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

Abstract

To provide a pan-genotypic agents against influenza virus and methods of using the same.SOLUTION: 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.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 302,548, filed Mar. 2, 2016, which is hereby incorporated by reference in its entirety.

Background Art

[0002] Influenza A virus (IAV) is a segmented RNA virus that causes significant morbidity and mortality worldwide. All currently approved IAV antiviral drugs are targeted against viral proteins, have limited subtypes, and suffer from rising antiviral resistance against all drug class members.

[0003] The IAV genome consists of eight single-stranded negative-sense viral RNA (vRNA) segments that encode at least 14 known viral proteins. The vRNA forms a complete viral ribonucleoprotein (vRNP) together with the nucleoprotein (NP) and a heterotrimeric polymerase complex that includes the PB2, PB1, and PA proteins. For full infectivity, an IAV virion must incorporate at least one vRNP of each segment. Each vRNP is likely to form a supramolecular complex that is maintained by inter-segment RNA-RNA and / or protein-RNA interactions that are hypothesized to interact with at least one other partner and induce the packaging process.

Summary of the Invention

Means for Solving the Problems

[0004] Aspects of the present disclosure provide a pan-genotypic composition designed to disrupt an RNA structural element of IAV called packaging stem loop 2 (PSL2) within the 5' packaging signal region of genomic segment PB2. Disruption of the PSL2 structure dramatically inhibits IAV. PSL2 is conserved across all tested influenza A subtypes.

[0005] A method for inhibiting influenza A virus in a sample is provided. Aspects of the method include contacting a sample containing viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds to the PSL2 motif to inhibit influenza A virus. In some cases, the vRNA is isolated from virions or cells. In some cases, the vRNA is within virions. In some cases, the vRNA is within infected cells. Also provided is a method for treating or preventing influenza A virus infection in a subject. Also provided is a method for screening a candidate agent for its ability to inhibit influenza A virus in cells, the method including contacting a sample with the candidate agent and determining whether the candidate agent specifically binds to the PSL2 motif of the vRNA. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to the PB2 vRNA region for use in the methods of the subject matter. In certain embodiments, for example, the following items are provided. (Item 1) An oligonucleotide compound comprising an oligonucleotide sequence complementary to the PB2 vRNA region, wherein the region comprises nucleotides 34 to 87 in the (-) sense notation of the 5' terminal coding region of the PB2 vRNA, or a salt thereof. (Item 2) The compound according to Item 1, comprising at least 8 nucleoside subunits complementary to the region of the packaging stem loop 2 (PSL2) motif of the region of the PB2 vRNA. (Item 3) The compound according to item 1, wherein the oligonucleotide contains internucleoside linkages selected from phosphorothioate, phosphorodithioate, phosphoramidate, and thiophosphoramidate linkages. (Item 4) The compound according to item 1, wherein the oligonucleotide contains locked nucleic acid (LNA) nucleotides. (Item 5) The oligonucleotide is 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 45), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 46), 5’ TAGCATACTTA 3’ (SEQ ID NO: 47), 5’ CCAAAAGA 3’ (SEQ ID NO: 48), 5’ CATACTTA 3’ (SEQ ID NO: 49), 5’ CAGACACGACCAAAA 3’ (SEQ ID NO: 50), 5’ TACTTACTGACAGCC 3’ (SEQ ID NO: 51), 5’ AGACACGACCAAAAG 3’ (SEQ ID NO: 52), 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 53), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 54), 5’ TAGCATACTTA 3’ (SEQ ID NO: 55), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 56), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 57), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 58), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 59), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 60), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 61), 5’ GAATTCGGATGGCCA 3’ (SEQ ID NO: 62), 5’ GGCCATCAATTAGTG 3’ (SEQ ID NO: 63), 5’ TTCGGATGGCCATCA 3’ (SEQ ID NO: 64), 5’ AGCCAGACAGCGA 3’ (SEQ ID NO: 65), and The compound according to item 1, comprising a sequence selected from 5’ GACAGCCAGACAGCA 3’ (SEQ ID NO: 66). (Item 6) The compound according to item 5, wherein the oligonucleotide contains at least 5 deoxyribonucleotide units and can recruit RNase. (Item 7) The oligonucleotide is LNA1: 5’ AccAaaAGaaT 3’ (SEQ ID NO: 67), LNA2: 5’ TggCcATcaaT 3’ (SEQ ID NO: 68), LNA3: 5’ TagCAtActtA 3’ (SEQ ID NO: 69), LNA4: 5’ CCAAAAGA 3’ (SEQ ID NO: 70), LNA5: 5’ CATACTTA 3’ (SEQ ID NO: 71), LNA6: 5’ CagaCaCGaCCaaAA 3’ (SEQ ID NO: 72), LNA7: 5’ TAcTtaCTgaCagCC 3’ (SEQ ID NO: 73), LNA8: 5’ AGACacgaccaAAAG 3’ (SEQ ID NO: 74), LNA9: 5’ TACTtactgacaGCC 3’ (SEQ ID NO: 75), LNA9.2: 5’ TACttactgacAGCC 3’ (SEQ ID NO: 76), LNA10: 5’ ACCaaaagAAT 3’ (SEQ ID NO: 77), LNA11: 5’ TGGccatcAAT 3’ (SEQ ID NO: 78), LNA12: 5’ TAGcatacTTA 3’ (SEQ ID NO: 79), LNA13: 5’ CgacCAaaAGaattC 3’ (SEQ ID NO: 80), LNA14: 5’ CGACcaaaagaATTC 3’ (SEQ ID NO: 81), LNA15: 5’ GaTGgCcATcaAttA 3’ (SEQ ID NO: 82), LNA16: 5’ GATGgccatcaATTA 3’ (SEQ ID NO: 83), LNA17: 5’ TcTAgCaTActTacT 3’ (SEQ ID NO: 84), LNA18: 5’ TCTAgcatactTACT 3’ (SEQ ID NO: 85), LNA19: 5’ GAAttcggatgGCCA 3’ (SEQ ID NO: 86), LNA20: 5’ GGCCatcaattaGTG 3’ (SEQ ID NO: 87), LNA21: 5’ TTCGgatggccaTCA 3’ (SEQ ID NO: 88), LNA22: 5’ AGCCagacagCGA 3’ (SEQ ID NO: 89), LNA23: 5’ GACAgccagacaGCA 3’ (SEQ ID NO: 90), LNA9.G74C: 5’ TACTtactgacaGTC 3’ (SEQ ID NO: 91), and LNA9.T80C: 5’ TACTtaccgacaGCC 3’ (SEQ ID NO: 92), and contains a sequence selected from The compound according to item 5, wherein the capital letters indicate LNA nucleotides and the lowercase letters indicate DNA nucleotides. (Item 8) A method for inhibiting influenza A virus in cells, comprising contacting a sample containing viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds to the PSL2 motif to inhibit the influenza A virus. (Item 9) The method according to item 8, wherein the agent is the oligonucleotide compound according to item 1. (Item 10) Contacting the sample with the agent results in a titer loss of at least 2 log 10 of the virus, and the agent destroys the overall structure of the PSL2 motif of the vRNA. The method according to item 8. (Item 11) The method according to item 8, wherein the vRNA is isolated from virions or cells. (Item 12) A method for treating or preventing influenza A virus infection in a subject, comprising administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of an agent that specifically binds to the PSL2 motif of viral RNA (vRNA). (Item 13) The method according to item 12, wherein the agent is a compound comprising an oligonucleotide sequence comprising at least 8 nucleoside subunits complementary to a region of PB2 vRNA. (Item 14) The method according to item 12, wherein the agent is the oligonucleotide compound according to item 1. (Item 15) The method according to item 14, wherein the subject is at risk of influenza A virus infection and the administration of the oligonucleotide compound protects the subject against infection for at least one week or more (e.g., two weeks or more, three weeks or more, one month or more, two months or more, three months or more, etc.). (Item 16) The method according to item 15, wherein the administration comprises administration of the oligonucleotide compound at an effective dose once a week, once every two weeks, or once a month. (Item 17) The method according to item 12, wherein the pharmaceutical composition further comprises an additional agent selected from a second oligonucleotide agent and an antiviral drug. (Item 18) The method according to item 12, wherein the subject is diagnosed as having or suspected of having influenza A virus infection.

[0006] Those skilled in the art will understand that the drawings described below are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way. BRIEF DESCRIPTION OF THE DRAWINGS

[0007]

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BEST MODE FOR CARRYING OUT THE INVENTION

[0008] Definitions Before describing exemplary embodiments in more detail, the following definitions are set forth to exemplify and define the meaning and scope of the terms used in the description.

[0009] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991) provide many of the ordinary meanings of the terms used herein to those of ordinary skill in the art. Nevertheless, certain terms are defined below for clarity and ease of reference.

[0010] As used herein and in the appended claims, it should be noted that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. It should further be noted that the claims may be drafted to exclude any optional element. As such, this description is intended to serve as a precedent for the use of exclusive terms such as “alone,” “only,” etc. in relation to the recitation of claim elements, or the use of “negative” limitations.

[0011] As used herein, the term “sample” relates to a substance or mixture of substances that contains one or more components of interest, which need not be, but typically is, in fluid form.

[0012] As used herein, the term “effective amount” refers to the amount of a substance (e.g., an agent of interest) that produces some desired local or systemic effect. The effective amount of the agent of interest varies depending on various factors including, but not limited to, the weight and age of the subject, the condition being treated, the severity of the condition, the method of administration, etc., and can be readily determined empirically using data such as that provided in the experimental section below.

[0013] As used herein, the term "sample" relates to a substance or mixture of substances, typically a fluid, i.e., in aqueous form, that contains one or more target components, although it need not be so. Samples can be derived from a variety of sources such as biological samples or solids, such as tissues or fluids isolated from an individual, including but not limited to, for example, plasma, serum, cerebrospinal fluid, semen, lymphatic fluid, external pieces of skin, airways, intestinal tract, and urogenital tract, tears, saliva, milk, blood cells, tumors, organs, and also (including but not limited to) samples of in vitro cell culture components such as cells in cell culture medium, putative virus-infected cells, recombinant cells, and conditioned media resulting from the growth of cell components. Components in the sample are herein referred to as "analytes". In many embodiments, the sample is a complex sample containing at least about 10 2 、5×10 2 、10 3 、5×10 3 、10 4 、5×10 4 、10 5 、5×10 5 、10 6 、5×10 6 、10 7 、5×10 7 、10 8 、10 9 、10 10 、10 11 、10 12 or more species of analytes.

[0014] "Antibody fragment" includes a portion of an intact antibody, e.g., the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments, diabodies, linear antibodies (Zapata et al., Protein Eng. 8(10):1057-1062 (1995)), single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments called "Fab" fragments, each having a single antigen-binding site, and a residual "Fc" fragment, a name reflecting its ability to crystallize readily. Pepsin treatment yields F(ab’)2 fragments that have two antigen-binding sites and can still cross-link antigens.

[0015] As used interchangeably herein, the terms "polypeptide" and "protein" refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term "fusion protein" or its grammatical equivalent refers to a protein consisting of multiple polypeptide components that are typically not joined in their natural state but are joined by their respective amino and carboxyl termini through peptide bonds that typically form a single continuous polypeptide. A fusion protein can be a combination of two, three, or even four or more different proteins. The term polypeptide includes, but is not limited to, fusion proteins having non-homologous amino acid sequences, fusions having non-homologous and homologous leader sequences, with or without an N-terminal methionine residue, immunologically tagged proteins, fusion proteins with a detectable fusion partner, e.g., a fusion protein including a fluorescent protein, β-galactosidase, luciferase, etc. as the fusion partner, etc., including fusion proteins.

[0016] Generally, a polypeptide can be of any length, for example, more than 2 amino acids, more than 4 amino acids, more than about 10 amino acids, more than about 20 amino acids, more than about 50 amino acids, more than about 100 amino acids, more than about 300 amino acids, and usually up to about 500 or more than 1000 amino acids. A "peptide" generally refers to more than 2 amino acids, more than 4 amino acids, more than about 10 amino acids, more than about 20 amino acids, and usually up to about 50 amino acids. In some embodiments, the peptide is from 5 to 30 amino acids in length.

[0017] The term "specific binding" refers to the ability of an agent to preferentially bind to a specific target (e.g., PSL2) present in a homogeneous mixture of different analytes. In some cases, the specific interaction will typically distinguish between the desired and undesired analytes in a sample by a factor of about 10 to 100-fold or more (e.g., more than about 1000-fold). In some cases, when they are specifically bound in a capture agent / analyte complex, the affinity between the agent of interest and the analyte (e.g., PSL2) is at least 10 -8 M, at least 10 -9 M, and usually up to about 10 -10 M. Specific binding can include hybridization, polypeptide-nucleic acid interactions, or small molecule-nucleic acid interactions.

[0018] "Oligonucleotide" refers to a polymer of ribose and / or deoxyribose nucleoside subunits having from about 2 to about 200 contiguous subunits. The nucleoside subunits can be joined by various subunit-linking bonds including, but not limited to, phosphodiester, phosphotriester, methylphosphonate, P3’→N5’ phosphoramidate, N3’→P5’ phosphoramidate, N3’→P5’ thiophosphoramidate, and phosphorothioate linkages. Further, "oligonucleotide" includes sugars (e.g., 2’-substituted), bases (see definition of "nucleoside" below), and modifications known to those of skill in the art to the 3’ and 5’ termini. In embodiments where the oligonucleotide moiety includes multiple subunit-linking bonds, each bond can be formed using the same chemistry or a mixture of linking chemistries can be used. The terms "oligonucleotide", "nucleic acid", "nucleic acid molecule", "nucleic acid fragment", "nucleic acid sequence or segment", or "polynucleotide" are used interchangeably and can also be used interchangeably with genes, cDNA, DNA, and RNA encoded by genes.

[0019] "Locked nucleic acid" (LNA) is a modified RNA nucleotide in which the ribose moiety is modified with an additional bridge that joins the 2’ oxygen and 4’ carbon. The bridge "locks" the ribose in the 3’-end conformation, which is commonly found in the A-form duplex. LNA nucleotides can be mixed with any convenient nucleotide or nucleotide analog, such as DNA or RNA residues in an oligonucleotide, as desired. LNA hybridizes to DNA or RNA according to the Watson-Crick base pairing principle. Such oligomers can be synthesized chemically. Generally, the locked ribose conformation improves base stacking and backbone pre-organization and increases the hybridization properties (melting temperature) of the oligonucleotide.

