Adeno-associated virus with site-directed mutagenesis and site-directed modification and preparation method and application thereof

Patent metadata

Field	Value
Publication number	US-10087217-B2
Application number	US-201415033723-A
Country	US
Kind code	B2
Filing date	Oct 30, 2014
Priority date	Oct 30, 2013
Publication date	Oct 2, 2018
Grant date	Oct 2, 2018

Abstract

The present invention relates to an adeno-associated virus with site-directed mutagenesis and site-specific modification, and a preparation method and uses thereof. Specifically, the present invention uses genetic code expansion techniques to incorporate non-natural amino acid into an adeno-associated virus capsid protein VP1 or fragment thereof, thereby obtaining an adeno-associated virus with site-directed mutagenesis using the non-natural amino acid. The adeno-associated virus with site-directed mutagenesis is equivalent to a wild-type virus in terms of production, transduction and mobility, can couple with other functional molecules, such as targeting molecules, and can carry a functional gene in a normal manner, which indicates that the adeno-associated virus with site-directed mutagenesis can be used as a tool adeno-associated virus, and applied in various aspects associated with adeno-associated virus such as finding adeno-associated virus binding proteins or using as target genetic therapy vector.

First claim

What is claimed is:

1. A site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof,
wherein an amino acid at a specific site of a corresponding wild type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated as an non-natural amino acid, and the specific site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or fragment thereof,
wherein the non-natural amino acid is Nε-2-azideoethyloxycarbonyl-L-lysine (NAEK),

 or the non-natural amino acid is an non-natural amino acid with structure DiZPK.

2. A site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof, wherein an amino acid at a specific site of a corresponding wild type adeno-associated virus capsid protein VP1 or a fragment thereof is mutated as an non-natural amino acid, and the specific site is at least one site selected from sites R447, G453, S578, N587, N587+1, S662 of VP1 or fragment thereof;
wherein the non-natural amino acid is an azido-containing non-natural amino acid, Nε-2-azideoethyloxycarbonyl-L-lysine (NAEK),

 or the non-natural amino acid is an non-natural amino acid with structure DiZPK;
wherein the NAEK and the amino acid sequence of VP1 or fragment thereof are linked as shown in Formula I:

wherein the direction from R1 to R2 is the direction from N-terminal to C-terminal of the amino acid sequence, in which amino acid at site N is one of amino acids at sites selected from site R447, site G453, site S578, site N587, site N587+1, site S662, R1 is an amino acid residue at site 1 to site N-1 of the amino acid sequence of VP1 or fragment thereof, R2 is an amino acid residue at site N+1 to C-terminal of the amino acid sequence of VP1 or fragment thereof;
wherein the DiZPK and the amino acid sequence of VP1 or fragment thereof are linked as shown in Formula II:

wherein the direction from R1 to R2 is the direction from N-terminal to C-terminal of the amino acid sequence, in which amino acid at site N is one of amino acids at sites selected from site R447, site G453, site S578, site N587, site N587+1, site S662, R1 is an amino acid residue at site 1 to site N-1 of the amino acid sequence of VP1 protein or fragment thereof, R2 is an amino acid residue at site N+1 to C-terminal of the amino acid sequence of VP1 protein or fragment thereof,
R3 is

3. The site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof according to claim 2 , wherein the adeno-associated virus is type II adeno-associated virus.

4. The site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof according to claim 2 , wherein the non-natural amino acid further links to a labeling group, fluorescence labeling group, or a labeling group capable of occurring a click chemistry with azide; or the non-natural amino acid further links to a targeting molecule, preferably, the targeting molecule further links to a group capable of occurring a click chemistry with azide.

5. A site-specifically mutated adeno-associated virus capsid protein, comprising the adeno-associated virus capsid protein VP1 or fragment thereof according to claim 2 .

6. A site-specifically mutated adeno-associated virus, comprising the adeno-associated virus capsid protein VP1 or fragment thereof according to claim 2 .

7. The adeno-associated virus according to claim 6 , wherein the non-natural amino acid further links to a labeling group, a fluorescence labeling group, or a labeling group capable of occurring a click chemistry with azide.

8. The adeno-associated virus according to claim 6 , wherein the non-natural amino acid further links to a targeting molecule, preferably, the targeting molecule further links to a group capable of occurring a click chemistry with azide.

9. The adeno-associated virus according to claim 6 , which carries a functional nucleic acid fragment or a nucleic acid fragment of a labeling molecule.

10. A composition or kit, which comprises the adeno-associated virus according to claim 2 .

11. A gene vaccine, which comprises the adeno-associated virus according to claim 2 .

12. A composition or kit, which comprises the adeno-associated virus according to claim 6 .

13. A gene vaccine, which comprises the adeno-associated virus according to claim 6 .

14. The site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof according to claim 1 , wherein the adeno-associated virus is type II adeno-associated virus.

15. The site-specifically mutated adeno-associated virus capsid protein VP1 or a fragment thereof according to claim 1 , wherein the non-natural amino acid further links to a labeling group, fluorescence labeling group, or a labeling group capable of occurring a click chemistry with azide; or the non-natural amino acid further links to a targeting molecule, preferably, the targeting molecule further links to a group capable of occurring a click chemistry with azide.

16. A site-specifically mutated adeno-associated virus capsid protein, comprising the adeno-associated virus capsid protein VP1 or fragment thereof according to claim 1 .

17. A site-specifically mutated adeno-associated virus, comprising the adeno-associated virus capsid protein VP1 or fragment thereof according to claim 1 .

18. The adeno-associated virus according to claim 17 , wherein the non-natural amino acid further links to a labeling group, a fluorescence labeling group, or a labeling group capable of occurring a click chemistry with azide.

19. The adeno-associated virus according to claim 17 , wherein the non-natural amino acid further links to a targeting molecule, preferably, the targeting molecule further links to a group capable of occurring a click chemistry with azide.

20. The adeno-associated virus according to claim 17 , which carries a functional nucleic acid fragment or a nucleic acid fragment of a labeling molecule.

21. A composition or kit, which comprises the adeno-associated virus according to claim 1 .

22. A gene vaccine, which comprises the adeno-associated virus according to claim 1 .

23. A composition or kit, which comprises the adeno-associated virus according to claim 17 .

24. A gene vaccine, which comprises the adeno-associated virus according to claim 17 .

Assignees

• Univ Beijing

Inventors

• Zhou Demin
• Zhang Chuanling
• Xiao Sulong
• Yao Tianzhuo
• Zheng Yongxiang
• Yu Fei
• Si Longlong
• Zhang Lihe

Classifications

• C12N2750/14134

  Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein · CPC title

• C12N2750/14151

  Methods of production or purification of viral material · CPC title

• C12N15/86

  Viral vectors · CPC title

• C12N2750/14145

  Special targeting system for viral vectors · CPC title

• C07K14/005 Primary

  from viruses · CPC title