Polynucleotides for use in recombinant adeno-associated virus virion production

Abstract

Accessory functions capable of supporting efficient recombinant AAV (rAAV) virion production in a suitable host cell are provided. The accessory functions are in the form of one or more vectors that are capable of being transferred between cells. Methods of producing rAAV virions are also provided. The methods can be practiced to produce commercially significant levels of rAAV particles without also generating significant levels of infectious helper virus or other contaminating by-products.

1 . A nucleic acid molecule which provides one or more accessory functions for supporting recombinant AAV (rAAV) virion production in a suitable host cell, said molecule comprising: 
 (a) an adenovirus VA RNA coding region;    (b) an adenovirus E4 ORF6 coding region;    (c) an adenovirus E2A 72 kD coding region;    (d) an adenovirus E1A coding region; and    (e) an adenovirus E1B region lacking an intact E1B55k coding region.    
     
     
         2 . An accessory function vector comprising the nucleic acid molecule of  claim 1 .  
     
     
         3 . The accessory function vector of  claim 2 , wherein said vector is a plasmid.  
     
     
         4 . The accessory function vector of  claim 3 , further comprising at least one heterologous promoter region operably linked to one or more of said coding regions.  
     
     
         5 . The accessory function vector of  claim 3  wherein an inducible promoter is operably linked to the E2A 72 kD coding region.  
     
     
         6 . The accessory fuiction vector of  claim 5  wherein the inducible promoter is a small molecule-regulated promoter.  
     
     
         7 . The accessory function vector of  claim 6  wherein the promoter is an ecdysone-inducible promoter.  
     
     
         8 . The accessory function vector of  claim 3  wherein an inducible promoter is operably linked to the E1A coding region.  
     
     
         9 . The accessory function vector of  claim 8  wherein the inducible promoter is a small molecule-regulated promoter.  
     
     
         10 . The accessory function vector of  claim 9  wherein the promoter is an ecdysone-inducible promoter.  
     
     
         11 . The nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule lacks adenoviral early gene regions E2B and E3.  
     
     
         12 . The nucleic acid molecule of  claim 1 , wherein said nucleic acid molecule provides accessory functions capable of supporting efficient rAAV virion production in an human 293 host cell.  
     
     
         13 . The nucleic acid molecule of  claim 12 , wherein one or more of (a)-(e) are derived from an adenovirus type-2 or type-5 genome.  
     
     
         14 . The nucleic acid molecule of  claim 1 , wherein the nucleic acid molecule provides accessory fluctions capable of supporting efficient recombinant AAV (rAAV) virion production in a suitable host cell that is not infectable by adenovirus or is not capable of supporting adenovirus replication.  
     
     
         15 . The nucleic acid molecule of  claim 14 , wherein one or more of (a)-(e) are derived from an adenovirus type-2 or type-5 genome.  
     
     
         16 . An accessory function vector comprising the nucleic acid molecule of  claim 11 .  
     
     
         17 . The accessory function vector of  claim 16 , wherein said vector is a plasmid.  
     
     
         18 . An accessory function vector system, comprising: 
 (a) a nucleic acid sequence that provides adenovirus VA RNAs;    (b) an adenovirus E4 ORF6 coding region;    (c) an adenovirus E2A 72 kD coding region;    (d) an adenovirus E1A coding region; and    (e) an adenovirus E1B region lacking an intact E1B55k coding region;    wherein (a)-(e) are included on more than one accessory function vector of said system.    
     
     
         19 . The accessory function vector system of  claim 18 , wherein said vectors are plasmids.  
     
     
         20 . A host cell comprising the accessory function vector of  claim 2 .  
     
     
         21 . A host cell comprising the accessory function vector of  claim 16 .  
     
     
         22 . A host cell comprising the accessory function vector system of  claim 18 .  
     
     
         23 . A cell capable of producing recombinant AAV (rAAV) virions when transfected with an AAV vector, said cell comprising the host cell of  claim 20 , wherein the host cell further comprises an AAV helper construct that is capable of being expressed in said cell to provide AAV helper functions.  
     
     
         24 . A system for the production of recombinant AAV (rAAV) comprising: 
 (a) a first nucleic acid comprising an SV40 large T-antigen coding region that is operably linked to an inducible promoter;    (b) a second nucleic acid comprising an adenovirus E1A coding region;    (c) a third nucleic acid comprising an adenovirus E1B coding region;    (d) a fourth nucleic acid comprising an Epstein-Barr virus nuclear antigen 1 coding region;    (e) a fifth nucleic acid comprising an adenovirus VA RNA coding region;    (f) a sixth nucleic acid comprising an adenovirus E4 ORF6 coding region;    (g) a seventh nucleic acid comprising AAV vector sequences; and    (h) an eighth nucleic acid comprising an AAV rep and cap coding region, an adenovirus E2A gene, an SV40 origin of replication, an Epstein-Barr virus latent origin of replication, and a selectable marker, wherein said eighth nucleic acid lacks an intact AAV p5 promoter region.    
     
     
         25 . A host cell comprising the system of  claim 24 .  
     
     
         26 . A system for the production of recombinant AAV (rAAV) comprising: 
 (a) a first nucleic acid comprising an SV40 large T-antigen coding region that is operably linked to an inducible promoter, an adenovirus E1A coding region, an adenovirus E1B coding region, an Epstein-Barr virus nuclear antigen 1 coding region, an adenovirus VA RNA coding region, an adenovirus E4 ORF6 coding region, and a selectable marker;    (b) a second nucleic acid comprising AAV vector sequences and a selectable marker; and    (c) a third nucleic acid comprising AAV rep and cap coding regions, an adenovirus E2A gene, an SV40 origin of replication, an Epstein-Barr virus latent origin of replication, and a selectable marker, wherein said third nucleic acid lacks an intact AAV p5 promoter region.    
     
     
         27 . A host cell comprising the system of  claim 26 .  
     
     
         28 . The system of  claim 26 , wherein the SV40 large T-antigen coding region is mutated to eliminate transforming activity.  
     
     
         29 . The system of  claim 26 , wherein the E1A coding region is operably linked to an inducible promoter.  
     
     
         30 . The system of  claim 26 , wherein the E4 ORF6 coding region is operably linked to an adenovirus E4 promoter.  
     
     
         31 . The system of  claim 26 , wherein the SV40 large T-antigen coding region is operably linked to an ecdysone-inducible promoter, the E1A coding region is operably linked to an ecdysone-inducible promoter, and the second nucleic acid further comprises ecdysone receptor subunit coding regions.  
     
     
         32 . The system of  claim 31 , wherein the E2A coding region is operably linked to an ecdysone-inducible promoter.