[0020] The disclosure encompasses isolated or substantially purified nucleic acid molecules and compositions containing such molecules. In the context of the present disclosure, an "isolated" or "purified" DNA or RNA molecule is a DNA or RNA molecule that exists separate from its natural environment and is thus not a natural product. An isolated DNA or RNA molecule can exist in a purified state or can exist in a non-natural environment, such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or a biologically active portion thereof is substantially free of other cellular material if produced by recombinant techniques, or substantially free of chemical precursors or other chemicals if chemically synthesized. In one embodiment, an "isolated" nucleic acid excludes sequences that naturally flank the nucleic acid in genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). For example, in various embodiments, an isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Fragments and variants of the disclosed nucleotide sequences are also encompassed by the present disclosure. A "fragment" or "portion" means less than the full length of the nucleotide sequence. The siRNAs of the present disclosure can be generated by any method known in the art, for example, by in vitro transcription, recombinantly, or by synthetic means. In one example, siRNAs can be generated in vitro by using recombinant enzymes such as T7 RNA polymerase and a DNA oligonucleotide template.

[0021] "Minor interference" or "short interfering RNA" or siRNA is a double-stranded RNA of nucleotides that is targeted to a gene of interest. "Double-stranded RNA" refers to a structure formed by complementary base pairing between two regions of an RNA molecule. siRNA is "targeted" to a gene where the nucleotide sequence of the double-stranded portion of the siRNA is complementary to the nucleotide sequence of the target gene. In some embodiments, the length of the double-stranded siRNA is less than 30 nucleotides. In some embodiments, the double-stranded can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 nucleotides in length. In some embodiments, the length of the double-stranded is 19 to 25 nucleotides in length. The double-stranded RNA portion of the siRNA can be part of a hairpin structure. In addition to the double-stranded portion, the hairpin structure can contain a loop portion located between the two sequences that form the double-stranded. The loop can vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13 nucleotides in length. The hairpin structure can also contain a 3' or 5' overhang portion. In some embodiments, the overhang is 0, 1, 2, 3, 4, or 5 nucleotides in length of a 3' or 5' overhang.

[0022] The term "lipid" is used broadly herein and encompasses substances that are soluble in organic solvents but are poorly soluble, if at all, in water. The term "lipid" includes, but is not limited to, carbohydrates, oils, fats (such as fatty acids, glycerides), sterols, steroids, and derivative forms of these compounds. Preferred lipids are fatty acids and their derivatives, carbohydrates and their derivatives, and sterols such as cholesterol. As used herein, the term "lipid" also includes amphiphilic compounds containing both a lipid and a hydrophilic moiety. Fatty acids typically contain an even number of carbon atoms in the straight chain (generally 12 to 24 carbons), may be saturated or unsaturated, and may contain or be modified to contain various substituents. Briefly, the term "fatty acid" also includes fatty acid derivatives such as, for example, fatty acid amides produced by conjugation reactions with modified ends of oligonucleotides.

[0023] Other definitions of terms may occur throughout the specification. (Mode for Carrying Out the Invention)

[0024] Before describing various embodiments, it is to be understood that the teachings of this disclosure are not limited to the specific embodiments described, and as such, may of course vary. It is also to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting, as the scope of the teachings is defined only by the appended claims.

[0025] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. Although the teachings are described with reference to various embodiments, the teachings are not intended to be limited to such embodiments. On the contrary, the teachings include various alternatives, modifications, and equivalents as would be understood by one of ordinary skill in the art.

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are described herein.

[0027] The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present claims have the right to antedate such publication by virtue of prior invention. Further, the provided publication dates may be different from the actual publication dates which can be independently confirmed.

[0028] As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has separate components and features that can be readily separated from, or combined with, any of the features of some other embodiments without departing from the scope or spirit of the present teachings. Any of the recited methods can be performed in the order of the recited events or in any other order that is theoretically possible.

[0029] All patents and publications, including all sequences disclosed in such patents and publications, as referred to herein are expressly incorporated by reference.

[0030] Method for inhibiting influenza A virus As summarized above, aspects of the present disclosure include a pan-genotypic composition designed to disrupt an RNA structural element of IAV, called packaging stem loop 2 (PSL2), within the 5' packaging signal region of genomic segment PB2. "Pan-genotypic" means that the composition is effective across a variety of different types of IAV in which the PSL2 structural element is conserved. In some cases, the subject composition can be referred to as having a broad spectrum. As used herein, the term "broad spectrum" refers to the antiviral activity of a single moiety that is active against two or more different viruses, such as three or more, four or more, five or more, six or more, eight or more, ten or more different viruses. The two or more different viruses can be selected from different virus subgroups (e.g., influenza A group 1 or influenza A group 2) or from within the same group (e.g., two or more of the H1, H2, H5, H6, H8, and H9 group 1 influenza A viruses, or two or more of the H3, H4, H7, and H10 group 2 influenza A viruses).

[0031] Disruption of the PSL2 structure dramatically inhibits IAV. PSL2 is conserved across all tested influenza A subtypes. Figure 1, panel a shows an example of a PSL2 structure that can be targeted in the subject method. Figure 4 illustrates the conservation of the nucleotide sequence of interest containing the PSL2 structure. In some cases, the subject composition has broad spectrum activity against IAV, such as activity against two or more IAVs selected from H1N1, H3N2, and H5N.

[0032] Aspects of the present disclosure include methods for inhibiting influenza A virus (IAV) in a sample. In some embodiments, the method includes contacting a sample containing viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds to the PSL2 motif to inhibit influenza A virus. In some cases, the sample is in vitro. In certain cases, the sample is in vivo. The vRNA in the sample can be contained within virions. In some cases, the vRNA is contained within cells, such as cells infected with viral particles.

[0033] Aspects of the present disclosure include methods for inhibiting influenza A virus (IAV) in a cell. In some embodiments, the method includes contacting a cell containing viral RNA (vRNA) having a PSL2 motif with an effective amount of an agent that specifically binds to the PSL2 motif to inhibit influenza A virus. In some cases, the cell is in vitro. In certain cases, the cell is in vivo.

[0034] In some embodiments, the vRNA in the sample (e.g., cell) includes PB2 vRNA. As used herein, "PB2 vRNA" means viral RNA (e.g., IAV RNA) that includes a conserved RNA structural element called packaging stem loop 2 (PSL2). The agent can bind to a specific site of the PSL2 motif to disrupt the overall structure of the vRNA, thereby inhibiting the virus (see, e.g., FIG. 14). For example, FIG. 1 illustrates the RNA secondary structures of wild-type PB2 and packaging mutant vRNA.

[0035] In some embodiments, contacting the sample (e.g., cell) with the agent results in at least a 2 log 10 reduction in viral titer, such as at least a 2 log 10resulting in loss of the virus titer. In some embodiments, the agent is an oligonucleotide compound (e.g., as described herein) or a salt thereof that comprises a sequence complementary to the PSL2 motif of vRNA.

[0036] In some examples of the method, the binding (e.g., via hybridization) of an oligonucleotide compound (e.g., one of the sequences described above) to a region of the PB2 vRNA disrupts the overall secondary RNA structure of the PB2 vRNA. In some cases, the subject compound targets at least a portion of the region defined by nucleotides 34 - 87 in the (-) sense notation of the 5' terminal coding region of the PB2 vRNA. In some cases, the compound targets at least a portion of the region defined by nucleotides 1 - 14 in the (-) sense notation of the 5' terminal coding region of the PB2 vRNA. In some examples of the method, the method further comprises recruiting an RNase to degrade PSL2 to the vRNA.

[0037] Aspects of the present disclosure include methods of treating or preventing influenza A virus infection in a subject. In some embodiments, the method comprises administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of an active agent that specifically binds to the PSL2 motif of viral RNA (vRNA) (e.g., as described herein). As such, in some cases, the subject is one that is infected with the virus. In certain cases, the subject is one that is at risk of infection with the virus or suspected of being infected. In some embodiments, the vRNA is PB2 vRNA.

[0038] Any convenient protocol for administering the agent to the subject can be used. The particular protocol used can vary depending on, for example, the site of administration and whether the agent is, for example, an oligonucleotide, antibody, protein, peptide, or small molecule. For in vivo protocols, any convenient administration protocol can be used. Depending on the identity and binding affinity of the agent, the desired response, the method of administration, such as topical or systemic, intraocular, periocular, retrobulbar, intramuscular, intravenous, intraperitoneal, subcutaneous, subconjunctival, intranasal, topical, eye drops, i.v.s.c., i.p., oral, etc., the half-life, cell number, or size of the implantation bed or implanted tissue, various protocols can be used.

[0039] Pharmaceutical compositions comprising the agent of the subject are also provided. Any convenient excipient, carrier, etc. can be used in the composition. Pharmaceutically acceptable carriers found useful in the compositions can include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases, etc. The pharmaceutical composition can also be lyophilized for subsequent reconstitution and use. The composition can also include carriers as described herein. Examples of carriers that can be used include, but are not limited to, alum, microparticles, liposomes, and nanoparticles. Any convenient additive can be included in the composition of the subject to enhance delivery of the active agent of the subject. Additives of interest include cell uptake enhancers, carrier proteins, lipids, dendrimer carriers, carbohydrates, etc.

[0040] In some cases, the pharmaceutical composition further comprises one or more additional active agents. Suitable active agents include the additional oligonucleotide compounds of the present disclosure and antiviral compounds or drugs for any convenient purpose. These include, but are not limited to, amantadine, rimantadine, zanamivir, oseltamivir, peramivir, and the like.

[0041] Agent Any convenient agent can be used as an agent for a target of interest (e.g., PSL2) in the subject methods and compositions. Suitable agents include, but are not limited to, ligands of PSL-2, PSL2-binding antibodies, scaffold proteins binding to PSL2, oligonucleotides, small molecules, and peptides, or fragments, variants, or derivatives thereof, or any combination of the foregoing.

[0042] Antibodies that can be used as agents in the present disclosure include, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)2 antibody fragments, Fv antibody fragments (e.g., V H or V L )), single-chain Fv antibody fragments, and dsFv antibody fragments. Further, the antibody molecule can be a fully human antibody, a humanized antibody, or a chimeric antibody. Antibodies that can be used in the present disclosure can include any mature or unprocessed antibody variable region bound to any immunoglobulin constant region. Minor changes in the amino acid sequence of an antibody or immunoglobulin molecule are encompassed by the present disclosure if the changes maintain more than 75%, e.g., 80% or more, 90% or more, 95% or more, or 99% or more of the sequence. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions occur within families of related amino acids in their side chains. Whether an amino acid change results in a functional peptide can be determined by analyzing the specific activity of the polypeptide derivative. In some embodiments, the agent is an antibody fragment (e.g., as described herein).

[0043] In some embodiments, the agent is a scaffolded polypeptide binder. A scaffold refers to the underlying peptidic framework (e.g., a consensus sequence or a structural motif) from which the polypeptide agent is derived. The underlying scaffold sequence includes residues that are fixed and a variety of residues that can confer different functions, such as specific binding to a target receptor, to the resulting polypeptide agent. Such structural motifs can be characterized and structurally compared as combinations of specific secondary and tertiary structural elements or, alternatively, as equivalent primary sequences of amino acid residues. Any convenient scaffold and scaffolded polypeptide can be used as an agent in the methods of the subject matter. In some embodiments, such agents can be identified using recombinant screening methods such as phage display screening. Exemplary scaffolded polypeptide binders include, but are not limited to, synthetic and recombinant small proteins such as affibodies.

[0044] In some cases, the agent is a small molecule that binds to PSL2. Exemplary small molecules include small organic or inorganic compounds having a molecular weight (MW) of greater than 50 to less than about 2,500 daltons (Da), such as greater than 50 to less than about 1,000 Da, or greater than 50 to less than about 500 Da. "Small molecule" encompasses a number of biological and chemical classes, including synthetic, semi-synthetic, or natural-occurring inorganic or organic molecules, including synthetic, recombinant, or natural-occurring polypeptides and nucleic acids. Exemplary small molecules can include functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl, or carboxyl group, and can contain at least two of the functional chemical groups. Small molecules can include cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecules are found among biomolecules and include peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0045] Oligonucleotide compound In some embodiments, the agent is an oligonucleotide or a derivative thereof, or a salt thereof (e.g., a pharmaceutically acceptable salt). In some examples, the oligonucleotide is complementary to a specific segment of the PSL2 motif (such as described herein). The complementary oligonucleotide that finds use in the subject methods will in some cases be at least 5, in some cases at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, about 12, at least 13, at least 14, at least 15, or more. In some cases, the complementary oligonucleotide will be of a length of 30 nucleotides or less, such as a length of 25 nucleotides or less, a length of 20 nucleotides or less, or a length of 15 nucleotides or less, the length being determined by factors such as efficiency of inhibition, specificity including absence of cross-reactivity, etc. The present disclosure provides short oligonucleotides that can be potent selective inhibitors of PSL2 function, e.g., of a length of 7 or 8 - 15 nucleotides. In some embodiments, the active agent is a compound comprising an oligonucleotide sequence comprising at least 8 nucleoside subunits complementary to a region of PB2 vRNA. In some embodiments, the active agent is a compound comprising an oligonucleotide sequence comprising at least 8 and 20 or fewer (e.g., 15 or fewer) nucleoside subunits complementary to a region of PB2 vRNA.

[0046] The specific region or region of the endogenous strand PSL2 sequence is selected to be complemented by the oligonucleotide agent. Selection of the specific sequence of the oligonucleotide can be based on structural analysis (such as described herein), and an empirical method can be used where several candidate sequences are analyzed for inhibition of the target IAV in vitro or in an animal model. Combinations of oligonucleotides and sequences can also be used where several regions of the target PSL2 are selected for antisense complementarity.

[0047] In some embodiments, the agent is an oligonucleotide compound comprising at least 5 nucleoside subunits (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20) complementary to the PSL2 motif of vRNA, or a salt thereof. In certain embodiments, one or more of the linkages of the oligonucleotide are selected from methylphosphonate, P3’→N5’ phosphoramidate, N3’→P5’ phosphoramidate, N3’→P5’ thiophosphoramidate, phosphorodithioate, and phosphorothioate linkages. In certain examples, the oligonucleotide sequence is a locked nucleic acid. In certain examples, the oligonucleotide sequence comprises one or more locked nucleic acid nucleotides such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more than this.

