JPWO2021011029A5

JPWO2021011029A5 -

Info

Publication number: JPWO2021011029A5
Application number: JP2022502941A
Authority: JP
Publication date: 2023-02-01

Description

非天然ＡＡＶ血清型プロモーター配列がｒＡＡＶの産生力価に影響を及ぼす能力をさらに示すために、そのようなプラスミドの天然ＡＡＶ２血清型Ｐ５（図１５Ａ；下向き縞模様の長方形）およびｐ４０（図１５Ａ；黒で塗られた長方形）プロモーターの代わりに非天然ＡＡＶ血清型プロモーター配列を含むＡＡＶヘルパープラスミドＡＡＶＲＣ６（ＡＡＶ２ｒｅｐ遺伝子およびＡＡＶ６血清型のＣａｐタンパク質をコードするｃａｐ遺伝子を有する）の誘導体を構築した（図１１）。構築物のＡＡＶ２Ｐ５プロモーターは変化せず、したがって天然ＡＡＶ２血清型プロモーター配列であった（図１５Ａ；黒で塗られた長方形）。 To further demonstrate the ability of non-native AAV serotype promoter sequences to influence rAAV production titers, native AAV2 serotypes P5 (Fig. 15A; down-striped rectangles) and p40 (Fig. 15A; A derivative of the AAV helper plasmid AAV RC6 (having the AAV2 rep gene and the cap gene encoding the Cap protein of the AAV6 serotype) containing a non-native AAV serotype promoter sequence in place of the black filled rectangle) promoter was constructed ( Figure 11). The AAV2 P5 promoter of the construct was unchanged and thus the native AAV2 serotype promoter sequence (Fig. 15A; filled rectangles).

非天然ＡＡＶ血清型プロモーター配列がｒＡＡＶの産生力価に影響を及ぼす能力をさらに示すために、そのようなプラスミドの天然ＡＡＶ２血清型Ｐ５（図１６Ａ；下向き縞模様の長方形）および／またはｐ４０（図１６Ａ；黒で塗られた長方形））プロモーターの代わりにＡＡＶヘルパープラスミドＡＡＶＲＣ１（ＡＡＶ２ｒｅｐ遺伝子およびＡＡＶ１血清型のＣａｐタンパク質をコードするｃａｐ遺伝子を有する）の誘導体、ＡＡＶヘルパープラスミドＡＡＶＲＣ５（ＡＡＶ２ｒｅｐ遺伝子およびＡＡＶ５血清型のＣａｐタンパク質をコードするｃａｐ遺伝子を有する）の誘導体、およびＡＡＶヘルパープラスミドＡＡＶＲＣ７（ＡＡＶ２ｒｅｐ遺伝子およびＡＡＶ７血清型のＣａｐタンパク質をコードするｃａｐ遺伝子を有する）の誘導体を、非天然ＡＡＶ血清型プロモーター配列を有するように構築した（図１１）。 To further demonstrate the ability of non-native AAV serotype promoter sequences to influence rAAV production titers, native AAV2 serotypes P5 (Fig. 16A; down-striped rectangles) and/or p40 (Fig. 16A; filled rectangle)) Derivatives of the AAV helper plasmid AAV RC1 (having the AAV2 rep gene and the cap gene encoding the Cap protein of the AAV1 serotype) instead of the promoter, AAV helper plasmid AAV RC5 (the AAV2 rep gene and of the AAV helper plasmid AAV RC7 (having the AAV2 rep gene and the cap gene encoding the Cap protein of the AAV7 serotype) were added to the non-natural AAV It was constructed with a serotype promoter sequence (Fig. 11).

Claims

A recombinant modified adeno-associated virus (AAV) helper vector comprising an AAV helper function-providing polynucleotide, said polynucleotide comprising a non-native AAV serotype P5 or P40 promoter sequence. vector.

2. The recombinant 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.

2. The recombinant 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.

The recombinant modified adeno-associated virus (AAV) helper vector of claim 1, wherein said vector is a plasmid vector.

2. The recombinant 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.

2. The recombinant modified adeno-associated virus (AAV) helper vector of claim 1, wherein said vector further comprises a non-AAV helper function providing polynucleotide.

