Chimeric adenoviruses

Abstract

Methods and vector systems for generating chimeric recombinant adenoviruses. These hybrid adenoviruses contain a genome that is derived from different adenovirus serotypes. In particular, novel hybrid adenoviruses are disclosed that have improved properties for gene therapy purposes. These properties include, but are not limited to, a decreased sensitivity towards neutralizing antibodies, a modified host range, a change in the titer to which adenovirus can be grown, the ability to escape trapping in the liver upon in vivo systemic delivery, and absence or decreased infection of antigen presenting cells of the immune system, such as macrophages or dendritic cells. These chimeric adenoviruses thus represent improved tools for gene therapy and vaccination, since they overcome the limitations observed with the currently used serotype subgroup C adenoviruses.

What is claimed is:  
     
         1 . A nucleic acid library comprising nucleic acid derived from different adenovirus serotypes.  
     
     
         2 . A method for selecting and producing a chimeric adenovirus having a desired host range determined by at least one part of a fiber of a first adenovirus serotype and immunological properties determined by at least one part of at least one of a hexon or penton of a second adenovirus serotype, said method comprising: 
 providing a recombinant vector derived from an adenovirus comprising at least one ITR and a packaging signal, said recombinant vector having a first insertion site for a gene of interest, said recombinant vector further having a second insertion site for a nucleic acid encoding at least one part of a fiber protein of the first adenovirus serotype and having a third insertion site for functionally inserting a nucleic acid encoding at least one part of at least one of a penton or a hexon of the second adenovirus serotype;    providing a nucleic acid library comprising a plurality of nucleic acids encoding a plurality of adenoviral proteins of a plurality of adenovirus serotypes, said plurality of nucleic acids flanked by restriction sites wherein said restriction sites correspond to said first, second, and third insertion sites in said recombinant vector;    inserting into said recombinant vector at least one first nucleic acid from said nucleic acid library, said at least one first nucleic acid obtained from the second adenovirus serotype and encoding at least one part of at least one of a penton or hexon protein, said penton or hexon protein having lower antigenicity relative to penton or hexon proteins of the first adenovirus serotype and conferring a viral particle having lower antigenicity;    inserting into said recombinant vector at least one second nucleic acid from said nucleic acid library, said at least one second nucleic acid obtained from the first adenovirus serotype and encoding at least one functional part of a fiber protein having the desired host range;    providing at least one packaging cell;    transfecting said recombinant vector into said at least one packaging cell; and    producing chimeric viral particles.    
     
     
         3 . The method according to  claim 2 , further comprising inserting said gene of interest into said recombinant vector prior to said transfecting.  
     
     
         4 . The method according to  claim 3 , wherein said providing a recombinant vector comprises providing an expression cassette for said gene of interest.  
     
     
         5 . The method according to  claim 2 , wherein said providing a nucleic acid library comprises providing a plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes, and wherein said plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes are flanked by uniform restriction sites.  
     
     
         6 . The method according to  claim 2 , wherein said providing a recombinant vector comprises providing a vector lacking the E1 adenoviral genome.  
     
     
         7 . The method according to  claim 6 , wherein said providing at least one packaging cell comprises providing at least one packaging cell selected from the group consisting of PER.C6, 911, 293, and E1 A549 cells.  
     
     
         8 . The method according to  claim 2 , wherein said providing a recombinant vector comprises providing a recombinant vector derived from an adenovirus Sub-Group C serotype.  
     
     
         9 . The method according to  claim 8 , wherein said adenovirus Sub-Group C serotype comprises one of Ad2 or Ad5.  
     
     
         10 . The method according to  claim 2 , wherein said at least one first nucleic acid is obtained from an adenovirus Sub-Group B or C serotype.  
     
     
         11 . The method according to  claim 2 , wherein said at least one second nucleic acid is obtained from an adenovirus Sub-Group B or C serotype.  
     
     
         12 . The method according to  claim 2 , wherein said providing a recombinant vector comprises providing a vector selected from the group consisting of viral, plasmid, and cosmid vectors.  
     
     
         13 . The method according to  claim 2 , wherein said at least one second nucleic acid obtained from the first adenovirus serotype comprises a nucleic acid encoding a knob protein of a fiber protein, and wherein said at least one first nucleic acid obtained from a second adenovirus serotype further comprises a nucleic acid encoding a base protein of a fiber protein and a shaft protein of a fiber protein.  
     