[0048] In some embodiments, the agent is an oligonucleotide that includes at least 5 deoxyribonucleotide units (e.g., units complementary to the PSL2 motif) (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20) and can recruit RNase. In some cases, the oligonucleotide recruits RNase to catalyze the degradation of the target vRNA into smaller components. Any convenient method and moiety for recruiting RNase can be incorporated into the subject agent (e.g., oligonucleotide). In some examples, the oligonucleotide agent further includes a sequence that recruits the RNase of interest. Unless otherwise indicated, oligonucleotide sequences as depicted herein include DNA sequences, (e.g., RNA sequences where U can optionally replace T), mixed RNA / DNA sequences, and analogs where one or more nucleotides of the sequence are modified nucleotides, such as LNA analogs, and / or analogs where one or more internucleoside linkages are replaced with non-naturally occurring linkages, such as phosphorothioate, phosphorodithioate, phosphoramidate, or thiophosphoramidate linkages.

[0049] In some embodiments, the oligonucleotide is 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 45), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 46), 5’ TAGCATACTTA 3’ (SEQ ID NO: 47), 5’ CCAAAAGA 3’ (SEQ ID NO: 48), 5’ CATACTTA 3’ (SEQ ID NO: 49), 5’ CAGACACGACCAAAA 3’ (SEQ ID NO: 50), 5’ TACTTACTGACAGCC 3’ (SEQ ID NO: 51), 5’ AGACACGACCAAAAG 3’ (SEQ ID NO: 52), 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 53), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 54), 5’ TAGCATACTTA 3’ (SEQ ID NO: 55), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 56), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 57), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 58), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 59), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 60), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 61), 5’ GAATTCGGATGGCCA 3’ (SEQ ID NO: 62), 5’ GGCCATCAATTAGTG 3’ (SEQ ID NO: 63), 5’ TTCGGATGGCCATCA 3’ (SEQ ID NO: 64), 5’ AGCCAGACAGCGA 3’ (SEQ ID NO: 65), and 5’ GACAGCCAGACAGCA 3’ (SEQ ID NO: 66).

[0050] In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 45). In certain embodiments, the oligonucleotide has the sequence: 5’ It contains 3’ (SEQ ID NO: 46). In certain embodiments, the oligonucleotide contains the sequence: 5’ TAGCATACTTA 3’ (SEQ ID NO: 47). In certain embodiments, the oligonucleotide contains the sequence: 5’ CCAAAAGA 3’ (SEQ ID NO: 48). In certain embodiments, the oligonucleotide contains the sequence: 5’ CATACTTA 3’ (SEQ ID NO: 49). In certain embodiments, the oligonucleotide contains the sequence: 5’ CAGACACGACCAAAA 3’ (SEQ ID NO: 50). In certain embodiments, the oligonucleotide contains the sequence: 5’ TACTTACTGACAGCC 3’ (SEQ ID NO: 51). In certain embodiments, the oligonucleotide contains the sequence: 5’ It contains 3’AGACACGACCAAAAG (SEQ ID NO: 52). In certain embodiments, the oligonucleotide contains the sequence: 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 53). In certain embodiments, the oligonucleotide contains the sequence: 5’ TGGCCATCAAT 3’ (SEQ ID NO: 54). In certain embodiments, the oligonucleotide contains the sequence: 5’ TAGCATACTTA 3’ (SEQ ID NO: 55). In certain embodiments, the oligonucleotide contains the sequence: 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 56). In certain embodiments, the oligonucleotide contains the sequence: 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 57). In certain embodiments, the oligonucleotide contains the sequence: 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 58). In certain embodiments, the oligonucleotide contains the sequence: 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 59). In certain embodiments, the oligonucleotide contains the sequence: 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 60). In certain embodiments, the oligonucleotide contains the sequence: 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 61). In certain embodiments, the oligonucleotide contains the sequence: 5’ GAATTCGGATGGCCA 3’ (SEQ ID NO: 62). In certain embodiments, the oligonucleotide contains the sequence: 5’ GGCCATCAATTAGTG 3’ (SEQ ID NO: 63). In certain embodiments, the oligonucleotide contains the sequence: 5’ TTCGGATGGCCATCA 3’ (SEQ ID NO: 64). In certain embodiments, the oligonucleotide contains the sequence: 5’ AGCCAGACAGCGA 3’ (SEQ ID NO: 65). In certain embodiments, the oligonucleotide contains the sequence: 5’ GACAGCCAGACAGCA contains 3’ (SEQ ID NO: 66).

[0051] In some examples, the binding of an oligonucleotide compound (e.g., one of the sequences described above) to the PB2 vRNA region (e.g., via hybridization) disrupts the overall secondary RNA structure of the PB2 vRNA.

[0052] The oligonucleotide sequences can contain any convenient number of DNA, RNA, and LNA nucleotides. In some examples of the oligonucleotide sequences described herein, the sequences are mixed RNA / DNA sequences. In some examples of the oligonucleotide sequences described herein, the sequences are mixed LNA / DNA sequences. In some examples of the oligonucleotide sequences described herein, the sequences are mixed LNA / RNA sequences. In some examples of the oligonucleotide sequences described herein, the sequences contain only LNA nucleotides. In some examples of the oligonucleotide sequences described herein, the sequences contain only DNA nucleotides. In some examples of the oligonucleotide sequences described herein, the sequences contain only RNA nucleotides.

[0053] In certain examples, the subject oligonucleotide has one of the following arrangements of nucleotide types in the sequence (e.g., one of SEQ ID NOs: 45-66): A is a sequence of 8 or more LNA nucleotides, A, B is a sequence of 6-8 DNA nucleotides and each A is a sequence of 3-4 LNA nucleotides, A-B-A, B is a sequence of 7-8 DNA nucleotides and each A is a sequence of 4 LNA nucleotides, A-B-A, Each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 2 DNA nucleotides, L-D-L-D-L-D-L, Each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-2 DNA nucleotides, L-D-L-D-L-D-L, Each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-3 DNA nucleotides, L-D-L-D-L-D-L, Each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-3 DNA nucleotides, L-D-L-D-L-D-L-D-L, and Each L is a sequence of 1 to 2 LNA nucleotides, and each D is a sequence of 1 to 2 DNA nucleotides, L-D-L-D-L-D-L-D-L.

[0054] The oligonucleotide sequence of the subject matter may further include one or more modified internucleoside linkages such as phosphorothioate, phosphorodithioate, phosphoramidate, and / or thiophosphoramidate linkages. The unnatural internucleoside linkages may be included at any convenient position in the sequence of the oligonucleotide of the subject matter.

[0055] In certain embodiments, the oligonucleotide has one of the following sequences, where capital letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides (i.e., deoxyribonucleotide units): LNA1: 5’ AccAaaAGaaT 3’ (SEQ ID NO: 67), LNA2: 5’ TggCcATcaaT 3’ (SEQ ID NO: 68), LNA3: 5’ TagCAtActtA 3’ (SEQ ID NO: 69), LNA4: 5’ CCAAAAGA 3’ (SEQ ID NO: 70), LNA5: 5’ CATACTTA 3’ (SEQ ID NO: 71), LNA6: 5’ CagaCaCGaCCaaAA 3’ (SEQ ID NO: 72), LNA7: 5’ TAcTtaCTgaCagCC 3’ (SEQ ID NO: 73), LNA8: 5’ AGACacgaccaAAAG 3’ (SEQ ID NO: 74), LNA9: 5’ TACTtactgacaGCC 3’ (SEQ ID NO: 75), LNA9.2: 5’ TACttactgacAGCC 3’ (SEQ ID NO: 76), LNA10: 5’ ACCaaaagAAT 3’ (SEQ ID NO: 77), LNA11: 5’ TGGccatcAAT 3’ (SEQ ID NO: 78), LNA12: 5’ TAGcatacTTA 3’ (SEQ ID NO: 79), LNA13: 5’ CgacCAaaAGaattC 3’ (SEQ ID NO: 80), LNA14: 5’ CGACcaaaagaATTC 3’ (SEQ ID NO: 81), LNA15: 5’ GaTGgCcATcaAttA 3’ (SEQ ID NO: 82), LNA16: 5’ GATGgccatcaATTA 3’ (SEQ ID NO: 83), LNA17: 5’ TcTAgCaTActTacT 3’ (SEQ ID NO: 84), LNA18: 5’ TCTAgcatactTACT 3’ (SEQ ID NO: 85), LNA19: 5’ GAAttcggatgGCCA 3’ (SEQ ID NO: 86), LNA20: 5’ GGCCatcaattaGTG 3’ (SEQ ID NO: 87), LNA21: 5’ TTCGgatggccaTCA 3’ (SEQ ID NO: 88), LNA22: 5’ AGCCagacagCGA 3’ (SEQ ID NO: 89), and LNA23: 5’ GACAgccagacaGCA 3’ (SEQ ID NO: 90).

[0056] Sequence variants of the oligonucleotide sequences described above are also encompassed by the present disclosure. It is understood that in any of the sequences described herein, one, two, three, four or more nucleotides can be mutated to provide desired properties such as improved inhibitory activity, complexation with modifiers, etc.

[0057] In some cases, any one of the sequences described herein (e.g., one of SEQ ID NOs: 45 - 96) is included within a longer sequence, for example, containing additional 5’ and / or 3’ nucleotides. In certain examples, the oligonucleotide of interest is 30 nucleotides or less in length, such as 25 nucleotides or less, 20 nucleotides or less, 19 nucleotides or less, 18 nucleotides or less, 17 nucleotides or less, 16 nucleotides or less, 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, 11 nucleotides or less, or 10 nucleotides or less.

[0058] In some cases, the oligonucleotide compound of the subject matter comprises a sequence having a deletion relative to one of the sequences described herein (e.g., one of SEQ ID NOs: 45-96). For example, the sequence is one in which 1, 2, or 3 nucleotides are deleted from the 5' and / or 3' end of a sequence, e.g., one of SEQ ID NOs: 45-96. In some cases, the deleted sequence has 1 nucleotide lost from the 5' end of one of SEQ ID NOs: 45-96. In some cases, the deleted sequence has 1 nucleotide lost from the 3' end of one of SEQ ID NOs: 45-96. In some cases, the deleted sequence has 2 nucleotides lost from the 5' end of one of SEQ ID NOs: 45-96. In some cases, the deleted sequence has 2 nucleotides lost from the 3' end of one of SEQ ID NOs: 45-96.

[0059] In certain cases, the oligonucleotide sequence can include mutations designed to cover a single nucleotide polymorphism (SNP) in the target PSL2 sequence. In some cases, the oligonucleotide is a modified version of LNA9 that includes a single mutation site that protects against nucleotide changes in the LNA9 target sequence in the PLS2 target sequence. It is understood that the SNP mutations of interest can be applied to any of the sequences described herein. Non-limiting examples of the mutant sequences of interest include the following: 5’ TACTTACTGACAGTC 3’ (SEQ ID NO: 91), 5’ TACTTACCGACAGCC 3’ (SEQ ID NO: 92), and 5’ GGATTTCGGATGGCCA 3’ (SEQ ID NO: 93).

[0060] In certain embodiments, the oligonucleotide has one of the following sequences, where uppercase letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides: LNA9.G74C: 5’ TACTtactgacaGTC 3’ (SEQ ID NO: 94), LNA9.T80C: 5’ TACTtaccgacaGCC 3’ (SEQ ID NO: 95), and LNA19.U56C: 5’ GGATttcggatggCCA 3’ (SEQ ID NO: 96).

[0061] In certain examples, the oligonucleotide has a maximum length corresponding to a particular region of the PSL2 structure (e.g., a sub-region corresponding to nucleotides 34-87). In certain examples, the length of the oligonucleotide is 20 or fewer nucleotides, such as 15 or fewer nucleotides, 14 or fewer nucleotides, 13 or fewer nucleotides, 12 or fewer nucleotides, 11 or fewer nucleotides, 10 or fewer nucleotides, 9 or fewer nucleotides, 8 or fewer nucleotides, 7 or fewer nucleotides, or even fewer.

[0062] Oligonucleotides can be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al., supra). Oligonucleotides can be chemically modified from their native phosphodiester structure to increase their intracellular stability and binding affinity. A number of such modifications that alter the chemistry of the backbone, sugar, or heterocyclic base are described in the literature.

[0063] Useful modifications in nucleic chemistry include phosphorothioates, phosphorodithioates in which both non-bridging oxygens are replaced by sulfur, phosphoramidites, alkyl phosphotriesters, and boranophosphates. As chiral phosphate derivatives, 3'-O'-5'-S-phosphorothioate, 3'-S-5'-O-phosphorothioate, 3'-CH2-5'-O-phosphonate, 3'-NH-5'-O-phosphoramidate, and thiophosphoramidate can be mentioned. Peptide nucleic acids replace the entire ribose phosphodiester backbone containing peptide bonds. Sugar modifications are also used to improve stability and affinity. The α-anomer of deoxyribose in which the base is inverted with respect to the natural β-anomer can be used. The 2'-OH of ribose sugar can be modified to form 2'-O-methyl or 2'-O-allyl sugars, which provide resistance to degradation without deteriorating affinity. Modifications of heterocyclic bases must maintain proper base pair formation. Some useful substitutions include the substitution of deoxythymidine for deoxyuridine and the substitution of deoxycytidine for 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine. 5-Propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted with deoxythymidine and deoxycytidine, respectively.

[0064] The oligonucleotide agent is any convenient modifier and can be derivatized, for example, by complexing the modifier with the 5'- and / or 3'-ends of the oligonucleotide sequence. In some cases, the modifier is a moiety that enhances cellular uptake (e.g., a lipid). Any convenient lipid can be complexed with the subject oligonucleotide. In some examples, the modifier is a fatty acid attached to the 5' or 3'-end via any linker. The lipid group can be an aliphatic hydrocarbon or fatty acid, including but not limited to derivatives of hydrocarbons and fatty acids, and examples include saturated straight-chain compounds having 14 to 20 carbons such as myristic (tetradecanoic) acid, palmitic (hexadecanoic) acid, and stearic (octadecanoic) acid, as well as their corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane, and octadecane. Examples of other suitable lipid groups that can be used include sterols such as cholesterol, substituted fatty acids, and hydrocarbons, particularly their polyfluorinated forms. The scope of lipid groups includes derivatives such as amine, amide, ester, and carbamate derivatives.

[0065] In some cases, the modifier is an additional nucleic acid sequence having a desired activity (e.g., recruitment of RNase as described herein). In certain examples, the modifier has a specific binding activity that provides delivery of the oligonucleotide to a specific target, such as a cell-specific protein. In some cases, the modifier is an antibody of interest that specifically binds to a cell-specific target of interest. In a particular example, the antibody modifier specifically binds to a hemagglutinin (HA) target.

[0066] The oligonucleotide agent can be used in any convenient form. In some examples, the oligonucleotide agent is single-stranded. In some examples, the oligonucleotide agent is double-stranded. In some examples, the oligonucleotide agent is siRNA. In some examples, the oligonucleotide agent is shRNA. In some examples, the oligonucleotide agent is ssRNA. In some examples, one or more nucleotides of the ssRNA can be replaced with LNA nucleotides. In some examples, the oligonucleotide agent is ssDNA. In some examples, one or more nucleotides of the ssDNA can be replaced with LNA nucleotides.

