Pan-genotypic agents against influenza virus and methods of using the same

1. An oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence comprising one or more modified ribose moieties and at least 8 nucleoside subunits complementary to a region of a Packaging Stem-Loop 2 (PSL2) motif of a PB2 viral RNA (vRNA) or mutant thereof, wherein said region includes nucleotides on both strands of the upper stem of the PSL2 motif, and wherein the oligonucleotide compound inhibits virus production.

2. The compound of claim 1 , wherein the oligonucleotide comprises an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages.

3. The compound of claim 2 , wherein the oligonucleotide comprises one or more chiral internucleoside linkages.

4. The compound of claim 1 , wherein the oligonucleotide comprises a bridged nucleic acid (BNA) nucleotide.

5. The compound of claim 4 , wherein the BNA nucleotide is selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

6. The compound of claim 1 , wherein the oligonucleotide comprises one or more 2′-modified nucleotides.

7. The compound of claim 1 , wherein the oligonucleotide comprises the sequence.

(SEQ ID NO: 56)

5′ CGACCAAAAGAATTC 3′;

8. The compound of claim 1 , wherein the oligonucleotide comprises a sequence selected from:

(SEQ ID NO: 98)

5′CGACCAAAAGAATT 3′; and

(SEQ ID NO: 99)

5′GACCAAAAGAATTCGG 3′.

9. The compound of claim 1 , comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from SEQ ID NOs. 56 and 98-99.

10. The compound of claim 8 , wherein the oligonucleotide comprises at least 5 deoxyribonucleotide units and is capable of recruiting an RNase.

11. The compound of claim 1 , wherein the oligonucleotide comprises a sequence selected from:

LNA14.1: 

 (SEQ ID NO: 133)

5′ GCGAccaaaagaATT 3′;

LNA14.2: 

(SEQ ID NO: 134)

5′ GCGAccaaaagAATT 3′;

LNA14.5: 

(SEQ ID NO: 135)

5′ CGACcaaaagaATT3′;

LNA14.7: 

(SEQ ID NO: 136)

5′ CGAccaaaagaATTC 3′;

LNA14.8: 

(SEQ ID NO: 137)

5′ CGACcaaaagaaTTC 3′;

LNA14.9: 

(SEQ ID NO: 138)

5′ CGAccaaaagaaTTC 3′;

LNA14.10: 

(SEQ ID NO: 139)

5′ GACcaaaagaaTTC 3′;

LNA14.14: 

(SEQ ID NO: 140)

5′ GCGAccaaaagaATTC 3′; 

LNA14.15: 

(SEQ ID NO: 141)

5′ GCGaccaaaagaATTC 3′; 

LNA14.16: 

(SEQ ID NO: 142)

5′ GCGaccaaaagaaTTC 3′; 

LNA14.17: 

(SEQ ID NO: 143)

5′ CGAccaaaagaaTTCG 3′; 

LNA14.19: 

(SEQ ID NO: 144)

5′ CGACcaaaagaaTTCG 3′; 

LNA14.22: 

(SEQ ID NO: 145)

5′ GACcaaaagaaTTC 3′;

LNA14.24: 

(SEQ ID NO: 146)

5′ ACCAaaagaaTTC 3′;

LNA14.27: 

(SEQ ID NO: 147)

5′ CGAccaaaagaatTCGG 3′;

LNA14.28: 

(SEQ ID NO: 148)

5′ GACcaaaagaatTCGG 3′;

LNA14.30: 

(SEQ ID NO: 149)

5′ GACCaaaagaattCGG 3′;

LNA14.31: 

(SEQ ID NO: 150)

5′ GACcaaaagaattCGG 3′;  

and

LNA14.32: 

(SEQ ID NO: 151)

5′ ACCAaaagaattCGGA 3′, 

wherein capitalized letters denote LNA nucleotides and lowercase letters denote DNA nucleotides.

12. The compound of claim 1 , comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from SEQ ID NOs. 133-151.

13. A method of inhibiting influenza A virus in a cell, the method comprising:

contacting a sample comprising viral RNA (vRNA) having a PSL2 motif with an effective amount of the oligonucleotide compound according to claim 1 .

14. The method of claim 13 , wherein contacting the sample with an agent results in at least 1 log 10 titer deficits of the virus or the agent disrupts the overall structure of the PSL2 motif of the vRNA.

15. The method of claim 13 , wherein the vRNA is isolated from a virion or a cell.

16. A method of treating or preventing influenza A virus infection in a subject, the method comprising:

administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the oligonucleotide compound according to claim 1 .

