Pan-Genotypic Agents Against Influenza Virus and Methods of Using the Same
Methods of inhibiting influenza A virus in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds the PSL2 motif to inhibit the influenza A virus. Also provided are methods of treating or preventing influenza A virus infection in a subject. Also provided are methods for screening a candidate agent for the ability to inhibit influenza A virus in a cell, the method comprising: contacting a sample with a candidate agent; and determining whether the candidate agent specifically binds to the PSL2 motif of vRNA. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a PB2 vRNA region that find use in the subject methods.
1 . An oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence comprising 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.
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 a sequence selected from:
(SEQ ID NO: 45)
5′ ACCAAAAGAAT 3′;
(SEQ ID NO: 46)
5′ TGGCCATCAAT 3′;
(SEQ ID NO: 47)
5′ TAGCATACTTA 3′;
(SEQ ID NO: 48)
5′ CCAAAAGA 3′;
(SEQ ID NO: 49)
5′ CATACTTA 3′;
(SEQ ID NO: 50)
5′ CAGACACGACCAAAA 3′;
(SEQ ID NO: 51)
5′ TACTTACTGACAGCC 3′;
(SEQ ID NO: 52)
5′ AGACACGACCAAAAG 3′;
(SEQ ID NO: 53)
5′ ACCAAAAGAAT 3′;
(SEQ ID NO: 54)
5′ TGGCCATCAAT 3′;
(SEQ ID NO: 55)
5′ TAGCATACTTA 3′;
(SEQ ID NO: 56)
5′ CGACCAAAAGAATTC 3′;
(SEQ ID NO: 57)
5′ CGACCAAAAGAATTC 3′;
(SEQ ID NO: 58)
5′ GATGGCCATCAATTA 3′;
(SEQ ID NO: 59)
5′ GATGGCCATCAATTA 3′;
(SEQ ID NO: 60)
5′ TCTAGCATACTTACT 3′;
(SEQ ID NO: 61)
5′ TCTAGCATACTTACT 3′;
(SEQ ID NO: 62)
5′ GAATTCGGATGGCCA 3′;
(SEQ ID NO: 63)
5′ GGCCATCAATTAGTG 3′;
(SEQ ID NO: 64)
5′ TTCGGATGGCCATCA 3′;
(SEQ ID NO: 65)
5′ AGCCAGACAGCGA 3′;
(SEQ ID NO: 66)
5′ GACAGCCAGACAGCA 3′;
(SEQ ID NO: 98)
5′CGACCAAAAGAATT 3′;
(SEQ ID NO: 99)
5′GACCAAAAGAATTCGG 3′;
(SEQ ID NO: 100)
5′ AGCATACTTACTGACA 3′;
(SEQ ID NO: 101)
5′ CATACTTACTGACA 3′;
(SEQ ID NO: 102)
5′ ATACTTACTGACAG 3′;
(SEQ ID NO: 103)
5′ CATACTTACTGACAGC 3′;
(SEQ ID NO: 104)
5′ AGACAGCGACCAAAAG 3′;
(SEQ ID NO: 105)
5′ ACAGCGACCAAAAG
(SEQ ID NO: 106)
5′CAGCCAGACAGCGAC 3′;
(SEQ ID NO: 107)
5′CAGCCAGACAGCGA 3′;
(SEQ ID NO: 108)
5′ACAGCCAGACAGCGA 3′;
(SEQ ID NO: 109)
5′GACAGCCAGACAGCG 3′;
(SEQ ID NO: 110)
5′ CATCAATTAGTGTCG 3′;
(SEQ ID NO: 111)
5′ CCATCAATTAGTGTCG 3′;
(SEQ ID NO: 112)
5′ GCCATCAATTAGTGTG 3′;
(SEQ ID NO: 113)
5′ AAGAATTCGGATGGC 3′;
(SEQ ID NO: 114)
5′ CAGACAGCGACCAA 3′;
and
(SEQ ID NO: 115)
5′ TGACAGCCAGACAGC 3′,
wherein the oligonucleotide comprises one or more modified nucleic acids (e.g., BNA, LNA, ENA, cEt, or 2′-modified).