[0067] Treatment method Aspects of the present disclosure include methods for treating or preventing influenza A virus infection in a subject. The subject oligonucleotide compounds have found use as a new class of antiviral therapeutics that can efficiently disrupt packaging and completely prevent a potentially lethal disease in vivo. As shown in the Examples section, in vivo intranasal administration of exemplary oligonucleotide compounds resulted in potent antiviral efficacy and prevented lethal IAV infection in mice.

[0068] Aspects of the method include administering to a subject in need thereof a therapeutically effective amount of the subject compound to treat the subject for an infectious disease or to prevent infection in the subject. "Therapeutically effective amount" means a concentration of the compound sufficient to induce the desired biological effect (e.g., treatment or prevention of a condition or disease, influenza A virus infection). "Treatment" means that at least an improvement in the symptoms associated with the condition affecting the host is achieved, and improvement is used in a broad sense to refer to at least a reduction in a parameter, e.g., the degree of symptoms associated with the condition being treated. As such, treatment includes situations where the pathological condition, or at least the symptoms associated therewith, are completely inhibited such that the host no longer suffers from that condition, or at least the symptoms characterizing that condition, e.g., the occurrence is prevented, or stopped, e.g., terminated. Thus treatment includes (i) prevention, i.e., reducing the risk of onset of clinical symptoms, including not causing clinical symptoms, e.g., preventing disease progression to a deleterious state, (ii) inhibition, i.e., stopping the onset or further progression of clinical symptoms, e.g., alleviating or completely inhibiting an active disease (e.g., an infectious disease), and / or (iii) alleviation, i.e., causing regression of clinical symptoms. In the context of influenza A virus infection, the term "treating" includes any or all of reducing the number of virus cells in a patient sample, inhibiting the replication of virus cells, and improving one or more symptoms associated with the infectious disease.

[0069] The subject to be treated can be one in need of a therapy for which the host to be treated is treatable using the subject compound. In some embodiments, the subject is suspected of having an influenza A virus infection. In certain embodiments, the subject is diagnosed as having an influenza A virus infection. As such, in some cases, the subject is infected with the virus.

[0070] In certain cases, the subject is at risk of or suspected of being infected with a virus. In some embodiments, the vRNA is PB2 vRNA. The subject methods can be used to prevent influenza A virus infection in a subject. "Prevention" means that a subject at risk of influenza A virus infection remains uninfected despite exposure to the virus under conditions that would normally cause infection. In some cases, administration of the subject agent (e.g., an oligonucleotide compound) protects the subject from infection for at least 1 week, at least 2 weeks, at least 3 weeks, at least 1 month, at least 2 months, at least 3 months, etc. Multiple doses of the subject compound can be administered according to the subject methods that provide long-term protection from infection in the subject. The timing and dosage can be readily determined using conventional methods.

[0071] In some cases, the subject treatment methods include the step of determining or diagnosing whether a subject has an influenza A virus infection. The determining step can be performed using any convenient method. In some cases, the determining step includes obtaining a biological sample from the subject and analyzing the sample for the presence of viral cells. The sample can be a cell sample. The determining step can include the identification of viral cells containing specific mutations.

[0072] Accordingly, various subjects can potentially be treated using the subject compounds and pharmaceutical compositions described herein. As used herein, the terms "subject" and "host" are used interchangeably. Generally, such subjects are "mammals" and the human is the intended one. Other subjects can include household pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, rats, e.g., animal models of disease), and non-human primates (e.g., chimpanzees and monkeys).

[0073] The amount of the compound of the subject being administered can be determined, using any convenient method, to be an amount sufficient to produce the desired effect in relation to a pharmaceutically acceptable diluent, carrier, or vehicle. The specifications for the unit dosage forms of the present disclosure will depend on the particular compound used and the effect to be achieved, as well as the pharmacodynamics associated with each compound in the host.

[0074] In some embodiments, the effective amount of the compound of the subject is an amount in the range of about 50 ng / ml to about 50 μg / ml (e.g., about 50 ng / ml to about 40 μg / ml, about 30 ng / ml to about 20 μg / ml, about 50 ng / ml to about 10 μg / ml, about 50 ng / ml to about 1 μg / ml, about 50 ng / ml to about 800 ng / ml, about 50 ng / ml to about 700 ng / ml, about 50 ng / ml to about 600 ng / ml, about 50 ng / ml to about 500 ng / ml, about 50 ng / ml to about 400 ng / ml, about 60 ng / ml to about 400 ng / ml, about 70 ng / ml to about 300 ng / ml, about 60 ng / ml to about 100 ng / ml, about 65 ng / ml to about 85 ng / ml, about 70 ng / ml to about 90 ng / ml, about 200 ng / ml to about 900 ng / ml, about 200 ng / ml to about 800 ng / ml, about 200 ng / ml to about 700 ng / ml, about 200 ng / ml to about 600 ng / ml, about 200 ng / ml to about 500 ng / ml, about 200 ng / ml to about 400 ng / ml, or about 200 ng / ml to about 300 ng / ml).

[0075] In some embodiments, an effective amount of the subject compound is in the range of about 10 pg to about 100 mg, such as about 10 pg to about 50 pg, about 50 pg to about 150 pg, about 150 pg to about 250 pg, about 250 pg to about 500 pg, about 500 pg to about 750 pg, about 750 pg to about 1 ng, about 1 ng to about 10 ng, about 10 ng to about 50 ng, about 50 ng to about 150 ng, about 150 ng to about 250 ng, about 250 ng to about 500 ng, about 500 ng to about 750 ng, about 750 ng to about 1 μg, about 1 μg to about 10 μg, about 10 μg to about 50 μg, about 50 μg to about 150 μg, about 150 μg to about 250 μg, about 250 μg to about 500 μg, about 500 μg to about 750 μg, about 750 μg to about 1 mg, about 1 mg to about 50 mg, about 1 mg to about 100 mg, or about 50 mg to about 100 mg. The amount can be a single dose or a total daily dose. The total daily dose can be in the range of 10 pg to 100 mg, or in the range of 100 mg to about 500 mg, or in the range of 500 mg to about 1000 mg.

[0076] In some embodiments, a single dose of the subject compound is administered. In other embodiments, multiple doses of the subject compound are administered. When multiple doses are administered over a period of time, the subject compound can be administered twice a day (qid), once a day (qd), every other day (qod), every two days, three times a week (tiw), or twice a week (biw) over a period of time. For example, the compound can be administered twice a day, once a day, every other day, three times a week, or twice a week over a period of from 1 day to about 2 years or more. For example, the compound can be administered for 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, or 2 years or more, depending on various factors, at any of the aforementioned frequencies.

[0077] Using any of a variety of methods, it is possible to determine whether a treatment method is effective. For example, a biological sample obtained from an individual treated by the subject method can be analyzed for the presence and / or level of viral cells. The evaluation of the effectiveness of a treatment method for a subject can include the evaluation of the subject before, during, and / or after treatment using any convenient method. Aspects of the subject method further include the step of evaluating the treatment response of the subject to the treatment.

[0078] In some embodiments, the method includes evaluating the condition of a subject, including diagnosing or assessing one or more symptoms of the subject associated with the disease or condition of interest being treated. In some embodiments, the method includes obtaining a biological sample from the subject and analyzing the sample for the presence of viral cells or components thereof associated with the disease or condition of interest (e.g., as described herein). The sample can be a cell sample. The evaluation step of the subject method can be performed one or more times before, during, and / or after administration of the subject compound using any convenient method. In certain cases, the evaluation step includes the identification and / or quantification of viral cells. In a specific example, evaluating the subject includes diagnosing whether the subject has a viral infection or symptoms thereof.

[0079] Screening method Aspects of the disclosure also include screening assays configured to identify agents that find use in the methods of the invention, as discussed above. Aspects of the disclosure include methods for screening candidate agents for their ability to inhibit influenza A virus in cells. In some examples, the method includes contacting a sample containing viral RNA (vRNA) comprising a PSL2 motif with a candidate agent and determining whether the candidate agent specifically binds to the PSL2 motif. In some cases, an agent that specifically binds to the PSL2 motif will treat a subject having influenza A virus infection. To evaluate or determine means to at least predict that a given test compound has the desired activity such that further testing of the compound in additional assays, such as animal models and / or clinical assays, is desirable.

[0080] Candidate agents are selected from small molecules, oligonucleotides, antibodies, and polypeptides. In some examples, the determining step includes detecting a cellular parameter, and a change in the parameter in the cell as compared to a cell not contacted with the candidate agent indicates that the candidate agent specifically binds to the PSL2 motif. In some cases, the screening method of interest is the method of SHAPE analysis (selective 2'-hydroxyl acylation analyzed by primer extension). In a particular example, the candidate agent is an oligonucleotide.

[0081] Drug screening can be performed using in vitro models, genetically modified cells or animals, or purified PSL2 protein. Ligands that compete with, modulate, or mimic the action of a lead drug can be identified. Drug screening identifies agents that bind to specific sites of the PSL2 motif. A wide variety of assays can be used for this purpose, including labeled in vitro binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, etc. Knowledge of the three-dimensional structure of PSL2 derived from the structural studies described herein can also lead to the rational design of small drugs that specifically inhibit IAV activity.

[0082] As used herein, the term "agent" refers to any molecule having the ability to bind to PSL2 and inhibit IAV, such as an oligonucleotide, protein, or pharmaceutical. Generally, multiple assay mixtures are analyzed in parallel at different agent concentrations to obtain specific responses to various concentrations. Typically, one of these concentrations acts as a negative control, i.e., at zero concentration or below the detection level.

[0083] Candidate agents include a number of chemical classes, such as oligonucleotides, antibodies, polypeptides, and organic molecules, such as small organic compounds having a molecular weight greater than 50 and less than about 2,500 daltons. Candidate agents contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and typically contain at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least two of the functional chemical groups. Candidate agents often contain cyclic carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are found among biomolecules, including peptides, sugars, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs, or combinations thereof.

[0084] Candidate agents are obtained from a wide variety of sources, including libraries of synthetic or natural compounds. For example, numerous means are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including the expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant, and animal extracts are available or can be readily prepared. Also, libraries and compounds made naturally or synthetically can be readily modified through conventional chemical, physical, and biochemical means and used to create combinatorial libraries. Directed or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., can be performed on known pharmacological agents to create structural analogs. The goal in certain embodiments is a compound that crosses the blood-brain barrier.

[0085] When the screening assay is a binding assay, one or more of the molecules can be bound to a member of the signal generation system, such as a label, which can directly or indirectly provide a detectable signal. Various labels include, but are not limited to, radioisotopes, fluorescent agents, chemiluminescent agents, enzymes, specific binding molecules, particles, such as magnetic particles, and the like. Specific binding molecules include pairs such as biotin and streptavidin, digoxin and anti-digoxin, and the like. For specific binding members, the complementary member will typically be labeled with a molecule that effects detection according to known procedures.

[0086] A variety of other reagents can be included in the screening assay. These include reagents such as salts, neutral proteins, such as albumin, detergents, etc., which are used to promote optimal protein-protein binding and / or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc., can be used. The components of the mixture are added in any order that results in the required binding. Incubation is carried out at any suitable temperature, typically 4 to 40 °C. The incubation period is selected for optimal activity but may be optimized to facilitate high-speed high-throughput screening. Typically, 0.1 to 1 hour will be sufficient.

Example

[0087] The following examples are set forth to provide a complete disclosure and description to those skilled in the art of how to make and use the invention and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or only the experiments that have been performed. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weights are weight average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure. Standard abbreviations, e.g., bp, base pair, kb, kilobase, pl, picoliter, s or sec, second, min, minute, h or hr, hour, aa, amino acid, kb, kilobase, bp, base pair, nt, nucleotide, i.m., intramuscular, i.p., intraperitoneal, s.c., subcutaneous, etc. may be used.

[0088] Materials and Methods Cells and Viruses: HEK293T and MDCK cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained in Dulbecco's modified Eagle's medium (Gibco) containing 10% fetal bovine serum and penicillin-streptomycin. Influenza A / PR / 8 / 34 (PR8) H1N1 virus was generated using an 8-plasmid reverse genetics system. The tissue culture adapted influenza A / Hong Kong / 8 / 68 (HK68) H3N2 virus was obtained from the ATCC (ATCC-VR-1679). Viruses were grown and amplified at 35 °C in 10-day-old specific pathogen-free chicken embryos (Charles River Laboratories, SPAEAS).

[0089] Plasmid constructs and cloning: Plasmids containing wild-type PB2 segments from influenza virus A / Puerto Rico / 8 / 34 (H1N1) [PR8], A / New York / 470 / 2004 (H3N2) [NY470], A / New York / 312 / 2001 (H1N1) [NY312], A / Brevig Mission / 1 / 1918 (H1N1)

[1918] , A / California / 04 / 2009 (H1N1) [CA09], A / Vietnam / 03 / 2004 (H5N1) [VN1203], and A / Anhui / 1 / 2013 (H7N9) were used. For the generation of the PR8 packaging mutant vRNA, we used the Stratagene QuickChange XL site-directed mutagenesis kit (Stratagene) for mutagenesis of the pDZ plasmid containing the PB2 gene of PR8. The sequences of each mutant construct were confirmed by automated sequencing.

[0090] Reverse genetics and virus titration: Influenza A / Puerto Rico / 8 / 34 (PR8) virus was generated using an 8-plasmid reverse genetics system (Hoffman et al., 2000). Briefly, to generate recombinant PR8 virus, 10 of 293T / MDCK co-cultures 6Cells were transfected with 1 μg of one of each of eight segments contained within a plasmid using a bidirectional dual PolI / II promoter system for simultaneous synthesis of genomic vRNA and mRNA, with Lipofectamine 3000 (Invitrogen). Cells were harvested 24 hours after transfection and inoculated into the chorioallantoic cavity of 10-day-old chicken embryos (Charles River, specific pathogen-free eggs for research grade). Rescue of recombinant virus was evaluated by hemagglutination activity. Each newly rescued virus was plaque titrated and mutations were confirmed by sequencing of the mutated genes. The plaque assay was performed as previously described (Szretter et al., 2006) on confluent MDCK cells. The hemagglutination (HA) assay was performed at room temperature in 96-well round-bottom plates using 50 μl of virus diluent and a 0.5% suspension of turkey red blood cells in 50 μl of phosphate-buffered saline (PBS).