     
         14 . The method according to  claim 2 , wherein said providing a nucleic acid library comprises providing nucleic acids carrying sequence mutations, and wherein said nucleic acids carrying sequence mutations encode proteins screened for characteristics selected from the group consisting of temperature stability, assembly, anchoring, redirected infection, and altered immune response.  
     
     
         15 . The method according to  claim 2 , wherein said providing a nucleic acid library comprises providing nucleic acids encoding a plurality of adenoviral proteins obtained from a plurality of adenovirus serotypes selected from the group consisting of adenovirus Sub-Groups A, B, C, D, E, F, and G.  
     
     
         16 . A library of chimeric adenovirus produced by the method according to  claim 2 .  
     
     
         17 . A method of generating a library of chimeric adenoviruses, said method comprising: 
 providing a plurality of recombinant vectors derived from an adenoviral genome, each of said plurality of recombinant vectors having an insertion site for a nucleic acid encoding at least one part of a fiber protein of an adenovirus serotype having a desired host range and having an insertion site for functionally inserting a nucleic acid encoding at least one part of at least one of a penton or a hexon protein of a differing adenovirus serotype having predetermined antigenic properties;    providing a nucleic acid library comprising a plurality of nucleic acids encoding a plurality of adenoviral proteins of a plurality of adenovirus serotypes, said plurality of nucleic acids flanked by restriction sites wherein said restriction sites correspond to said insertion sites in said recombinant vector;    inserting into each of said plurality of recombinant vectors at least one first nucleic acid from said nucleic acid library encoding at least one functional part of a fiber protein obtained from an adenovirus serotype having a desired host range;    inserting into each of said plurality of recombinant vectors at least one second nucleic acid from said nucleic acid library encoding at least one functional part of a penton or hexon protein of a differing adenovirus serotype having predetermined antigenic properties, said penton or hexon protein of each respective recombinant vector having lower antigenicity relative to penton or hexon proteins of the adenovirus serotype conferring the desired host range and resulting in a viral particle having lower antigenicity; 
 providing a plurality of packaging cells;  
   transfecting said plurality of recombinant vectors into said plurality of packaging cells; and    producing a library of chimeric viral particles defined by differing fiber protein and penton or hexon protein adenovirus serotypes.    
     
     
         18 . The method according to  claim 17 , wherein said producing a library of chimeric viral particles comprises generating a library of chimeric capsids.  
     
     
         19 . The method according to  claim 17 , further comprising screening the produced chimeric viral particles for properties selected from the group consisting of target cell specificity, immunogenicity, re-directed neutralization, re-directed hemagglutination, infection efficiency, toxicity, and pharmacokinetics.  
     
     
         20 . The method according to  claim 17 , wherein said providing a nucleic acid library comprises providing a plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes, and wherein the plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes are flanked by uniform restriction sites.  
     
     
         21 . A method for selecting and producing a chimeric adenovirus having a desired host range determined by at least one part of a fiber of a first adenovirus serotype, immunological properties determined by at least one part of at least one of a hexon or penton of a second adenovirus serotype, said method comprising: 
 providing a recombinant vector derived from the genome of adenovirus serotype 5, said recombinant vector comprising at least one ITR and a packaging signal and having an insertion site for a gene of interest, said recombinant vector further having an insertion site for a nucleic acid encoding at least one part of a fiber protein of the first adenovirus serotype and having an insertion site for functionally inserting a nucleic acid encoding at least one part of at least one of a penton or a hexon protein of the second serotype of adenovirus;    providing a nucleic acid library comprising a plurality of nucleic acids encoding a plurality of adenoviral proteins of a plurality of adenovirus serotypes, at least some of said plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes;    providing said plurality of nucleic acids flanked by restriction sites wherein said restriction sites correspond to said insertion sites in said recombinant vector and wherein the at least some of the plurality of nucleic acids encoding proteins of like functions for differing adenovirus serotypes are flanked by uniform restriction sites;    inserting into said recombinant vector at least one first nucleic acid from said nucleic acid library, said at least one first nucleic acid obtained from the second adenovirus serotype and encoding at least one part of at least one of a penton or hexon protein, said penton or hexon protein having lower antigenicity relative to penton or hexon proteins of the first adenovirus serotype and resulting in a viral particle having lower antigenicity;    inserting into said recombinant vector at least one second nucleic acid from said nucleic acid library, said at least one second nucleic acid obtained from the first adenovirus serotype and encoding at least one functional part of a fiber protein having the desired host range;    inserting said gene of interest into said recombinant vector;    providing at least one packaging cell;    transfecting said recombinant vector into said at least one packaging cell; and 
 producing chimeric viral particles.