
 under conditions wherein recombinant adenovirus having an E1 deletion and a gp19K deletion is generated.  
 
     
     
         34 . The method according to  claim 33 , wherein said adapter plasmid further comprises a first heterologous nucleotide sequence inserted into said E1 region deletion and said recombinant nucleic acid further comprises a second heterologous nucleotide sequence inserted into said gp19K region.  
     
     
         35 . The method according to  claim 33 , wherein said at least one second nucleotide sequence comprises a first and second molecule wherein said first molecule has said partial overlap with said adapter plasmid at the 3′ end, and said second molecule comprises said Inverted Terminal Repeat and region including deletion of said gp19K sequences.  
     
     
         36 . A method of generating a recombinant adenovirus, said method comprising: growing a cell comprising adenovirus complementing sequences transfected with: 
 i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and    ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said second nucleotide sequence comprises sufficient adenovirus sequences for replication, wherein said second nucleotide sequence comprises a deletion of at least the E1 region and encapsulating signal of said adenovirus;    wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus, under conditions wherein recombinant adenovirus is generated.    
     
     
         37 . A method of generating a recombinant adenovirus, said method comprising: growing a cell comprising adenovirus complementing sequences transfected with: 
 i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and    ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said second nucleotide sequence comprises a sequence for adenovirus-independent replication, and sufficient adenoviral sequences necessary for replication, wherein said second nucleotide sequence comprises at least a deletion of the E1 region and encapsulating signal of said adenovirus;    wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus,    under conditions wherein recombinant adenovirus is generated.    
     
     
         38 . The method according to  claim 37 , wherein said cell comprises at least one nucleic acid molecule and wherein said cell expresses SV40 Large T antigen proteins or functional fragments of.SV40 Large T antigen proteins having the activity thereof.  
     
     
         39 . The method according to  claim 38 , wherein said second recombinant nucleic acid molecule is replicated.  
     
     
         40 . A replication defective adenovirus comprising: 
 an adenoviral genome comprising at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, and lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in a cell into which said replication defective adenovirus is transferred.    
     
     
         41 . The replication defective adenovirus of  claim 40 , wherein the adenoviral genome further comprises at least one expression cassette.  
     
     
         42 . The replication defective adenovirus of  claim 41 , wherein said at least one expression cassette comprises a gene functionally linked to transcription regulatory sequences.  
     
     
         43 . The replication defective adenovirus of  claim 40 , further comprising one or more non-adenoviral nucleic acid sequences.  
     
     
         44 . The replication defective adenovirus of  claim 43 , wherein said one or more non-adenoviral nucleic acid sequences are inserted into the E1 region or into the E3 region gp19K gene.  
     
     
         45 . A cell comprising: 
 a replication defective adenovirus comprising an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said cell into which said replication defective adenovirus has been transferred.    
     
     
         46 . The cell of  claim 45 , wherein said cell is a mammalian cell.  
     
     
         47 . A method of generating recombinant adenovirus incapable ofreplicating, said method comprising:  
       growing a cell comprising adenovirus complementing sequences and 
 i) a first recombinant nucleic acid comprising a first nucleotide sequence based on or derived from an adenovirus, wherein said first nucleotide sequence comprises a functional encapsulating signal and two functional Inverted Terminal Repeats or functional fragments or derivatives thereof, and wherein said first recombinant nucleic acid has no functional adenoviral genes and,  
 ii) a second recombinant nucleic acid comprising a second nucleotide sequence based on or derived from an adenovirus, wherein said nucleotide sequence comprises at least all adenovirus sequences or functional fragments or derivatives thereof necessary for replication and capsid gene expression, and a complementary sequence to an upstream part of the same strand of said nucleic acid,  
 wherein said complementary sequence can base-pair with said upstream part so that it functions as a start-site for a nucleic acid polymerase, wherein said second nucleotide sequence comprises a deletion of one Inverted Terminal Repeat, the E1 region and the encapsulating signal of said adenovirus; wherein, said complementing sequences, said first nucleotide sequence and said second nucleotide sequence have no overlapping sequences which allow for homologous recombination leading to replication competent virus,  
 under conditions wherein recombinant adenovirus is generated.  
 
     
     
         48 . A cell comprising the recombinant nucleic acid of  claim 1 .  
     
     
         49 . A method of replacing a defective gene in a genome of a host cell, said method comprising: 
 growing said host cell with a recombinant nucleic acid molecule derived from a replication defective adenovirus comprising a functional version or part of said defective gene under conditions wherein at least one allele of said defective gene in said host cell genome is replaced.    
     
     
         50 . The method according to  claim 49 , wherein said replication defective adenovirus expresses no adenoviral genes.  
     
     
         51 . The method according to  claim 49 , wherein said defective gene is a defective tumor suppressor gene.  
     
     
         52 . An isolated cell comprising: 
 an adenoviral genome of a replication defective adenovirus, said adenoviral genome having at least a functional encapsulating signal and two functional Inverted Terminal Repeats or fragments or derivatives thereof having the activity of a functional Inverted Terminal Repeat, said adenoviral genome lacking functional adenoviral genes and having no overlapping sequences allowing for homologous recombination leading to replication competent virus in said isolated cell.    
     
     
         53 . The isolated cell of  claim 52 , wherein said cell is a human cell.  
     
     
         54 . A replication-defective adenovirus comprising an adenoviral genome, wherein said adenoviral genome has a first deletion in an adenoviral E1 region and a second deletion in an adenoviral gp19K region.  
     
     
         55 . The replication-defective adenovirus of  claim 54 , wherein the adenoviral genome further comprises a transgene.  
     
     
         56 . The replication-defective adenovirus of  claim 55 , wherein transcription of said transgene is directed by an E3 promoter.  
     
     
         57 . A cell comprising the recombinant nucleic acid of  claim 17 .  
     
     
         58 . A cell comprising the recombinant nucleic acid of  claim 24 .  
     
     
         59 . A cell comprising the recombinant nucleic acid of claim  26 .