[0091] Virus growth rate: The growth rate for the PR8 virus was determined by inoculating 10-day-old chicken eggs with 100 plaque-forming units (PFU) of virus. 72 hours after inoculation, the virus titer in the chorioallantoic fluid was determined by plaque titration against MDCK cells.

[0092] Isolation of packaged vRNA: To analyze the packaged vRNA for the PR8 mutant virus, 10-day-old eggs were inoculated with approximately 1000 PFU of recombinant virus and incubated for 72 hours. Chorioallantoic fluid was collected and the supernatant was purified by low-speed centrifugation. The purified supernatant was then layered on a 30% sucrose cushion and ultracentrifuged at 30,000 RPM for 2.5 hours (Beckman Rotor SW41). The pelleted virus was resuspended in PBS and extracted with TRIzol (Invitrogen). The precipitated vRNA was resuspended in 10 mM Tris-HCl (pH 8.0) with a final volume of 20 μl and stored at -80°C.

[0093] qPCR analysis of packaged vRNA: Approximately 200 ng of extracted vRNA was reverse transcribed using a universal 3’ primer (5’-AGGGCTCTTCGGCCAGCRAAAGCAGG) (SEQ ID NO: 97) and Superscript III reverse transcriptase (RT) (Invitrogen). The RT product was diluted 10,000-fold and used as a template for quantitative PCR (qPCR). Separate PCRs were then performed with segment-specific primers as previously described (Marsh et al., 2007). The 10 ul reaction mixture contained 1 ul of diluted RT product, 0.5 uM primer concentrate, and SYBR GreenER dye, 200 uM deoxynucleoside triphosphates, heat-labile UDG, optimized SYBR Green Select Buffer, and AmpliTaq DNA polymerase UP enzyme in SYBR Select Master Mix (Applied Biosystems). Relative vRNA concentrations were determined by cycle threshold analysis, and after normalizing the total vRNA amount by equalizing the levels of HAvRNA, the incorporation rate was calculated relative to the level of wild-type vRNA packaging. Virus packaging results represent the mean levels of vRNA incorporation ± standard deviation from two independent virus purifications at three quantified vRNA levels, n = 6.

[0094] Mouse infection: Groups of 6 - 8 week-old female BALB / C mice (Jackson Laboratory) were lightly anesthetized with isoflurane and infected intranasally with 50 ul of 1000 PFU of wild-type mouse-adapted PR8 (H1N1) virus (ATCC), PB2 mutant PR8 recombinant virus, or sterile PBS. Body weights were measured daily, and animals were humanely sacrificed on day 10 or when weight loss exceeded 20%. All animal husbandry and experimental procedures were in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the Stanford University Administrative Approved by the Panel on Laboratory Animal Care.

[0095] Locked nucleic acid (LNA) design and preparation: Oligonucleotides containing locked nucleic acid (LNA) were custom synthesized from Exiqon. Capital letters indicate LNA. Lowercase letters indicate typical (unlocked) DNA nucleotides. All oligonucleotides contained phosphorothioate internucleoside linkages. The LNA was designed to be complementary to different sequences contained in the PSL2 structure of segment PB2. LNA8 and 9 were designed to contain a continuous stretch of 6 - 8 DNA nucleotides for RNase-H recruitment. The sequences of all LNAs are shown below. LNA1: 5’ AccAaaAGaaT 3’ (SEQ ID NO: 67) LNA2: 5’ TggCcATcaaT 3’ (SEQ ID NO: 68) LNA3: 5’ TagCAtActtA 3’ (SEQ ID NO: 69) LNA4: 5’ CCAAAAGA 3’ (SEQ ID NO: 70) LNA5: 5’ CATACTTA 3’ (SEQ ID NO: 71) LNA6: 5’ CagaCaCGaCCaaAA 3’ (SEQ ID NO: 72) LNA7: 5’ TAcTtaCTgaCagCC 3’ (SEQ ID NO: 73) LNA8: 5’ AGACacgaccaAAAG 3’ - containing RNase-H activity (SEQ ID NO: 74) LNA9: 5’ TACTtactgacaGCC 3’ - containing RNase-H activity (SEQ ID NO: 75) LNA9.2: 5’ TACttactgacAGCC 3’ (SEQ ID NO: 76) LNA10: 5’ ACCaaaagAAT 3’ (SEQ ID NO: 77) LNA11: 5’ TGGccatcAAT 3’ (SEQ ID NO: 78) LNA12: 5’ TAGcatacTTA 3’ (SEQ ID NO: 79) LNA13: 5’ CgacCAaaAGaattC 3’ (SEQ ID NO: 80) LNA14: 5’ CGACcaaaagaATTC 3’ (SEQ ID NO: 81) LNA15: 5’ GaTGgCcATcaAttA 3’ (SEQ ID NO: 82) LNA16: 5’ GATGgccatcaATTA 3’ (SEQ ID NO: 83) LNA17: 5’ TcTAgCaTActTacT 3’ (SEQ ID NO: 84) LNA18: 5’ TCTAgcatactTACT 3’ (SEQ ID NO: 85) LNA19: 5’ GAAttcggatgGCCA 3’ (SEQ ID NO: 86) LNA20: 5’ GGCCatcaattaGTG 3’ (SEQ ID NO: 87) LNA21: 5’ TTCGgatggccaTCA 3’ (SEQ ID NO: 88) LNA22: 5’ AGCCagacagCGA 3’ (SEQ ID NO: 89) LNA23: 5’ GACAgccagacaGCA 3’ (SEQ ID NO: 90)

[0096] The following oligonucleotides were designed to cover single nucleotide polymorphisms (SNPs) in the PSL2 sequence. The following exemplary sequences are modified versions of LNA9 that contain single mutation sites that protect against several bird and bat strains whose PLS2 target sequences contain nucleotide changes at the LNA9 target sequence. It is understood that a similar design can be applied to any of the sequences described herein.

[0097] LNA9.G74C: 5’ TACTtactgacaGTC 3’ (SEQ ID NO: 94) LNA9.T80C: 5’ TACTtaccgacaGCC 3’ (SEQ ID NO: 95) LNA19.U56C: 5’ GGATttcggatggCCA 3’ (SEQ ID NO: 96)

[0098] Antiviral assay: LNAs were reconstituted at 100 μM in RNase-free water, aliquoted, and stored at -20 °C prior to single use. Using Lipofectamine 3000 (Life Technology), LNAs were transfected into cells at final concentrations of 1 μM, 100 nM, 10 nM, and 1 nM according to the manufacturer's protocol. For the prophylactic antiviral assay, 10 6 MDCK cells were seeded in 6-well plates 24 h before transfection with the designated LNA. Cells were then infected with 0.01 MOI of PR8 (H1N1) or HK68 (H3N2) virus 4 h, 2 h, or 1 h after transfection. For the post-infection therapeutic antiviral evaluation, MDCK cells were infected with PR8 or HK68 as described. LNAs were then transfected 4 h, 2 h, or 1 h after infection. Forty-eight hours after infection, the supernatant was harvested and the virus titer was determined by plaque assay.

[0099] In vitro transcription of vRNA: For each wild-type isolate (PR8, 1918, VN1203, NY470, NY312, CA09, and A / Anhui / 1 / 2013 H7N9) and PR8 packaging mutant clone, PB2 cDNA was amplified from plasmid using segment-specific primers under the T7 promoter. The amplified cDNA was gel purified using an Invitrogen DNA gel kit. vRNA was then generated by in vitro transcription using T7-MEGAscript. vRNA for SHAPE was purified by MEGAclear (Thermofisher, cat. no. AM1908) to a purity and length confirmed by capillary electrophoresis.

[0100] sf-SHAPE analysis of vRNA: PB2 vRNA was folded in 100 mM HEPES, pH = 8 (100 mM NaCl, 2.5 mM MgCl, 1 min at 65 °C, cooled at room temperature for 5 min, 20 - 30 min at 37 °C). Acylation for 2 min with NMIA (Wilkinson et al., 2006) and reverse transcription (RT) primer extension were performed at 45 °C for 1 min, 52 °C for 25 min, and 65 °C for 5 min as previously described (Mortimer and Weeks, 2009). 6FAM was used for all labeled primers. Exceptions to these protocols were as follows: (i) RNA purification after acylation was performed using an RNA C&C column (Zymo Research) instead of ethanol precipitation, (ii) before and after the addition of SHAPE primer buffer, the mixture was placed at room temperature for 2 - 5 min, which significantly improved RT transcription yield, (iii) DNA purification was performed using 96-well format Sephadex G-50 size exclusion resin and then concentrated by vacuum centrifugation, resulting in more significant removal of primers, (iv) 2 pmol of RNA was used in the ddGTP RNA sequencing reaction.

[0101] The ABI 3100 Genetic Analyzer (50 cm capillary filled with POP6 matrix) was set to the following parameters: voltage 15 kV, T = 60 °C, injection time = 15 s. Using the GeneScan program, data were acquired for each sample consisting of purified DNA resuspended in 9.75 ul of Hi-Di formamide with 0.25 ul of ROX 500 internal size standard (ABI Cat. 602912) added. The PeakScanner parameters were set to the following: smoothing = none, window size = 25, size calling = local Southern, baseline window = 51, peak threshold = 15. Fragments 250 and 340 were computationally excluded from the ROX500 standard (Akbari et al., 2008). The data from PeakScanner were then processed into SHAPE data using FAST (Fast Analysis of SHAPE Traces), a custom program (Pang et al., 2011). FAST automatically corrects for signal differences due to handling errors, adjusts for signal decay, and converts fragment lengths to nucleotide positions using a ddGTP ladder as an external sizing standard and the local Southern method (Pang et al., 2011 and Pang et al., 2012).

[0102] RNA structure parameters: Gradient and intercept parameters of 2.6 and -0.8 kcal / mol were first tried as proposed (Deigan et al., 2009), and small intercepts close to 0.0 kcal / mol (e.g., ~ -0.3) were found to produce fewer non-optimal structures (within 10% of the maximum energy difference). This parameter differential may be due to the accurate fitting achieved between experimental and control data sets by the automated FAST algorithm. In its current implementation, FAST is incorporated into RNAstructure and requires MFC (Microsoft Foundation Classes). RNA structures were drawn and colored using RNAViz 2 (De Rijk et al., 2003) and finished in Adobe Illustrator.

[0103] Composition design, RNA synthesis, and chemical modification for mutate-and-map experiments: Double-stranded DNA templates were prepared by PCR assembly of DNA oligomers designed by an automated MATLAB script (NA_Thermo, available at "https: / / github.com / DasLab / NA_thermo" as previously described) (Kladwang and Cordero et al., 2011). The compositions for mutate-and-map (M 2 ) included all single mutants to Watson-Crick equivalents. Compensatory mutants for mutation / rescue were designed based on base pairing in the proposed secondary structure (Tian et al., 2014). In vitro transcription reactions, RNA purification, and quantification steps were as previously described (Kladwang and Cordero et al., 2011). One-dimensional chemical mapping, mutate-and-map (M 2 ), and mutation / rescue were performed in 96-well format as previously described (Kladwang and VanLang et al., 2011, Kladwang and Cordero et al., 2011, Cordero et al., 2013). Briefly, RNA was heated and cooled to remove secondary structure heterogeneity, then appropriately folded and incubated with SHAPE reagent (5 mg / mL of 1-methyl-7-nitroisatoic anhydride (1M7)) (Mortimer and Weeks, 2007), the modification reaction was quenched, the RNA was recovered by poly(dT) magnetic beads (Ambion) and FAM-labeled Tail2-A20 primer, the RNA was washed twice with 70% ethanol (EtOH), resuspended in ddH2O, followed by reverse transcription into cDNA and heat NaOH treatment to remove the RNA. The final cDNA library was recovered by magnetic bead separation, rinsed, eluted with ROX-350 ladder in Hi-Di formamide (Applied Biosystems), and loaded onto a capillary electrophoresis sequencer (ABI3100). Data processing, structure modeling, and data accumulation: The HiTRACE software package version 2.0 was used to analyze the CE data (both MATLAB toolbox and web server are available (Yoon et al., 2011, Kim et al., 2013)). Trace alignment, baseline subtraction, sequence assignment, profile fitting, attenuation correction, and normalization were achieved as previously described (Kim et al., 2009, Kladwang et al., 2014). Sequence assignment was manually achieved by matching from the sequencing ladder. The data-driven secondary structure model was obtained using the Fold program of the RNA Structure Package version 5.4 (Mathews et al., 2004) with pseudo energy gradient and intercept parameters of 2.6 kcal / mol and -0.8 kcal / mol. M 2 The two-dimensional Z-score matrix for the dataset, and the helical bootstrap confidence values were calculated as previously described (Tian et al., 2014, Kladwang and VanLang et al., 2011). The Z-score matrix was used as the base-pairwise pseudo free energy at gradients and intercepts of 1.0 kcal / mol and 0 kcal / mol. The secondary structure images were generated by VARNA (Darty et al., 2009). All chemical mapping datasets, including one-dimensional mapping, mutate-and-map, and mutation / rescue, are stored in the RNA Mapping Database (「http: / / rmdb.stanford.edu」) (Cordero et al., 2012).

[0104] SHAPE analysis of LNA-targeted vRNA: The DNA template of the PR8 segment PB2 was prepared by PCR assembly of DNA oligomers, and the in vitro transcription reaction, RNA purification, and quantification steps were as previously described (Kladwang and VanLang et al., 2011). One-dimensional SHAPE chemical mapping was performed in 96-well plate format as described above, with the following exceptions: After the RNA was denatured and refolded as described, 100 nM of each prepared LNA was added to the folded RNA and incubated with 5 mg / mL of the SHAPE reagent 1M7 (1-methyl-7-nitroisatoic anhydride). Modification quenching, RNA recovery, resuspension, reverse transcription, cDNA sequencing, and data processing were performed as described. See Kladwang and VanLang et al., 2011.

