Potent and balanced bidirectional promoter

Abstract

The invention provides a bidirectional hCMV-CAG4 promoter and recombinant vectors and recombinant virus comprising the bidirectional hCMV-CAG4 promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction. The invention also provides methods of making and using such recombinant vectors and recombinant virus.

1 - 16 . (canceled) 
     
     
         17 . A method of producing a genetically stable recombinant adenovirus comprising a first transgene and a second transgene that each are potently expressed when the adenovirus infects a target cell, the method comprising:
 a) preparing a construct comprising a bidirectional promoter comprising:
 a cytomegalovirus major immediate early enhancer as an enhancer building block, flanked by 
 a human cytomegalovirus major immediate early promoter (hCMV promoter) as a first promoter building block, and 
 a chicken beta actin promoter as a second promoter building block, with 
 a hybrid chicken beta actin/rabbit beta globin intron as a first intron building block adjacent to and downstream of the chicken beta actin promoter, and 
 a human apolipoprotein A-1 intron as a second intron building block directly contiguous with and downstream of the hCMV promoter building block, 
 wherein the bidirectional promoter comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 4, and 
 wherein the bidirectional promoter is operably linked to the first transgene in one direction and to the second transgene in the opposite direction; and 
   b) incorporating said construct into the genome of the recombinant adenovirus.   
     
     
         18 . The method according to  claim 17 , wherein the bidirectional promoter comprises SEQ ID NO: 4. 
     
     
         19 . The method according to  claim 17 , wherein the first transgene and the second transgene are different and at least one of the first transgene and the second transgene encodes an antigen 
     
     
         20 . The method according to  claim 17 , wherein the recombinant adenovirus has a deletion in the E1 region of its genome. 
     
     
         21 . The method according to  claim 17 , wherein the recombinant adenovirus is a human adenovirus serotype 35 or a human adenovirus serotype 26. 
     
     
         22 . A method for expressing at least two transgenes in a cell, the method comprising:
 providing a cell with a recombinant vector or a recombinant virus comprising a recombinant nucleic acid molecule comprising a bidirectional promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction, wherein the bidirectional promoter comprises:
 a cytomegalovirus major immediate early enhancer as an enhancer building block, flanked by 
 a human cytomegalovirus major immediate early promoter (hCMV promoter) as a first promoter building block, and 
 a chicken beta actin promoter as a second promoter building block, with 
 a hybrid chicken beta actin/rabbit beta globin intron as a first intron building block adjacent to and downstream of the chicken beta actin promoter, and 
 a human apolipoprotein A-1 intron as a second intron building block directly contiguous with and downstream of the hCMV promoter building block, 
 wherein the bidirectional promoter comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 4. 
   
     
     
         23 . The method according to  claim 22 , wherein the bidirectional promoter comprises SEQ ID NO: 4. 
     
     
         24 . The method according to  claim 22 , wherein the first transgene and the second transgene are different and at least one of the first transgene and the second transgene encodes an antigen. 
     
     
         25 . The method according to  claim 22 , wherein the recombinant virus is an adenovirus. 
     
     
         26 . The method according to  claim 25 , wherein the recombinant adenovirus has a deletion in the E1 region of its genome. 
     
     
         27 . The method according to  claim 25 , wherein the recombinant virus is a human adenovirus serotype 35 or a human adenovirus serotype 26. 
     
     
         28 . A method for inducing an immune response against at least two antigens, the method comprising administering to a subject a recombinant vector or a recombinant virus comprising a recombinant nucleic acid molecule comprising a bidirectional promoter operably linked to a first transgene in one direction and to a second transgene in the opposite direction, wherein the bidirectional promoter comprises:
 a cytomegalovirus major immediate early enhancer as an enhancer building block, flanked by   a human cytomegalovirus major immediate early promoter (hCMV promoter) as a first promoter building block, and   a chicken beta actin promoter as a second promoter building block, with a hybrid chicken beta actin/rabbit beta globin intron as a first intron building block adjacent to and downstream of the chicken beta actin promoter, and   a human apolipoprotein A-1 intron as a second intron building block directly contiguous with and downstream of the hCMV promoter building block,   wherein the bidirectional promoter comprises a sequence that is at least 90% identical to the sequence of SEQ ID NO: 4.   
     
     
         29 . The method according to  claim 28 , wherein the bidirectional promoter comprises SEQ ID NO: 4. 
     
     
         30 . The method according to  claim 28 , wherein the bidirectional promoter comprises SEQ ID NO: 4. 
     
     
         31 . The method according to  claim 28 , wherein the first transgene and the second transgene are different and at least one of the first transgene and the second transgene encodes an antigen 
     
     
         32 . The method according to  claim 28 , wherein the recombinant virus is an adenovirus. 
     
     
         33 . The method according to  claim 28 , wherein the recombinant adenovirus has a deletion in the E1 region of its genome. 
     
     
         34 . The method according to  claim 28 , wherein the recombinant virus is a human adenovirus serotype 35 or a human adenovirus serotype 26.