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

Abstract

Presented are ways to address the problem of replication competent adenovirus in adenoviral production for use with, for example, gene therapy. Packaging cells having no overlapping sequences with a selected vector and are suited for large scale production of recombinant adenoviruses. A system for use with the invention produces adenovirus incapable of replicating. The system includes a primary cell containing a nucleic acid based on or derived from adenovirus and an isolated recombinant nucleic acid molecule for transfer into the primary cell. The isolated recombinant nucleic acid molecule is based on or derived from an adenovirus, and further has at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat. The isolated recombinant nucleic acid molecule lacks overlapping sequences with the nucleic acid of the cell. Otherwise, the overlapping sequences would enable homologous recombination leading to replication competent adenovirus in the primary cell into which the isolated recombinant nucleic acid molecule is to be transferred.

What is claimed is:  
     
         1 . A method for producing a recombinant adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising: 
 providing a cell, said cell harboring nucleic acid based on or derived from adenovirus;    transferring recombinant nucleic acid into said cell, said recombinant nucleic acid comprising: 
 another nucleic acid based on or derived from adenovirus, and further including at least one functional encapsidating signal, and at least one functional Inverted Terminal Repeat, said recombinant nucleic acid lacking overlapping sequences with the cellular nucleic acid leading to replication competent adenovirus;  
   culturing said cell; and    harvesting recombinant adenovirus produced from said cell.    
     
     
         2 . The method according to  claim 1  wherein said recombinant nucleic acid is in linear form and comprises functional Inverted Terminal Repeats at or near both termini.  
     
     
         3 . The method according to  claim 1  wherein said cell is a primary cell.  
     
     
         4 . The method of  claim 1  wherein said recombinant nucleic acid is DNA.  
     
     
         5 . The method of  claim 2  wherein said recombinant nucleic acid is DNA.  
     
     
         6 . The method of  claim 3  wherein said recombinant nucleic acid is DNA.  
     
     
         7 . A method of producing, in a producer cell, a recombinant adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising: 
 culturing, in a suitable medium, a producer cell comprising one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus, wherein said producer cell expresses at least adenoviral E1A region gene products; and    harvesting recombinant adenovirus produced from said cell.    
     
     
         8 . The method according to  claim 7  wherein at least one or more of said recombinant nucleic acid molecules of said producer cell further comprises a marker gene.  
     
     
         9 . The method according to  claim 8  wherein said marker gene is under control of an E1A responsive promoter, said E1A being of an adenovirus of the family Adenoviridae.  
     
     
         10 . A method of producing a recombinant adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising: 
 culturing a producer cell in a suitable medium and harvesting said adenovirus therefrom, wherein said producer cell comprises: 
 one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus in said producer cell, and wherein said producer cell expresses adenoviral E1 and E2A region gene products; and  
   harvesting recombinant adenovirus produced from said producer cell.    
     
     
         11 . A method according to  claim 10  wherein said E2A region is under the control of an inducible promoter.  
     
     
         12 . A method according to  claim 10  wherein said E2A region is mutated so that at least one of its products is temperature sensitive.  
     
     
         13 . A method according to  claim 11  wherein said E2A region is mutated so that at least one of its products is temperature sensitive.  
     
     
         14 . The method according to  claim 7  wherein said producer cell is a diploid cell.  
     
     
         15 . The method according to  claim 10  wherein said producer cell is a diploid cell.  
     
     
         16 . The method according to  claim 11  wherein said producer cell is a diploid cell.  
     
     
         17 . The method according to  claim 7  wherein said producer cell is of non-human origin.  
     
     
         18 . The method according to  claim 10  wherein said producer cell is of non-human origin.  
     
     
         19 . The method according to  claim 11  wherein said producer cell is of non-human origin.  
     
     
         20 . The method according to  claim 14  wherein said producer cell is of monkey origin.  
     
     
         21 . The method according to  claim 15  wherein said producer cell is of monkey origin.  
     
     
         22 . The method according to  claim 16  wherein said producer cell is of monkey origin.  
     
     
         23 . A method according to  claim 7  wherein one or more of said recombinant nucleic acid molecule of said producer cell further has a mutated B2A region of an adenovirus of the family Adenoviridae.  
     
     
         24 . A method according to  claim 10  wherein one or more of said recombinant nucleic acid molecule of said producer cell further has a mutated E2A region of an adenovirus of the family Adenoviridae.  
     
     
         25 . A method according to  claim 11  wherein one or more of said recombinant nucleic acid molecule of said producer cell further has a mutated E2A region of an adenovirus of the family Adenoviridae.  
     
     
         26 . A method of producing a recombinant adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising: 
 culturing a producer cell in a suitable medium, said producer cell comprising: 
 one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another which would otherwise allow for homologous recombination leading to replication competent adenovirus in said producer cell, said producer cell further expressing adenoviral E1 and E2A region gene products, wherein said E2A region is mutated so that at least one of its products is temperature sensitive; and  
   harvesting said recombinant adenovirus from said cell.    
     
     
         27 . A method of producing a recombinant adenovirus comprising a gene of interest, without the concomitant production of replication competent adenovirus through homologous recombination, said method comprising: 
 culturing a producer cell in a suitable medium, said producer cell comprising: 
 one or more recombinant nucleic acid molecules having no overlapping sequences with respect to one another, and DNA sequences encoding the adenoviral E1A and E1B region gene products; and  
   harvesting recombinant adenovirus from said cell.    
     
     
         28 . The method according to  claim 27  wherein said recombinant nucleic acid molecule further comprises DNA sequences encoding adenoviral E2A region gene products.  
     
     
         29 . The method according to  claim 28  wherein one of said DNA sequences encoding the E2A region gene product is selected from the group consisting of a DNA sequence encoding the wild-type E2A region operably linked to an inducible promoter and a DNA sequence encoding a temperature sensitive 125 mutation.