[0105] Example 1: SHAPE characterization of the IAV segment PB2 packaging signal identifies conserved structures Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and computational modeling were applied to IAV segment PB2 genomic vRNA to search for structured RNA domains. In vitro transcribed full-length (−) sense PB2 vRNA from strain A / Puerto Rico / 8 / 1934 (H1N1) “PR8” was folded in solution (Pang et al., 2011) and investigated using an electrophilic SHAPE reagent that preferentially reacts with nucleotides present in a flexible single-stranded state (Wilkinson et al., 2006) (Figure 1). This analysis, consistent with recent bioinformatics studies (Priore et al., 2012, Moss et al., 2011) that found a high likelihood of RNA secondary structure conservation in the (+) sense for (−) sense RNA for all segments, including PB2, revealed that most of the 2341 nt vRNA was not extensively structured (Figure 2). These previous studies did not analyze the terminal coding region (TCR) and instead stopped at 80 nucleotides just before the end of the PB2 5’TCR. SHAPE-derived modeling indicated several areas within this region containing a stable RNA secondary structure, most notably a stem-loop motif, herein referred to as packaging stem-loop 2 (PSL2) (Figure 1A), that includes nucleotides 34–87 ((−) sense notation). This segment included a set of nucleotides previously shown to be involved in PB2 packaging through mutagenesis analysis via an unexplained mechanism (Figure 1A–1B, cyclic nucleotide reference) (Gao et al., 2012, Marsh (et al., 2008, Liang et al., 2008, Gog et al., 2007). These conventional mutations support the hypothesis that they act through disruption of the PSL2 structure, and SHAPE analysis of the mutants resulted in different conformations that all discarded the wild-type PSL2 structure (Figure 1C, Figure 3). The 60-nucleotide region encompassing PSL2 showed nearly 100% sequence conservation at the single-nucleotide level among seasonal and pandemic strains from different subtypes and species (Figure 4), indicating the existence of stringent biological requirements to maintain an intact PSL2 structure. Since different downstream sequences within PB2 vRNA can alter the secondary structure of PSL2, the structural conservation of PSL2 was investigated by performing SHAPE analysis on full-length wild-type PB2 vRNA isolated from various IAV strains and subtypes, including highly pathogenic avian H5N1 and pandemic 1918 H1N1 strains. Despite the presence of two branching nucleotides within the stem-loop and prominent branching in the adjacent sequences, the PSL2 stem-loop structure was recovered in SHAPE-derived modeling of PB2 RNA across these diverse species and subtypes (Figure 1D - 1F).

[0106] Figures 1A - 1F show SHAPE chemical mapping performed on full - length (-) sense wild - type PB2 vRNA. Colors indicate SHAPE reactivity and are proportional to the probability that a nucleotide is single - stranded. All structures are cropped to emphasize the 5’ - terminal sequence structure. Energy = ΔG free - energy value of the determined structure generated by an RNA structure modeling algorithm using SHAPE pseudo - free energy parameters. (Figure 1A) Wild - type PB2 RNA secondary structure from strain A / Puerto Rico / 8 / 1934 “PR8” (H1N1). Colored circles correspond to nucleotide sites where synonymous mutations have been reported to affect PB2 packaging (Gao et al., 2012, Marsh et al., 2011). (Figure 1B) Packaging efficiency of the synonymous mutants in (Figure 1A) determined by qPCR. Results were performed in triplicate. Error bars = ±SD. The lower frame shows the mutant name and corresponding mutational change. Nucleotide numbering is shown in the genome (-) sense orientation. (Figure 1C) SHAPE - determined structures of PB2 packaging - defective mutant vRNAs, m757 (G44C) and m745 (A80U). Black frames = sites of synonymous mutations. (Figures 1D - 1F) SHAPE - determined structures of wild - type PB2 from pandemic and highly pathogenic strains, including different subtypes: (Figure 1D) 1918 pandemic (A / Brevig Mission / 1 / 1918 (H1N1)), (Figure 1E) highly pathogenic avian (A / Vietnam / 1203 / 2004 (H5N1)), (Figure 1F) 2009 pandemic “swine” (A / California / 04 / 2009 (H1N1)).

[0107] Figures 2, panels A - B show the SHAPE reactivity of the full - length PB2 vRNA. (Figure 2, panel A) Mean SHAPE reactivity as a function of nucleotide position for the full - length (-) sense PB2 vRNA from the IAV strain A / Puerto Rico / 8 / 1934 (H1N1). The PSL2 region (highlighted in blue, nt 34 - 86) encompasses a 5’ packaging signal domain with a high density of codons whose third position is conserved and has one of the lowest SHAPE reactivities within the vRNA. The region after PSL2 is relatively unstructured but contains another potential site for the presence of an RNA structure between nucleotides 1400 and 1500. Interestingly, this second internal region was also predicted to contain structural elements in a 2011 bioinformatics study (reference 19). (Figure 2, panel B) Enlargement of the PSL2 region from (Figure 2a). Window bin size = 10 nt.

[0108] Figures 3A - 3E show that packaging - defective mutations disrupt the wild - type SHAPE reactivity. Left: Mutant SHAPE reactivity plotted as a change relative to WT. Nucleotide numbering starts from the 5’ end of the (-) sense vRNA. Orange bars indicate the sites of the mutations. Energy values represent the ΔG free energy of the predicted structure generated by an RNA structure modeling algorithm using SHAPE pseudo - free energy parameters. Right: SHAPE - determined structures of the full - length (-) sense mutant PB2 vRNAs from the PR8 strain (H1N1). Images are cropped to highlight the 5’ end region. (Figure 3A) Wild - type. Packaging - defective mutants: (Figure 3B) m744b (AG83, 85UA). (Figure 3C) m745 (A80U). (Figure 3D) m55c (CU35, 36UC). (Figure 3E) m757 (G44C).

[0109] Figure 4 shows the conservation of nucleotide sequences containing the PSL2 structure. Graphical representation of nucleic acid sequence alignments across diverse influenza A virus subtypes and strains (「weblogo.」 followed by 「berkeley」 followed by 「.edu」). The overall height represents sequence conservation at that nucleotide position, and the height of the symbols within each position indicates the relative frequency of each nucleotide at that site. Black frame = PSL2 region. Sequences included in the alignment: highly pathogenic A / Brevig Mission / 1 / 1918 (H1N1), pandemic 「swine influenza」 A / California / 04 / 2009 (H1N1), contemporary human A / New York / 470 / 2004 (H3N2), human A / Puerto Rico / 8 / 1934 (H1N1), highly pathogenic avian A / Vietnam / 03 / 2004 (H5N1), human A / Hong Kong / 8 / 1968 (H3N2), and human A / New York / 312 / 2001 (H1N1). RNA nucleotides numbered in the (-) sense orientation. Sequence alignment of the 5’ terminal region of PB2 corresponding to the above sequences. The shaded blue frame encompasses the PSL2 RNA secondary structure elements. Black spots indicate branched nucleotide sites.

[0110] The Mutate-and-Map strategy validates the PSL2 structure and predicts novel packaging variants. To further test the SHAPE analysis of the PSL2 RNA structure and identify mutations that provide additional information required for in vivo studies, the multidimensional chemical mapping (Kladwang and Das, 2010) method was applied to the PSL2 segment. First, the mutate-and-map (M 2 ) measurements confirmed disruption of the chemical reactivity pattern after global mutagenesis of each stem residue, including changes at nucleotides previously found to be important for PB2 packaging (Figure 5A, see legend) (Marsh et al., 2008, Gog et al., 2007). These M 2Automated computational analysis based on the data recovered the SHAPE-induced PSL2 structure with high confidence (Figures 1C, 3, 5B–5D) and further validated the structural model. Second, as a predictive test, compensatory mutations were designed to restore base pairs in the wild-type stem-loop structure disrupted by the initial packaging-deficient mutations (Figure 6, panels A–B). These mutation-rescue variants actually restored the PSL2 SHAPE pattern, provided base-pair resolution in in vitro validation of the modeled structure, and proposed an array variant for testing the role of the PSL2 structure in vivo.

[0111] Figures 5A - 5D show the two - dimensional Mutate - and - Map (M2) analysis of the PSL2 RNA secondary structure. (Figure 5A) The mapping of overall single - nucleotide mutations and the resulting chemical accessibility reveals interactions in the 3 - dimensional structure of the RNA. Chemical accessibility plotted on a greyscale (black = highest SHAPE reactivity) over 88 single mutations at single - nucleotide resolution of the PSL2 element from the PR8 strain PB2. Reactivity peaks (left to right) correspond to nucleotides from the 5’ to 3’ end of the PB2 RNA. Nucleotide sites corresponding to known packaging mutations (as reported by Marsh et al., 2008) are indicated in blue to the right. Red arrows indicate mutant sites with significant packaging - deficit mutations predicted by the M2 analysis. (Figure 5B) Strong features of the isolated mutate - and - map data from Z - score analysis (number of standard deviations from the mean at each residue). The Z - score was calculated by subtracting the mean reactivity of this nucleotide over all mutants from the reactivity of each nucleotide and dividing by the standard deviation (output_Zscore_from_rdat in HiTRACE). Squares indicate secondary - structure models derived from the mutate - and - map data. Dark symbols highlight evidence of structured nucleotide - pair formation. (Figure 5C) RNA secondary structure for the 5’ packaging signal region (nt30 - 93) derived from incorporating the Z - score into an RNA - structure - modeling algorithm: bootstrap confidence estimates given as green percentage values. Bootstrap values provide a numerically accurate indicator of structural confidence. Low bootstrap confidence values indicate the presence of alternative structure models. (Figure 5D) Bootstrap values support the values for each base - pair shown in greyscale shading.

[0112] Figure 6 shows the design of compensatory mutations for previously described PR8PB2 mutants. (Figure 6, Panel A) Previously described synonymous mutants (m757, m745, m55c) map to the PSL2 structure. (Figure 6, Panel B) Compensatory mutations (m55c-comp, m745-comp, and m757-comp) were designed at sites predicted to restore the wild-type PSL2 structure based on SHAPE and mutate-and-map chemical analyses. Nucleotides within the black frames indicate the sites of compensatory mutations. (-) Sense vRNA orientation is shown. For mutations where nonsynonymous changes were required to restore the structure, changes in the encoded protein sequence are shown.

[0113] To test whether the PSL2 stem-loop structure observed in solution was associated with viral packaging in the cellular environment, the same nine synonymous mutations reported by Gog et al., 2007 and Marsh et al., 2008 (Figures 1A - 1B, Figure 7 Panel A) as well as M 2 four new synonymous mutations characterized by the analysis (Figure 7, Panel B) were cloned into a pDZ plasmid containing the PR8PB2 gene (Marsh et al., 2008, Liang et al., 2008, Gog et al., 2007) (Figure 8). Here, the packaging efficiency of nine previously known mutants in the PR8 background was equivalent to that originally described in the WSN33 virus 15 (Figure 7, Panel C). Of these, mutants m55c, m757, m745, and m744b were predicted to show the most significant impairment based on their positions within the stem region of PSL2 (Figure 1C, Figures 3A - 3F, Figure 7). In contrast, published mutations that had no effect on PB2 packaging (e.g., m731) mapped to unstructured apical loops or fell outside of PSL2 and did not alter its structural integrity (Figure 9A) (Marsh et al., 2008). M 2Three novel synonymous variants (m74-1, m74-2, and m68) (Figure 5A) identified by analysis to have a significant impact on the in vitro PSL2 structure showed a significant decrease in PB2 packaging, and the mutated sites that resulted in negligible changes in SHAPE reactivity compared to the wild-type PSL2 structure resulted in wild-type-like packaging efficiency levels (e.g., m56) (Figure 7, panel D).

[0114] Figure 7 shows synonymous mutations of a single highly conserved codon of PR8 PB2 vRNA. (Figure 7, panel A) Previously published synonymous mutations involved in PB2 packaging. The upper line is the parental PR8 vRNA sequence ((+) sense orientation), and the mutated single nucleotide is the red bold character on the underline. The numbering and terminology system of the introduced mutations are based on the reports by Marsh et al., 2008 and Gog et al., 2007. The yellow highlighted region indicates the sequence containing the PSL2 structure. (Figure 7, panel B) Design of primer sequences for cloning of synonymous mutations identified from M2 analysis (see Supplementary Figure 4a) into the pDZ plasmid. The sequence is in the (+) sense orientation. Emphasized nucleotides = mutated sites. (Figure 7, panels C-D) (Figure 7, panel C) Previously published synonymous variants, and (Figure 7, panel D) Packaging efficiency representing the ratio of variant PB2 packaging to parental wild-type PB2 for synonymous variants identified by M2 analysis. Results from two independent experiments, assayed three ways (n = 6). Error bars represent ±SD.

[0115] Figure 8 shows the PB2 packaging mutant terminology system and the corresponding mutated sites. 1) Previously published synonymous mutations involved in PB2 packaging (shown in blue), based on reports by Marsh et al., 2008 and Gog et al., 2007, and 2) a mutation terminology system chart showing the names and sites of mutations from the PSL2 structural design single mutants (shown in black) and the double compensatory mutants (shown in red). The numbering and terminology system of the introduced mutations are based on genomic (-) sense vRNA. Examples where mutations result in protein coding changes are indicated by the synonymous (SYN) or non-synonymous (non-SYN) fields.

[0116] Figures 9A - 9C show the effects of synonymous mutations on the PSL2 structure. Left: Predicted RNA secondary structure of PB2 packaging mutants determined by sf-SHAPE analysis for full-length (-) sense PB2 vRNA from the PR8 strain. For clarity only, the wild-type structure is shown in the upper right corner box. Right: SHAPE reactivity graph shown as the change in mutant reactivity relative to the wild-type. Energy values and packaging efficiency percentages are shown below the figure headings. Mutants: (Figure 9A) m731. (Figure 9B) m751. (Figure 9C) m748. The packaging efficiency percentages of PB2 incorporation for each of the previously described mutants are highlighted in blue.

[0117] Compensatory mutations rescued not only viral packaging for segment PB2 (Figure 10, panels A–C, Figure 6, panels A–B), but also other segments previously reported to be affected by deleterious mutations, consistent with the proposed hierarchical role of PB2 in IAV packaging (Muramoto et al., 2006, Gao et al., 2012, Marsh et al., 2008) (Figure 10, panels D–F). In addition to the recovery of PB2 packaging, compensatory mutations resulted in a complete or near-complete rescue of the virus titer reduction caused by deletion mutations (Figure 10, panels G–I). Some nonsynonymous compensatory mutations were able to restore PB2 packaging better than others (m745-comp and m55c-comp compared to m757-comp) (Figure 10, panels A–C), or reflected an incomplete restoration of PB2 protein function through exogenous addition. Such exogenous addition was necessary because several nonsynonymous mutations affected both the PSL2 structure and protein sequence. The most sensitive test of the PSL2 structure was from packaging experiments that did not require the addition of a supplemental wild-type PB2 protein. Based on computational enumeration and multidimensional mutagenesis-rescue experiments (Tian et al., 2014), a single mutation-rescue substitution pair that was both synonymous and eliminated the need for wild-type PB2 protein addition was found (m52 / m65, Figure 11, panels A–B, Figure 12, panel s, Figure 13). Performing each mutation alone (m52 and m65) resulted in a packaging efficiency of less than 4% PB2 incorporation and a titer reduction of more than 4 log 10 above that previously reported for packaging-deficient viruses (i.e., 2 log 10 ), resulting in a severe impairment (Figure 11, panels C–D, Figure 7, panel c, Figure 10). When introduced together into the double-mutant m52 / 65-comp strain that restored the PSL2 structure, despite having the altered sequences, the compensatory mutations restored both packaging efficiency and virus titer to wild-type levels.

