Production of recombinant AAV virions

Abstract

Stocks of infectious rAAV are generated using yeast strains, bacterial strains, and bacteriophages engineered to express the required AAV proteins and harboring rAAV vector sequences. Stocks of rAAV virions of all serotypes and pseudotypes can be generated in prokaryotic and eukaryotic cells using the methods described herein.

What is claimed is:  
     
         1 . A nucleic acid comprising: 
 (A) a polynucleotide interposed between a first AAV inverted terminal repeat and second AAV inverted terminal repeat;    (B) a promoter operably linked to the polynucleotide; and    (C) a yeast origin of replication.    
     
     
         2 . The nucleic acid of  claim 1 , wherein the nucleic acid is comprised within a plasmid.  
     
     
         3 . The nucleic acid of  claim 2 , wherein the first AAV terminal repeat is an AAV serotype 2 terminal repeat.  
     
     
         4 . The nucleic acid of  claim 3 , wherein the second AAV terminal repeat is an AAV serotype 2 terminal repeat.  
     
     
         5 . The nucleic acid of  claim 1 , wherein the polynucleotide comprises a reporter gene.  
     
     
         6 . The nucleic acid of  claim 5 , wherein the reporter gene encodes a green fluorescent protein.  
     
     
         7 . The nucleic acid of  claim 1 , wherein the promoter is a cytomegalovirus promoter.  
     
     
         8 . The nucleic acid of  claim 1 , wherein the nucleic acid further comprises a selectable marker.  
     
     
         9 . The nucleic acid of  claim 8 , wherein the selectable marker is selected from the group consisting of: TRP, URA, HIS, LEU, and LYS.  
     
     
         10 . The nucleic acid of  claim 1 , wherein the origin of replication is 2 micron.  
     
     
         11 . The nucleic acid of  claim 1 , wherein the origin of replication is an autonomously replicating sequence.  
     
     
         12 . The nucleic acid of  claim 1 , further comprising a centromere sequence.  
     
     
         13 . The nucleic acid of  claim 12 , wherein the centromere sequence is CEN6.  
     
     
         14 . A nucleic acid comprising: 
 (A) a polynucleotide that encodes a protein selected from the group consisting of an AAV Rep protein and an AAV capsid protein;    (B) a promoter operably linked to the polynucleotide; and    (C) a yeast origin of replication.    
     
     
         15 . The nucleic acid of  claim 14 , wherein the nucleic acid comprises both a first nucleotide sequence encoding an AAV Rep protein and a second nucleotide sequence encoding an AAV capsid protein.  
     
     
         16 . The nucleic acid of  claim 15 , wherein the Rep protein is Rep52 or Rep 78.  
     
     
         17 . The nucleic acid of  claim 15 , wherein the nucleotide sequence encoding the AAV Rep protein is operably linked to the promoter.  
     
     
         18 . The nucleic acid of  claim 15 , wherein the nucleotide sequence encoding the AAV Rep protein is operably linked to a transcription termination region.  
     
     
         19 . The nucleic acid of  claim 15 , wherein the AAV capsid protein is selected from the group consisting of VP1, VP2, and VP3.  
     
     
         20 . The nucleic acid of  claim 15 , wherein the nucleotide sequence encoding the AAV capsid protein is operably linked to the promoter.  
     
     
         21 . The nucleic acid of  claim 15 , wherein the nucleotide sequence encoding the AAV capsid protein is operably linked to a transcription termination region.  
     
     
         22 . The nucleic acid of  claim 21 , wherein the transcription termination region is from ADH1.  
     
     
         23 . The nucleic acid of  claim 21 , wherein the transcription termination region is from CYC1.  
     
     
         24 . The nucleic acid of  claim 14 , wherein the promoter is selected from the group consisting of: GAL1, GAL10, ADH, and CYC1.  
     
     
         25 . The nucleic acid of  claim 14 , wherein the nucleic acid further comprises a selectable marker.  
     
     
         26 . The method of  claim 25 , where in the selectable marker is selected from the group consisting of: TRP, URA, HIS, LEU, and LYS.  
     
     
         27 . The nucleic acid of  claim 14 , wherein the origin of replication is 2 micron.  
     
     
         28 . The nucleic acid of  claim 14 , wherein the origin of replication is an autonomously replicating sequence.  
     
     
         29 . The nucleic acid of  claim 14 , further comprising a centromere sequence.  
     
     
         30 . The nucleic acid of  claim 29 , wherein the centromere sequence is CEN6.