8 . The compound of claim 1 , wherein the oligonucleotide comprises a sequence selected from:
(SEQ ID NO: 62)
5′ GAATTCGGATGGCCA 3′;
(SEQ ID NO: 65)
5′ AGCCAGACAGCGA 3′;
(SEQ ID NO: 98)
5′CGACCAAAAGAATT 3′;
(SEQ ID NO: 99)
5′GACCAAAAGAATTCGG 3′;
(SEQ ID NO: 100)
5′ AGCATACTTACTGACA 3′;
(SEQ ID NO: 101)
5′ CATACTTACTGACA 3′;
(SEQ ID NO: 102)
5′ ATACTTACTGACAG 3′;
(SEQ ID NO: 103)
5′ CATACTTACTGACAGC 3′;
(SEQ ID NO: 104)
5′ AGACAGCGACCAAAAG 3′;
(SEQ ID NO: 105)
5′ ACAGCGACCAAAAG
(SEQ ID NO: 106)
5′CAGCCAGACAGCGAC 3′;
(SEQ ID NO: 107)
5′CAGCCAGACAGCGA 3′;
(SEQ ID NO: 108)
5′ACAGCCAGACAGCGA 3′;
(SEQ ID NO: 109)
5′GACAGCCAGACAGCG 3′;
(SEQ ID NO: 110)
5′ CATCAATTAGTGTCG 3′;
(SEQ ID NO: 111)
5′ CCATCAATTAGTGTCG 3′;
(SEQ ID NO: 112)
5′ GCCATCAATTAGTGTG 3′;
(SEQ ID NO: 113)
5′ AAGAATTCGGATGGC 3′;
(SEQ ID NO: 114)
5′ CAGACAGCGACCAA 3′;
and
(SEQ ID NO: 115)
5′ TGACAGCCAGACAGC 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: 45-66) and (SEQ ID NOs: 98-115)
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 7 , wherein the oligonucleotide comprises a sequence selected from:
LNA19:
(SEQ ID NO: 86)
5′GAAttcggatgGCCA 3′;
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′;
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′;
LNA22.14:
(SEQ ID NO: 126)
5′GACAgccagacAGCG
LNA24:
(SEQ ID NO: 127)
5′CATcaattagtgTCG 3′;
LNA25:
(SEQ ID NO: 128)
5′CCAtcaattagtgTCG 3′;
LNA26:
(SEQ ID NO: 129)
5′GCCatcaattagtGTG 3′;
LNA27:
(SEQ ID NO: 130)
5′AAGAattcggaTGGC 3′;
LNA28:
(SEQ ID NO: 131)
5′ CAGacagcgacCAA 3′;
LNA29:
(SEQ ID NO: 132)
5′ TGAcagccagacAGC 3′;
LNA14.5:
(SEQ ID NO: 135)
5′CGACcaaaagaATT 3′;
LNA14.8:
(SEQ ID NO: 137)
5′CGACcaaaagaaTTC 3′;
LNA14.28:
(SEQ ID NO: 148)
5′GACcaaaagaatTCGG 3′;
LNA14.30:
(SEQ ID NO: 149)
5′GACCaaaagaattCGG 3′;
LNA9.1:
(SEQ ID NO: 159)
5′ AGCAtacttactGACA 3′;
LNA9.2a:
(SEQ ID NO: 160)
5′ CATacttactgACA 3′;
LNA9.8:
(SEQ ID NO: 164)
5′ ATActtactgACAG
LNA9.12:
(SEQ ID NO: 167)
5′ CATActtactgacAGC
LNA8a:
(SEQ ID NO: 188)
5′ AGAcagcgaccaaAAG
LNA8a.1:
(SEQ ID NO: 189)
5′ AGACagcgaccaAAAG
and
LNA8a.2:
(SEQ ID NO: 190)
5′ ACAGcgaccaAAAG,
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 LNA1-LNA29 (SEQ ID NOs: 67-92 and SEQ ID NOs: 116-191).
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.