[0118] Figures 10, panels A-I, show the effect of compensatory mutations in the PR8 PB2 packaging-deficient mutant on virus packaging and titer. (Figure 10, panels A-C) Packaging efficiency of packaging-deficient and compensatory mutant PB2 vRNA. For compensatory mutations that require nonsynonymous changes, the wild-type PB2 protein expression plasmid was co-transfected during virus rescue. pWT = expression plasmid encoding for wild-type PR8 PB2 protein. Values are given as the percentage of PB2 vRNA packaging in comparison to the wt parental PR8 virus. Results from two independent experiments, assays were performed in triplicate (n = 6). (Figure panels D-F) Packaging efficiency of packaging-deficient and compensatory mutant viruses and their effect on the packaging of other interacting segments, PB1, PA, NP, and Mx. Assays were performed in triplicate (n = 6). (Figure 10, panels G-I) Virus titer by plaque assay. Results in PFU / mL, assays were performed in triplicate.

[0119] Figure 11 shows that multidimensional chemical mapping reveals novel PB2 packaging defects and compensatory variant partners. (Figure 11, panel A) Electropherogram results from global single nucleotide mutation mapping in a Mutate-Map-Rescue analysis to test base pair formation from a 1D data-derived model and identify individual and compensatory double mutants predicted to be good synonymous PSL2 defects and compensatory variant pairs. Chemical accessibility plotted on a greyscale (black = highest SHAPE reactivity) across 88 single mutations at single nucleotide resolution of the PSL2 element from the PR8 strain PB2. Reactivity peaks (left to right) correspond to nucleotides from the 5’ to 3’ end of the PB2 RNA. See Figure 12 for a complete list of Mutate-Rescue pairs. (Figure 11, panel B) Mutation design of single mutants m52 (G52U) and m65 (C65A) on the PSL2 structure, and the double m52 / 65 rescue pair. (Figure 11, panel C) Packaging efficiency of synonymous single and double mutant mutate-and-rescue pairs. Values given as the percentage of PB2 vRNA packaging in comparison to the wt parental PR8 virus. Results from two independent experiments, assayed three ways (n = 6). (Figure 11, panel D) Virus titers in PFU / mL, three-way results. Error bars represent ±SD.

[0120] Panels a - t in Figure 12 show two - dimensional Mutate - Map - Rescue (M2R) analysis. The mutation / rescue results verify the PSL2 RNA secondary structure. Electropherograms of SHAPE analysis in compensatory double mutations to test base - pair formation from a 1D data - derived model and to identify good PSL2 - deficient and compensatory mutant pairs. Chemical accessibility plotted on a greyscale (black = highest SHAPE reactivity) across 88 single mutations at single - nucleotide resolution of the PSL2 element from the PR8 strain PB2. For each tested pair, the “tetrad” of wild - type, single - mutant 1, single - mutant 2, and compensatory double - mutant is grouped for comparison. (Figure 12, panels a - r) Nonsynonymous mutate - and - rescue pairs. All unframed electropherograms are pairs where no disruption and / or rescue was observed. Blue frames indicate good deficiency and rescue mutations. (Figure 12, panel s) Double - synonymous mutate - and - rescue pairs. Green frames = good synonymous deficiency and rescue pairs. (Figure 12, panel t) Packaging efficiency for nonsynonymous mutate - and - rescue pairs. Values given as the ratio of PB2 vRNA packaging compared to the wt parental PR8 virus. Results from two independent experiments, three assays performed (n = 6). Error bars represent ±S.D.

[0121] Figure 13 shows the design of primer sequences for two - dimensional Mutate - Map - Rescue (M2R) mutants. From top to bottom of SEQ ID NOs: (28 - 43). Primer sequences used for QuickChange mutagenic cloning of M2R mutants into the pDZ plasmid. The sequences are in the (+) sense orientation. The left field indicates synonymous (Syn.) or nonsynonymous (non - syn.) changes. Highlighted nucleotides = mutation sites. The mutant primer sets within the frame indicate the double - synonymous mutant partners, m52 and m65.

[0122] To test the relevance of the PSL2 structure in an in vivo model, 6- to 8-week-old BALB / C mice were inoculated intranasally with 1000 PFU of wild-type PR8 virus or strains with mutations predicted to disrupt or restore the PSL2 structure. Mice infected with the PSL2-disrupting mutation - m745 mutant strain (20% packaging efficiency) or the severely packaging-deficient single mutant virus, m52 (<4% packaging efficiency) - showed either reduced clinical signs of disease or none in either weight loss or survival rate, respectively, compared to the PBS control (Figure 14, panels A - B). Notably, the inclusion of compensatory mutations that restored the PSL2 structure rescued viral pathogenicity: animals infected with m52 / 65-comp and m745-comp showed a death profile and survival curve equivalent to those of mice infected with wild-type PR8 (Figure 14, panels A - B). Consistent with APLAC guidelines, all mice were humanely sacrificed after reaching a weight loss of more than 20%.

[0123] Figure 14, panels A - B show that packaging-deficient viruses are attenuated in vivo. Weight loss and percent survival of mice infected with single PSL2-disrupting and compensatory PSL2-restoring double mutant viruses. 6- to 8-week-old BALB / C female mice were infected intranasally with 1000 PFU of PR8 wild-type (wt) virus, packaging-deficient single mutant viruses, m52 and m745, compensatory double mutant viruses, m52 / 65 and m745-comp, or a PBS control. Mice were monitored daily for percent weight loss and percent survival on day 0. Results are presented as the mean of 6 mice per condition from 2 independent experiments. (Figure 14, panel A) Percent weight loss. (Figure 14, panel B) Kaplan - Meier survival plot of the individual cohorts depicted in (Figure 14, panel A).

[0124] Example 2: Therapeutic design and targeting of the PSL2 structure inhibits IAV infection in vitro and in vivo To explore the therapeutic potential targeting PSL2-mediated viral packaging, nine locked nucleic acids (LNAs) containing phosphorothioate nucleoside linkages (Vester and Wengel, 2004) were designed against the major residues predicted to disrupt the overall RNA secondary structure of the element and thereby inhibit virus production (Figure 15, panel A). Two of the designed LNAs, LNA8 and LNA9, are identical to LNA6 and LNA7, respectively, in sequence but have 6 - 7 unmodified (unlocked) DNA nucleotides optimized for RNase-H activation. First, to evaluate the effect of LNA binding on the PSL2 RNA secondary structure, toe-printing and SHAPE chemical mapping were performed on PB2 vRNA in the presence of LNAs. As demonstrated by the antiviral assay results, the sequences encoded by LNA6 - 9 showed the greatest ability to bind and disrupt the wild-type PSL2 structure (Figure 16).

[0125] Figures 15, panels A - D show that locked nucleic acids targeting the PSL2 RNA structure exhibit potent antiviral activity in vitro and in vivo. (Figure 15, panel A) Positions of compensatory locked nucleic acids (LNAs) designed against different regions of the PSL2 structure. (Figure 15, panel B) To screen LNAs for antiviral activity, MDCK cells were pretreated with each indicated 100 nM LNA by Lipofectamine transfection for 1 hour prior to infection with 0.01 MOI of PR8 (H1N1) virus or A / Hong Kong / 8 / 68 (H3N2) virus. Forty-eight hours after infection, supernatants were collected and virus titers were determined by plaque assay. Results from two independent experiments, three assays performed (n = 6). (Figure 15, panel C) Time course of pretreatment (RX) vs. post-infection treatment with LNA9 at titrated concentrations (100 nM, 10 nM, 1 nM). WT+Lipo = infection with Lipofectamine control. Pretreatment: Confluent MDCK cells in 6-well plates were treated with LNA9 either 2 hours or 4 hours before infection. Treated supernatants were removed at the indicated time points and cells were infected with 0.01 MOI of wtPR8 virus for 1 hour. For post-infection treatment: MDCK cells were infected with 0.01 MOI of PR8 virus for 1 hour, then supernatants were replaced and cells were treated with LNA9 either 2 or 4 hours before infection. Supernatants were collected 48 hours later and virus titers were determined by plaque assay three times. Figure 15, panel D shows the effect of intranasal LNA treatment on the survival of virus-infected mice. Mice were intranasally administered 20 μg of LNA9, scrambled LNA, or PBS (non-infected control) 12 hours before PR8 virus infection. All mice received two additional treatments at 8 hpi and 36 hpi (n = 7 mice per condition).

[0126] Figures 16A - 16B show SHAPE analysis for LNA - RNA binding. (Figure 16A) Electrophoresis profiles of SHAPE analysis performed on LNA1, 2, 4, 5, 6 / 8, and 7 / 9 (100 nM) in the presence of PR8 PB2 vRNA. S1, a small molecule that interacts with hepatitis C virus IRES RNA, was added as a control for RNA binding. Left column, SHAPE reagent, unlabeled reaction without 1M7. Right: with labeled reagent. (Figure 16B) Electrophoresis profiles of SHAPE performed on LNA - vRNA combinations at titrated concentrations of LNA. For each LNA, the left set of columns is without labeled reagent.

[0127] Next, in a first pilot experiment to determine the antiviral potential of LNA - mediated targeting of PSL2 across two different IAV subtypes, MDCK cells were pretreated with 100 nM of each LNA and delivered by Lipofectamine™ transfection for 1 hour prior to infection with either 0.01 MOI of wild - type PR8 (H1N1) virus or tissue - culture adapted A / Hong Kong / 8 / 68 (HK68) (H3N2) virus. Forty - eight hours post - infection, the supernatant was harvested and virus production was measured by plaque assay (Figure 15, panel B). LNAs (LNA1, LNA4) specific only to the apical loop of PSL2 had little to no effect on virus titers. Similarly, LNAs 3 and 5 targeting the 3’ bases of PSL2 also did not inhibit virus production. In contrast, nucleotide coverage of both the apical loop and the middle bulge by LNA6 resulted in a more than 2 log 10 titer loss for PR8 (Figure 15, panels A - B). The RNase - H - activating copy of LNA8, LNA6, resulted in an even greater antiviral activity of up to 3 log for both viruses. Most surprisingly, the RNase - H - activating copy of LNA9, LNA7, had the strongest antiviral ability and reduced virus production by nearly 5 log and 4 log for PR8 and HK68, respectively.

[0128] Since the optimal candidate LNA was identified, the treatment time course and concentration parameters of the antiviral activity of LNA9 were further investigated. MDCK cells were treated with 10-fold dilutions of a single dose of LNA9 either 2 or 4 hours before infection with wild-type PR8 virus at 0.01 MOI, or at either 2 or 4 hours post-infection. Cells pretreated with LNA had the most potent antiviral response (greater than 4 logs) and showed strong virus inhibition (greater than 2 logs) even at the lowest dilution (1 nM) (Figure 15, panel C). There was a tendency for antiviral activity to decrease as the time of post-infection treatment increased, but greater than 3 log suppression of virus titer was achieved even at the latest time point of addition tested.

[0129] Example 3: In vivo efficacy experiment: Long-term single-dose prophylaxis Balb / C female mice (5 mice / group) were pre-treated intranasally with a single dose of 20 μg LNA9 either 3 days before (-3 days) or 1 day before (-1 day) infection with a lethal dose of wild-type PR8 virus. Mice were monitored daily for weight loss, clinical score, and survival. Figure 17, panel A shows the percentage of mice surviving over time. Figure 17, panel B shows the percentage of weight loss over time after administration.

[0130] A single dose of 20 μg LNA9 administered 3 days before infection completely protected the mice from lethal influenza disease. Untreated control mice were humanely sacrificed at an average of 5.5 days when they had lost more than 25% of their body weight. In contrast, the pretreatment group showed minimal weight loss, few to no clinical signs of the disease, and complete recovery to pre-infection body weight.

[0131] These results show that a single inhalable dose of LNA9 administered several days before infection can provide long-term protection against lethal disease, indicating that the subject compounds may find use in prophylactic treatment during influenza outbreaks and pandemics.

[0132] Example 4: Susceptibility of Influenza Virus to Oseltamivir and LNA9 after Serial Passages in the Presence of Drugs: Drug Selection Experiments Oseltamivir (Tamiflu) is the most widely used and stockpiled neuraminidase inhibitor (NAI) on the market. Similar to all NAIs, oseltamivir requires a conformational rearrangement in the viral neuraminidase (NA) protein that accommodates the drug. Any mutation in the NA protein that affects this rearrangement reduces the binding affinity of oseltamivir and thus drug efficacy. In particular, the H274Y variant (also known as the H275Y mutation depending on the nomenclature system) is most commonly associated with oseltamivir resistance. The rapid selection of the H274Y mutation in immunocompromised patients can lead to the clinical failure of the last-resort NAI drug, peramivir, indicating that the selection of multidrug-resistant viruses in immunocompromised hosts may be more common than previously thought. This, along with the recent spread of oseltamivir-resistant and NAI-resistant viruses, indicates the need to reevaluate the use of common NAIs. The development of new classes of antivirals is urgent to reduce the harmful effects of future influenza pandemics on human health.

[0133] The sequence region in segment PB2 containing the PSL2 stem-loop is highly conserved across IAV subtypes, strains, and isolates from a wide range of host species, likely reflecting stringent biological conditions for its conservation. SHAPE analysis of this region confirmed the maintenance of the PSL2 structure among seasonal and pandemic viruses from different subtypes and hosts, strongly indicating that this structural element could be a novel pan-genotype therapeutic target (thus LNA9 has the potential for a broad-spectrum against IAV isolates). Also, since the subject LNA targets a highly conserved viral genomic RNA target that clearly has strong constraints on its mutability, the subject LNA targeting PSL2 is expected to have a higher barrier to the development of resistance compared to NAIs.

[0134] The susceptibility of influenza virus to LNA9 versus oseltamivir after serial passage under drug pressure was investigated. Oseltamivir had an initial IC 50 of 41 nM against PR8 at passage 1 of drug treatment as determined by plaque reduction assay. After only 6 virus passages with increasing amounts of drug, the IC 50 of oseltamivir increased sharply up to a 50 μM - 1000-fold increase rate. See Figure 18, Panels A - D. In comparison, after 10 passages of the virus in the presence of LNA9, the IC 50 remained stable from 18 to 16 pM. Figure 19, Panels A and B.

