Recombinantly-modified adeno-associated virus helper vectors and their use to improve the packaging efficiency of recombinantly-modified adeno-associated virus

Abstract

The present invention is directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that are capable of increasing the packaging efficiency of recombinantly-modified adeno-associated virus (rAAV) and their use to improve the packaging efficiency of such rAAV. The present invention is particularly directed to recombinantly-modified adeno-associated virus (AAV) helper vectors that have been further modified to replace (or augment) the P5 and/or P40 promoter sequences that are natively associated with the Rep proteins encoded by such rAAV with AAV P5 and/or P40 promoters that are associated with the Rep proteins of an rAAV of different serotype. The use of such substitute or additional promoter sequences causes increased production of recombinantly-modified adeno-associated virus.

What is claimed is: 
     
         1 . A recombinantly-modified adeno-associated virus (AAV) helper vector that comprises an AAV helper function-providing polynucleotide, wherein said polynucleotide comprises a non-native AAV serotype P5 or P40 promoter sequence. 
     
     
         2 . The recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 1 , wherein said AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P5 promoter sequence. 
     
     
         3 . The recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 1 , wherein said AAV helper function-providing polynucleotide vector comprises a non-native AAV serotype P40 promoter sequence. 
     
     
         4 . The recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 1 , wherein said vector is a plasmid vector. 
     
     
         5 . The recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 1 , wherein said non-native AAV serotype P5 or P40 promoter sequence replaces a native AAV serotype promoter sequence. 
     
     
         6 . The recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 1 , wherein said vector additionally comprises a non-AAV helper function-providing polynucleotide. 
     
     
         7 . A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein said method comprises culturing cells that have been transfected with:
 (1) said rAAV; and   (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of  claim 6 ;   wherein said culturing is conducted in a culture medium under conditions sufficient to permit the production of said rAAV and wherein the presence of said non-native AAV serotype P5 or P40 promoter sequence causes said cells to produce said rAAV at an increased production titer relative to that which would be attained if said AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.   
     
     
         8 . The method of  claim 7 , wherein said transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition. 
     
     
         9 . The method of  claim 7 , wherein:
 (A) said AAV helper function-providing polynucleotide of said vector encodes an AAV1 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (C) said AAV helper function-providing polynucleotide of said vector encodes an AAV3 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of said serotypes; or   (H) said AAV helper function-providing polynucleotide of said vector encodes an AAV8 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of said serotypes.   
     
     
         10 . The method of  claim 7 , wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells. 
     
     
         11 . The method of  claim 10 , wherein said cells are HEK293 human embryonic kidney cells. 
     
     
         12 . The method of  claim 10 , wherein said cells are BHK21 baby hamster kidney cells. 
     
     
         13 . A method for increasing the production titer of a recombinantly-modified adeno-associated virus (rAAV) that comprises a transgene cassette, wherein said method comprises culturing cells that have been transfected with:
 (1) said rAAV;   (2) the recombinantly-modified adeno-associated virus (AAV) helper vector of  claims 1 ; and   ( 3 ) an additional vector that comprises a non-AAV helper function-providing polynucleotide;   wherein said culturing is conducted in a culture medium under conditions sufficient to permit the production of said rAAV and wherein the presence of said non-native AAV serotype P5 or P40 promoter sequence causes said cells to produce said rAAV at an increased production titer relative to that which would be attained if said AAV helper function-providing polynucleotide contained native serotype P5 and P40 promoters.   
     
     
         14 . The method of  claim 13 , wherein said transgene cassette encodes a protein, or comprises a transcribed nucleic acid, that is therapeutic for a genetic or heritable disease or condition. 
     
     
         15 . The method of  claim 13 , wherein:
 (A) said AAV helper function-providing polynucleotide of said vector encodes an AAV1 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (C) said AAV helper function-providing polynucleotide of said vector encodes an AAV3 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or a hybrid of one or more of said serotypes;   (G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or a hybrid of one or more of said serotypes; or   (H) said AAV helper function-providing polynucleotide of said vector encodes an AAV8 Cap protein, and said non-native AAV serotype promoter sequence is a promoter sequence of an AAV of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid of one or more of said serotypes.   
     
     
         16 . The method of  claim 13 , wherein said cells are human embryonic kidney cells, baby hamster kidney cells or sf9 insect cells. 
     
     
         17 . The method of  claim 16 , wherein said cells are HEK293 human embryonic kidney cells. 
     
     
         18 . The method of  claim 16 , wherein said cells are BHK21 baby hamster kidney cells. 
     
     
         19 . A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of  claim 6 , and a pharmaceutically acceptable carrier. 
     
     
         20 . A pharmaceutical composition that comprises recombinantly-modified adeno-associated virus (rAAV) produced by the method of  claim 13 , and a pharmaceutically acceptable carrier.