17. The method of claim 16 , wherein the subject is at risk of influenza A virus infection and the administering of the oligonucleotide compound protects the subject against infection for 1 week or more.

18. The method of claim 17 , wherein the administering comprises weekly, biweekly, or monthly administration of an effective dose of the oligonucleotide compound.

19. The method of claim 16 , wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral drug.

20. The method of claim 16 , wherein the subject has been diagnosed with or suspect of having an influenza A virus infection.

21. The compound of claim 7 , wherein the oligonucleotide comprises a bridged nucleic acid (BNA) nucleotide.

22. The compound of claim 21 , wherein the BNA nucleotide is selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

23. The compound of claim 8 , wherein the oligonucleotide comprises a bridged nucleic acid (BNA) nucleotide.

24. The compound of claim 23 , wherein the BNA nucleotide is selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

25. The compound of claim 1 , wherein said region includes nucleotides of the upper stem region of the PSL2 motif corresponding to positions 52 and 65 of the wild type PB2 sequence set forth as SEQ ID NO: 1.

26. An oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence comprising one or more modified ribose moieties and at least 8 nucleoside subunits complementary to a region of a Packaging Stem-Loop 2 (PSL2) motif of a PB2 viral RNA (vRNA) or mutant thereof, wherein the oligonucleotide compound has a length of 15 nucleotides or less, comprises the sequence 5′ AGCCAGACAGCGA 3′ (SEQ ID NO: 65), and inhibits virus production.

27. The compound of claim 26 , wherein the oligonucleotide comprises:

an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages, and/or

a bridged nucleic acid (BNA) selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

28. The compound of claim 26 , wherein the oligonucleotide comprises a sequence selected from:

(SEQ ID NO: 106)

5′ CAGCCAGACAGCGAC 3′;

(SEQ ID NO: 107)

5′ CAGCCAGACAGCGA 3′;

and 

(SEQ ID NO: 108)

5′ ACAGCCAGACAGCGA 3′.

29. The compound of claim 28 , wherein the oligonucleotide comprises:

an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages, and/or

a bridged nucleic acid (BNA) selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

30. The compound of claim 26 , wherein the oligonucleotide comprises a sequence selected from:

LNA22:

(SEQ ID NO: 89)

5′ AGCCagacagCGA 3′;

LNA22.2:

(SEQ ID NO: 116)

5′ CAGCcagacagCGAC 3′;

LNA22.3:

(SEQ ID NO: 117)

5′ CAGccagacagCGAC 3′;

LNA22.5:

(SEQ ID NO: 118)

5′ CAGccagacaGCGA 3′;

LNA22.6:

(SEQ ID NO: 119)

5′ CAGccagacagCGA 3′;

LNA22.7:

(SEQ ID NO: 120)

5′ CAGCcagacagCGA 3′;

LNA22.8:

(SEQ ID NO: 121)

5′ ACAgccagacagCGA 3′;

LNA22.9:

(SEQ ID NO: 122)

5′ ACAGccagacaGCGA 3′;

and

LNA22.10:

(SEQ ID NO: 123)

5′ ACAgccagacaGCGA 3′,

wherein capitalized letters denote LNA nucleotides and lowercase letters denote DNA nucleotides.

31. An oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence comprising one or more modified ribose moieties and at least 8 nucleoside subunits complementary to a region of a Packaging Stem-Loop 2 (PSL2) motif of a PB2 viral RNA (vRNA) or mutant thereof, wherein the oligonucleotide compound inhibits virus production and comprises the sequence ACAGCCAGACAGCGA (SEQ ID NO: 108) or GACAGCCAGACAGCG (SEQ ID NO: 109).

32. The compound of claim 31 , wherein the oligonucleotide comprises:

an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages, and/or

a bridged nucleic acid (BNA) selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

33. The compound of claim 31 , wherein the oligonucleotide comprises a sequence selected from:

LNA22.8:

(SEQ ID NO: 121)

5′ ACAgccagacagCGA 3';

LNA22.9:

(SEQ ID NO: 122)

5′ ACAGccagacaGCGA 3';

LNA22.10:

(SEQ ID NO: 123)

5′ ACAgccagacaGCGA 3';

LNA22.11:

(SEQ ID NO: 124)

5′ GACAgccagacaGCG 3';

LNA22.13:

(SEQ ID NO: 125)

5′ GACagccagacaGCG 3';

and

LNA22.14:

(SEQ ID NO: 126)

5′ GACAgccagacAGCG,

wherein capitalized letters denote LNA nucleotides and lowercase letters denote DNA nucleotides.