Methods and compositions for treating negative-sense single-stranded RNA virus

Patent metadata

Field	Value
Publication number	US-12227769-B2
Application number	US-201917309038-A
Country	US
Kind code	B2
Filing date	Oct 18, 2019
Priority date	Oct 19, 2018
Publication date	Feb 18, 2025
Grant date	Feb 18, 2025

Abstract

The current disclosure relates to methods, compositions and kits for detecting modified adenosine in a target RNA molecule. Aspects relate to a method for detecting modified adenosine in a target ribonucleic acid (RNA) comprising contacting the target RNA with an adenosine deaminase enzyme (adenosine deaminase, RNA-specific) to generate a target RNA with deaminated adenosines and sequencing the target RNA with deaminated adenosines; wherein the modified adenosine is detected when the nucleotide sequence includes adenosine within a m6A motif.

First claim

The invention claimed is:

1. An attenuated negative-sense single-stranded RNA virus of the family Pneumoviridae comprising sequence alterations in the viral genome or antigenome corresponding to the G gene encoding one or more N 6 -methyladenosine (m6A) consensus sites, wherein the alterations result in reduction of m6A modifications of viral mRNA, antigenome or genome.

2. The attenuated virus of claim 1 , wherein the sequence alterations change at least two nucleotides of at least one m6A consensus site.

3. The attenuated virus of claim 1 , wherein at least one sequence alteration comprises a change of an adenine or a cytosine in an m6A consensus site in virus mRNA and/or the virus antigenome.

4. The attenuated virus of claim 3 , wherein there are at least two sequence alterations and the two sequence alterations comprise a change of an adenine and cytosine in the same m6A consensus site in virus mRNA and/or the virus antigenome.

5. The attenuated virus of claim 1 , wherein at least one of the sequence alterations changing an m6A consensus site does not alter the amino acid sequence of an encoded polypeptide.

6. The attenuated virus of claim 5 , wherein the virus comprises alterations in encoded m6A consensus sites in at least three different positions in the genome or antigenome.

7. The attenuated virus of claim 1 , wherein the virus is respiratory syncytial virus (RSV).

8. The attenuated RSV of claim 7 , wherein the mutated m6A consensus sites are in regions 392-467 nt, 567-660 nt, and/or 716-795 nt of the G gene in reference to SEQ ID NO: 1.

9. The attenuated RSV of claim 8 , wherein mutated consensus sites are in regions 392-467 nt, 567-660 nt, and 716-795 nt of the G gene in reference to SEQ ID NO: 1.

10. The attenuated virus of claim 1 , wherein the virus comprises metapneumovirus (MPV).

11. The attenuated virus of claim 10 , wherein mutated m6A consensus sites comprise one or more consensus sites selected from the following consensus sites in the MPV antigenome: site 1, 171-AAm 6 AC»TA-175; site 2, 187-GAm 6 A»GCA-191; site 3, 227-AAm 6 ACT»G-231; site 4, 246-AGm 6 AC»TA-250; site 5, 255-AGm 6 AC»TA-259; site 6, 341-AGm 6 ACA»G-345; site 7, 346-GAm 6 A»GCC-351; site 8, 422-GAm 6 ACA»G-426; site 9, 428-AGm 6 ACA»G-432; site 10, 453-AAm 6 AC»TA-457; site 11, 464-GGm 6 ACA»G-468; site 12, 476-GAm 6 ACA»G-480; site 13, 518-GAm 6 ACC»G-522; and site 14, 553-AGm 6 A»GCC-557 in reference to SEQ ID NO: 3.

12. The attenuated virus of claim 11 , wherein mutated m6A consensus sites comprise one or more consensus sites selected from the following consensus sites in the MPV genome: site 1, 237-G»CGm 6 TC»GC-241; site 2, 290-AG»Am 6 TCC»A-294; site 3, 433-AGm 6 T»C CC-437; site 4, 441-A»C Gm 6 TC»GC-445; site 5, 570-AGm 6 T»C CC-574; and site 6, 616-AG»Am 6 TCC»G-620 in reference to the complement of SEQ ID NO: 3.

13. The attenuated virus of claim 1 , wherein the alterations do not change an encoded amino acid.

14. The attenuated virus of claim 1 , wherein replication of the attenuated virus is at least 3-fold reduced compared to a virus without mutations in the viral genome encoding one or more N 6 -methyladenosine (m6A) consensus sites in viral mRNA, the viral antigenome, or the viral genome.

15. A method for increasing immunity to a negative-sense single-stranded RNA virus of the family Pneumoviridae in a patient comprising administering to the patient a composition comprising the attenuated virus of claim 1 .

16. The method of claim 15 , wherein the patient is a pediatric patient.

17. The method of claim 15 , wherein the patient does not have symptoms of a viral infection.

18. A method for producing an attenuated negative-sense single-stranded RNA virus of the family Pneumoviridae comprising infecting a cell line with the attenuated virus of claim 1 ; culturing the cell line under conditions to promote virus replication; and collecting virus particles.

19. The method of claim 18 , wherein culturing the cell line under conditions to promote virus replication comprises culturing under serum-free conditions.

20. The method of claim 18 , wherein the cell line comprises cells that are reduced in endogenous expression of one or more m6A writer proteins.

21. The method of claim 20 , wherein the writer proteins comprise methyltransferase-like 3 (METTL3) and/or methyltransferase-like 14 (METTL14).

22. The method of claim 18 , wherein the cell line comprises cells that are in reduced in expression of one or more m6A eraser proteins.

23. The method of claim 22 , wherein the eraser proteins comprise fat mass and obesity-associated (FTO) and/or AlkB homolog 5 (ALKBH5).

24. The attenuated virus of claim 1 , wherein the virus comprises respiratory syncytial virus (RSV) or metapneumovirus (MPV).

25. The attenuated virus of claim 1 , wherein at least one sequence alteration comprises a change of a cytosine in an m6A consensus site in virus mRNA and/or the virus antigenome.

Assignees

• Univ Chicago
• Nationwide Childrens Hospital
• Univ Ohio State

Inventors

• Li Jianrong
• Peeples Mark E
• He Chuan
• Niewiesk Stefan
• Lu Mijia
• Xue Miaoge
• Zhang Zijie
• Zhao Boxuan

Classifications

• C12N2760/18522

  New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes · CPC title

• A61K35/76

  Viruses; Subviral particles; Bacteriophages · CPC title

• A61K2039/543

  intranasal · CPC title

• A61K2039/5254

  avirulent or attenuated · CPC title

• C12N2760/18534

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