Production of recombinant AAV using adenovirus comprising AAV rep/cap genes

Abstract

This invention relates to novel adenoviruses useful in the production of high titers of recombinant adeno-associated virus (rAAV) comprising a foreign DNA inert and methods of making these adenoviruses. The adenovirus comprises the AAV rep gene in which the p5 promoter of rep is replaced by a minimal promoter or by no promoter. The invention also provides methods of producing high levels of rAAV as a substantially homogeneous preparation and compositions of rAAV.

1 . An adenovirus vector for the manufacture of rAAV, wherein the adenovirus vector comprises an AAV rep gene, and wherein the AAV p5 promoter is deleted upstream of the AAV rep gene.  
     
     
         2 . The adenovirus vector of  claim 1 , wherein the AAV rep gene comprises a minimal promoter in place of the p5 promoter.  
     
     
         3 . The adenovirus vector of  claim 1 , wherein the AAV rep gene contains no promoter in place of the p5 promoter.  
     
     
         4 . The adenovirus vector of  claim 1 , wherein the adenovirus vector further comprises an AAV cap gene.  
     
     
         5 . The adenovirus vector of  claim 2 , wherein the minimal promoter contains a TATA box.  
     
     
         6 . The adenovirus vector of  claim 2 , wherein the minimal promoter is a minimal  Drosophila  heat shock promoter.  
     
     
         7 . The adenovirus vector of  claim 2 , wherein the minimal promoter is a minimal adenoviral E1b promoter.  
     
     
         8 . The adenovirus vector of  claim 4 , wherein the rep gene and the cap gene are inserted in place of at least a portion of one or more of the E1, E3 or E4 genes of adenovirus in a locus of the adenovirus vector.  
     
     
         9 . The adenovirus vector of  claim 8 , wherein both the rep gene and the cap gene are inserted within the same locus of the adenovirus vector.  
     
     
         10 . The adenovirus vector of  claim 8 , wherein the rep gene and the cap gene are inserted within different loci of the adenovirus vector.  
     
     
         11 . The adenovirus vector of  claim 4 , wherein the rep and cap genes are derived from different AAV serotypes from each other.  
     
     
         12 . A method for producing recombinant rAAV, comprising the steps of: 
 a) infecting the adenovirus vector according to  claim 1  into a host cell containing an rAAV genome;    b) growing the host cells under conditions in which rAAV is produced; and    c) collecting the rAAV from the host cells.    
     
     
         13 . The method according to  claim 12 , wherein the rAAV genome is stbly integrated in a chromosome of the host cells.  
     
     
         14 . The method according to  claim 12 , wherein an adenovirus vector comprises the rAAV genome and is co-infected with the adenovirus vector comprising the rep gene into the host cell.  
     
     
         15 . The method according to  claim 12 , wherein the adenovirus vector provides necessary helper functions for rAAV production.  
     
     
         16 . The method according to  claim 12 , wherein the host cell is 293 cells.  
     
     
         17 . The method according to  claim 12 , further comprising the step of purifying the rAAV.  
     
     
         18 . The method according to  claim 12 , wherein the host cells are selected from CHO, BHK, MDCK, 10T1/2, WEHI cells, COS, BSC 1, BSC 40, BMT 10, VERO, WI38, MRC5, A549, HT1080, 293, B-50, 3T3, NIH3T3, HepG2, Saos-2, Huh7, HER, HEK, HEL, or HeLa cells.  
     
     
         19 . A lysate or supernatant comprising the rAAV produced by the method according to  claim 12 .  
     
     
         20 . A purified rAAV produced by the method according to  claim 19 .  
     
     
         21 . A pharmaceutical composition comprising the rAAV according to  claim 20  and further comprising a pharmaceutically acceptable carrier.  
     
     
         22 . A method for transient or stable gene transfer of a desired transgene to a mammalian cell, comprising the step of infecting the mammalian cell with the rAAV according to  claim 20 .  
     
     
         23 . The method according to  claim 22 , wherein said transient or stable gene transfer is for genetic immunization, correction of genetic defects or production of proteins in vitro, in vivo, or ex vivo.  
     
     
         24 . The vector according to  claim 15 , wherein said helper functions are encoded by at least one gene product selected from the group consisting of adenoviral genes E1A, E1B, E2A, E4orf6 and VAI, or at least one gene product selected from the group consisting of HSV type 1 genes UL5, UL8, UL52, and UL29.