A method of increasing the production titer of a recombinant modified adeno-associated virus (rAAV) containing a transgene cassette, said method comprising culturing cells transfected with:
(1) the rAAV; and (2) the recombinant modified adeno-associated virus (AAV) helper vector of claim 6,
The culturing is carried out in a medium under conditions sufficient to permit production of the rAAV, and the presence of the non-native AAV serotype P5 or P40 promoter sequence indicates that the cells provide the AAV helper function. A method that causes production of said rAAV with increased production titers compared to those achieved when the polynucleotide contains the native serotype P5 and P40 promoters.

8. The transgene cassette of claim 7, wherein said transgene cassette comprises a protein-encoding or transcribed nucleic acid, said protein or said transcribed nucleic acid being therapeutic for a genetic or hereditary disease or condition. A recombinant modified adeno-associated virus (rAAV) as described.

8. The method of claim 7, wherein:
(A) the AAV helper function-providing polynucleotide of the vector encodes an AAV1 Cap protein and the non-native AAV serotype promoter sequence is serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or the serotype is one or more hybrid AAV promoter sequences of
(B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence comprises serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8; AAV promoter sequences of one or more hybrid 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 of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or said serotype or (H) said AAV helper function-providing polynucleotide of said vector encodes AAV8 Cap protein and said non-native AAV serotype promoter sequence is serotype AAV1 , AAV3, AAV4, AAV5, AAV6 or AAV7, or the promoter sequence of AAV that is a hybrid of one or more of said serotypes;
Method.

8. 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.

11. The method of claim 10, wherein said cells are BHK21 baby hamster kidney cells.

A method of increasing the production titer of a recombinant modified adeno-associated virus (rAAV) containing a transgene cassette, said method comprising culturing cells transfected with:
(1) the rAAV;
(2) a recombinant modified adeno-associated virus (AAV) helper vector according to claim 1; and (3) a further vector, in particular a plasmid vector, comprising a non-AAV helper function providing polynucleotide;
The culturing is carried out in a medium under conditions sufficient to permit production of the rAAV, and the presence of the non-native AAV serotype P5 or P40 promoter sequence indicates that the cells provide the AAV helper function. A method that causes production of said rAAV with increased production titers compared to those achieved when the polynucleotide contains the native serotype P5 and P40 promoters.

14. The transgene cassette of claim 13, wherein said transgene cassette comprises a protein-encoding or transcribed nucleic acid, said protein or said transcribed nucleic acid being therapeutic for a genetic or hereditary disease or condition. described method.

14. The method of claim 13, wherein
(A) the AAV helper function-providing polynucleotide of the vector encodes an AAV1 Cap protein and the non-native AAV serotype promoter sequence is serotype AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, or the serotype is one or more hybrid AAV promoter sequences of
(B) said AAV helper function-providing polynucleotide of said vector encodes an AAV2 Cap protein, and said non-native AAV serotype promoter sequence comprises serotype AAV1, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8; AAV promoter sequences of one or more hybrid 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 of serotype AAV1, AAV4, AAV5, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(D) said AAV helper function-providing polynucleotide of said vector encodes an AAV4 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV5, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(E) said AAV helper function-providing polynucleotide of said vector encodes an AAV5 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV6, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(F) said AAV helper function-providing polynucleotide of said vector encodes an AAV6 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV5, AAV7 or AAV8, or said serotype is one or more hybrid AAV promoter sequences of
(G) said AAV helper function-providing polynucleotide of said vector encodes an AAV7 Cap protein and said non-native AAV serotype promoter sequence is of serotype AAV1, AAV3, AAV4, AAV5, AAV6 or AAV8, or said serotype or (H) said AAV helper function-providing polynucleotide of said vector encodes AAV8 Cap protein and said non-native AAV serotype promoter sequence is serotype AAV1 , AAV3, AAV4, AAV5, AAV6 or AAV7, or a hybrid AAV of one or more of said serotypes.
Method.

14. 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.

17. The method of claim 16, wherein said cells are BHK21 baby hamster kidney cells.

7. A pharmaceutical composition comprising the recombinant modified adeno-associated virus (rAAV) of claim 6 and a pharmaceutically acceptable carrier.

14. A pharmaceutical composition comprising the recombinant modified adeno-associated virus (rAAV) of claim 13 and a pharmaceutically acceptable carrier.