IP Library › Granted Patent US 12,227,769
Granted Patent B2
US 12,227,769 · App. 17/309,038 · Granted Feb 18, 2025

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

Inventors: Jianrong Li (Dublin, OH); Mark E. Peeples (Bexley, OH); Chuan He (Chicago, IL); Stefan Niewiesk (Dublin, OH); Mijia Lu (Columbus, OH); Miaoge Xue (Columbus, OH); Zijie Zhang (Chicago, IL); Boxuan Zhao (Palo Alto, CA)
Assignees: The University of Chicago; Nationwide Children's Hospital; The Ohio State University
C12N7/00A61K35/76C12N2760/18522
View Patent ↗
Loading inventors, assignments & file history…
Monitor This Case
Get email alerts when status or documents change.
Order Certified Copies
Most orders are placed with the USPTO same day — all within 24 business hours.
Order via The Patent Place →
Pre-filled with this patent's details
Quick Facts
Patent No.
US 12,227,769
App. No.
17/309,038
Granted
Feb 18, 2025
Kind
B2
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.

Claims (25)

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.

Assignments (4)
CONFIRMATORY LICENSE Recorded Dec 5, 2023
From: UNIVERSITY OF CHICAGO
To: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT
Reel/Frame 065774/0812 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 16, 2021
From: HE, CHUAN; ZHANG, ZIJIE; ZHAO, BOXUAN
To: THE UNIVERSITY OF CHICAGO
Reel/Frame 055941/0056 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 16, 2021
From: PEEPLES, MARK E.
To: NATIONWIDE CHILDREN'S HOSPITAL
Reel/Frame 055941/0090 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Apr 16, 2021
From: LI, JIANRONG; NIEWIESK, STEFAN; LU, MIJIA; XUE, MIAOGE
To: THE OHIO STATE UNIVERSITY
Reel/Frame 055941/0148 →
Continuity (2)
Provisional Application 62748175 · Oct 19, 2018
Related Publication 20220033783A1 · Feb 3, 2022
References Cited (26)
WO WO9818324 · 1998 [cited by applicant]
WO WO2017177029 · 2017 [cited by applicant]
Gokhale et al. N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection. Cell Host & Microbe 20, 654-665, Nov. 9, 2016. [cited by examiner]
Imam et al. N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle. PNAS, 115(35): 8829-8834, Aug. 28, 2018. [cited by examiner]
Gonzales-van Horn et al. Making the Mark: The Role of Adenosine Modifications in the Life Cycle of RNA Viruses. Cell Host & Microbe 21, Jun. 14, 2017, p. 661-669. [cited by examiner]
Zhang et al. Rational Design of Human Metapneumovirus Live Attenuated Vaccine Candidates by Inhibiting Viral mRNA Cap Methyltransferase. Journal of Virology, 2014, 88(19): 11411-11429. [cited by examiner]
“17th NSV 2018.” [cited by applicant]
Canaani et al., “Identification and mapping of N6-methyladenosine containing sequences in simian virus 40 RNA.” [cited by applicant]
Collins et al., “Production of infectious human respiratory syncytial virus from cloned cDNA confirms an essential role for the transcription elongation factor from the 5′ proximal open reading frame of the M2 mRNA in g… [cited by applicant]
Furuichi et al., “Blocked, methylated 5′-terminal sequence in avian sarcoma virus RNA” [cited by applicant]
Gokhale et al., “N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection” [cited by applicant]
Hesser et al., “N6-methyladenosine modification and the YTHDF2 reader protein play cell type specific roles in lytic viral gene expression during Kaposi's sarcoma-associated herpesvirus infection.” [cited by applicant]
International Search Report and Written Opinion issued in Corresponding PCT Application No. PCT/US2019/056942, dated Mar. 17, 2020. [cited by applicant]
Kane et al., “Inhibition of Methylation at Two Internal N [cited by applicant]
Kane et al., “Precise localization of m6A in Rous sarcoma virus RNA reveals clustering of methylation sites: implications for RNA processing.” [cited by applicant]
Kennedy et al., “Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression.” [cited by applicant]
Krug et al., “Influenza viral mRNA contains internal N6-methyladenosine and 5′-terminal 7-methylguanosine in cap structures.” [cited by applicant]
Li, A., et al., “Cytoplasmic m6A reader YTHDF3 promotes mRNA translation.” [cited by applicant]
Moss et al., “5′-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.” [cited by applicant]
Shi, H., et al., “YTHDF3 facilitates translation and decay of N6-methyladenosinemodified RNA.” [cited by applicant]
Tirumuru et al., “N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression.” [cited by applicant]
Tsai et al., “Addition of m6A to SV40 late mRNAs enhances viral structural gene expression and replication” [cited by applicant]
Winkler et al., “m(6)A modification controls the innate immune response to infection by targeting type I interferons.” [cited by applicant]
Xue et al., “Viral N6-methyladenosine upregulates replication and pathogenesis of human respiratory syncytial virus” [cited by applicant]
Ye et al., “Kaposi's Sarcoma-Associated Herpesvirus Utilizes and Manipulates RNA N(6)-Adenosine Methylation To Promote Lytic Replication.” [cited by applicant]
Yue et al., “RNA N6-methyladenosine methylation in posttranscriptional gene expression regulation.” [cited by applicant]