[0135] LNA9 can also be used to treat drug-resistant viruses. A drug-resistant mutant of the A / WSN / 33 (H1N1) virus was generated using a reverse genetics virus rescue system in which the NA gene was mutated to contain the H274Y resistance mutation. Against this virus, oseltamivir had an IC 50 of 53 μM. Importantly, LNA9 maintained its potency and efficacy against the WSN H274Y virus at picomolar activity. Figure 19, Panel C. This result is strong evidence for the therapeutic treatment of NA-I resistant viruses with PSL2-targeted LNAs. This result also highlights the activity of LNA9 against different IAV isolates.

[0136] References 1. M.J. Memoli, R.J. Hrabal, A. Hassantoufighi, M.C. Eichelberger, J.K. Taubenberger, Rapid selection of oseltamivir- and peramivir-resistant pandemic H1N1 virus during therapy in 2 immunocompromised hosts. Clin Infect Dis 50, 1252 - 1255 (2010); published online Epub May 1 (10.1086 / 651605). 2. R. Hai, M. Schmolke, V. H. Leyva-Grado, R. R. Thangavel, I. Margine, E. L. Jaffe, F. Krammer, A. Solorzano, A. Garcia-Sastre, P. Palese, N. M. Bouvier, Influenza A(H7N9) virus gains neuraminidase inhibitor resistance without loss of in vivo virulence or transmissibility. Nature communications 4, 2854(2013)10.1038 / ncomms3854). 3. F. G. Hayden, M. D. de Jong, Emerging influenza antiviral resistance threats. J Infect Dis 203, 6 - 10(2011); published online EpubJan 1(10.1093 / infdis / jiq012). 4. X. Liu, T. Li, Y. Zheng, K. W. Wong, S. Lu, H. Lu, Poor responses to oseltamivir treatment in a patient with influenza A(H7N9) virus infection. Emerging microbes & infections 2, e27(2013); published online EpubMay(10.1038 / emi.2013.30). 5. J. Parsons, M. P. Castaldi, S. Dutta, S. M. Dibrov, D. L. Wyles, T. Hermann, Conformational inhibition of the hepatitis C virus internal ribosome entry site RNA. Nat Chem Biol 5, 823 - 825(2009); published online EpubNov(10.1038 / nchembio.217). 6. C. Romero-Lopez, A. Berzal-Herranz, Unmasking the information encoded as structural motifs of viral RNA genomes: a potential antiviral target. Rev Med Virol 23, 340 - 354 (2013); published online EpubNov (10.1002 / rmv.1756). 7. P. S. Palese, M. L., Fields Virology. D. M. e. a. Knipe, Ed., Orthomyxoviridae (Lippincott Williams & Wilkins, ed. 5th, 2007). 8. R. W. Compans, J. Content, P. H. Duesberg, Structure of the ribonucleoprotein of influenza virus. J Virol 10, 795 - 800 (1972); published online EpubOct ( 9. E. C. Hutchinson, J. C. von Kirchbach, J. R. Gog, P. Digard, Genome packaging in influenza A virus. J Gen Virol 91, 313 - 328 (2010); published online EpubFeb (10.1099 / vir.0.017608 - 0). 10. T. Noda, Y. Kawaoka, Structure of influenza virus ribonucleoprotein complexes and their packaging into virions. Rev Med Virol 20, 380 - 391 (2010); published online EpubNov (10.1002 / rmv.666). 11. Y. Muramoto, A. Takada, K. Fujii, T. Noda, K. Iwatsuki-Horimoto, S. Watanabe, T. Horimoto, H. Kida, Y. Kawaoka, Hierarchy among viral RNA (vRNA) segments in their role in vRNA incorporation into influenza A virions. J Virol 80, 2318 - 2325 (2006); published online Epub Mar (10.1128 / jvi.80.5.2318 - 2325.2006). 12. Q. Gao, Y. Y. Chou, S. Doganay, R. Vafabakhsh, T. Ha, P. Palese, The influenza A virus PB2, PA, NP, and M segments play a pivotal role during genome packaging. J Virol 86, 7043 - 7051 (2012); published online Epub Jul (10.1128 / jvi.00662 - 12). 13. G. A. Marsh, R. Rabadan, A. J. Levine, P. Palese, Highly conserved regions of influenza a virus polymerase gene segments are critical for efficient viral RNA packaging. J Virol 82, 2295 - 2304 (2008); published online Epub Mar (10.1128 / jvi.02267 - 07). 14. E. Fournier, V. Moules, B. Essere, J. C. Paillart, J. D. Sirbat, C. Isel, A. Cavalier, J. P. Rolland, D. Thomas, B. Lina, R. Marquet, A supramolecular assembly formed by influenza A virus genomic RNA segments. Nucleic Acids Res 40, 2197 - 2209 (2012); published online Epub Mar (10.1093 / nar / gkr985). 15. C. Gavazzi, C. Isel, E. Fournier, V. Moules, A. Cavalier, D. Thomas, B. Lina, R. Marquet, An in vitro network of intermolecular interactions between viral RNA segments of an avian H5N2 influenza A virus: comparison with a human H3N2 virus. Nucleic Acids Res 41, 1241 - 1254 (2013); published online Epub Jan (10.1093 / nar / gks1181). 16. J. R. Gog, S. Afonso Edos, R. M. Dalton, I. Leclercq, L. Tiley, D. Elton, J. C. von Kirchbach, N. Naffakh, N. Escriou, P. Digard, Codon conservation in the influenza A virus genome defines RNA packaging signals. Nucleic Acids Res 35, 1897 - 1907 (2007) 10.1093 / nar / gkm087). 17. W. N. Moss, S. F. Priore, D. H. Turner, Identification of potential conserved RNA secondary structure throughout influenza A coding regions. Rna 17, 991 - 1011 (2011); published online Epub Jun (10.1261 / rna.2619511). 18. Y. Liang, T. Huang, H. Ly, T. G. Parslow, Mutational analyses of packaging signals in influenza virus PA, PB1, and PB2 genomic RNA segments. J Virol 82, 229 - 236 (2008); published online Epub Jan (10.1128 / JVI.01541 - 07). 19. K. A. Wilkinson, E. J. Merino, K. M. Weeks, Selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nature protocols 1, 1610 - 1616 (2006) 10.1038 / nprot.2006.249). 20. P. S. Pang, M. Elazar, E. A. Pham, J. S. Glenn, Simplified RNA secondary structure mapping by automation of SHAPE data analysis. Nucleic Acids Res 39, e151 (2011); published online Epub Dec (10.1093 / nar / gkr773). 21. S.F. Priore, W.N. Moss, D.H. Turner, Influenza A virus coding regions exhibit host-specific global ordered RNA structure. PLoS One 7, e35989 (2012) 10.1371 / journal.pone.0035989). 22. W. Kladwang, R. Das, A mutate-and-map strategy for inferring base pairs in structured nucleic acids: proof of concept on a DNA / RNA helix. Biochemistry 49, 7414 - 7416 (2010); published online Epub Sep 7 (10.1021 / bi101123g). 23. S. Tian, P. Cordero, W. Kladwang, R. Das, High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states. Rna 20, 1815 - 1826 (2014); published online Epub Nov (10.1261 / rna.044321.114). 24. B. Vester, J. Wengel, LNA (locked nucleic acid): high-affinity targeting of complementary RNA and DNA. Biochemistry 43, 13233 - 13241 (2004); published online Epub Oct 26 (10.1021 / bi0485732). 25. K. Klumpp, R. W. Ruigrok, F. Baudin, Roles of the influenza virus polymerase and nucleoprotein in forming a functional RNP structure. Embo J 16, 1248 - 1257 (1997); published online Epub Mar 17 (10.1093 / emboj / 16.6.1248). 26. R. Coloma, J. M. Valpuesta, R. Arranz, J. L. Carrascosa, J. Ortin, J. Martin - Benito, The structure of a biologically active influenza virus ribonucleoprotein complex. PLoS Pathog 5, e1000491 (2009); published online Epub Jun (10.1371 / journal.ppat.1000491). 27. F. Baudin, C. Bach, S. Cusack, R. W. Ruigrok, Structure of influenza virus RNP. I. Influenza virus nucleoprotein melts secondary structure in panhandle RNA and exposes the bases to the solvent. Embo J 13, 3158 - 3165 (1994); published online Epub Jul 1 ( 28.T. Coelho, D. Adams, A. Silva, P. Lozeron, P.N. Hawkins, T. Mant, J. Perez, J. Chiesa, S. Warrington, E. Tranter, M. Munisamy, R. Falzone, J. Harrop, J. Cehelsky, B.R. Bettencourt, M. Geissler, J.S. Butler, A. Sehgal, R.E. Meyers, Q. Chen, T. Borland, R.M. Hutabarat, V.A. Clausen, R. Alvarez, K. Fitzgerald, C. Gamba-Vitalo, S.V. Nochur, A.K. Vaishnaw, D.W. Sah, J.A. Gollob, O.B. Suhr, Safety and efficacy of RNAi therapy for transthyretin amyloidosis. N Engl J Med 369, 819 - 829 (2013); published online Epub Aug 29 (10.1056 / NEJMoa1208760). 29. K. Fitzgerald, M. Frank-Kamenetsky, S. Shulga-Morskaya, A. Liebow, B. R. Bettencourt, J. E. Sutherland, R. M. Hutabarat, V. A. Clausen, V. Karsten, J. Cehelsky, S. V. Nochur, V. Kotelianski, J. Horton, T. Mant, J. Chiesa, J. Ritter, M. Munisamy, A. K. Vaishnaw, J. A. Gollob, A. Simon, Effect of an RNA interference drug on the synthesis of proprotein convertase subtilisin / kexin type 9 (PCSK9) and the concentration of serum LDL cholesterol in healthy volunteers: a randomised, single-blind, placebo-controlled, phase 1 trial. Lancet 383, 60 - 68 (2014); published online Epub Jan 4 (10.1016 / S0140-6736(13)61914-5). 30. J. Gottlieb, M. R. Zamora, T. Hodges, A. W. Musk, U. Sommerwerk, D. Dilling, S. Arcasoy, J. DeVincenzo, V. Karsten, S. Shah, B. R. Bettencourt, J. Cehelsky, S. Nochur, J. Gollob, A. Vaishnaw, A. R. Simon, A. R. Glanville, ALN-RSV01 for prevention of bronchiolitis obliterans syndrome after respiratory syncytial virus infection in lung transplant recipients. J Heart Lung Transplant 35, 213 - 221 (2016); published online Epub Feb (10.1016 / j.healun.2015.08.012). 31. E. Hoffmann, G. Neumann, Y. Kawaoka, G. Hobom, R. G. Webster, A DNA transfection system for generation of influenza A virus from eight plasmids. Proc Natl Acad Sci U S A 97, 6108 - 6113 (2000); published online Epub May 23 (10.1073 / pnas.100133697). 32. K. J. Szretter, A. L. Balish, J. M. Katz, Influenza: propagation, quantification, and storage. Current protocols in microbiology Chapter 15, Unit 15G 11 (2006); published online Epub Dec (10.1002 / 0471729256.mc15g01s3). 33. G. A. Marsh, R. Hatami, P. Palese, Specific residues of the influenza A virus hemagglutinin viral RNA are important for efficient packaging into budding virions. J Virol 81, 9727 - 9736 (2007); published online Epub Sep (10.1128 / jvi.01144 - 07). 34. S. A. Mortimer, K. M. Weeks, Time - resolved RNA SHAPE chemistry: quantitative RNA structure analysis in one - second snapshots and at single - nucleotide resolution. Nature protocols 4, 1413 - 1421 (2009) 10.1038 / nprot.2009.126). 35. A. Akbari, G. Marthinsen, J. T. Lifjeld, F. Albregtsen, L. Wennerberg, N. C. Stenseth, K. S. Jakobsen, Improved DNA fragment length estimation in capillary electrophoresis. Electrophoresis 29, 1273 - 1285 (2008); published online Epub Mar (10.1002 / elps.200700523). 36. P. S. Pang, E. A. Pham, M. Elazar, S. G. Patel, M. R. Eckart, J. S. Glenn, Structural map of a microRNA-122:hepatitis C virus complex. J Virol 86, 1250 - 1254 (2012); published online Epub Jan (10.1128 / JVI.06367-11). 37. K. E. Deigan, T. W. Li, D. H. Mathews, K. M. Weeks, Accurate SHAPE-directed RNA structure determination. Proc Natl Acad Sci U S A 106, 97 - 102 (2009); published online Epub Jan 6 (10.1073 / pnas.0806929106). 38. P. De Rijk, J. Wuyts, R. De Wachter, RnaViz 2: an improved representation of RNA secondary structure. Bioinformatics 19, 299 - 300 (2003); published online Epub Jan 22. 39. W. Kladwang, C. C. Van Lang, P. Cordero, R. Das, A two-dimensional mutate-and-map strategy for non-coding RNA structure. Nature chemistry 3, 954-962 (2011); published online Epub Dec (10.1038 / nchem.1176). 40. W. Kladwang, P. Cordero, R. Das, A mutate-and-map strategy accurately infers the base pairs of a 35-nucleotide model RNA. Rna 17, 522-534 (2011); published online Epub Mar (10.1261 / rna.2516311). 41. P. Cordero, W. Kladwang, C. C. Van Lang, R. Das, in RNA Folding (Methods in Molecular Biology), C. Waldsich, Ed. (2013), pp. in press. 42. S. A. Mortimer, K. M. Weeks, A fast-acting reagent for accurate analysis of RNA secondary and tertiary structure by SHAPE chemistry. J Am Chem Soc 129, 4144-4145 (2007); published online Epub Apr 11 (10.1021 / ja0704028). 43. S. Yoon, J. Kim, J. Hum, H. Kim, S. Park, W. Kladwang, R. Das, HiTRACE: high-throughput robust analysis for capillary electrophoresis. Bioinformatics 27, 1798-1805 (2011); published online Epub Jul 1 (10.1093 / bioinformatics / btr277). 44. H. Kim, P. Cordero, R. Das, S. Yoon, HiTRACE-Web: an online tool for robust analysis of high-throughput capillary electrophoresis. Nucleic Acids Research 41, W492-W498 (2013); published online Epub July 1, 2013 (10.1093 / nar / gkt501). 45. J. Kim, S. Yu, B. Shim, H. Kim, H. Min, E.-Y. Chung, R. Das, S. Yoon, A robust peak detection method for RNA structure inference by high-throughput contact mapping. Bioinformatics 25, 1137-1144 (2009); published online Epub May 1, 2009 (10.1093 / bioinformatics / btp110). 46. W. Kladwang, T. H. Mann, A. Becka, S. Tian, H. Kim, S. Yoon, R. Das, Standardization of RNA chemical mapping experiments. Biochemistry 53, 3063-3065 (2014); published online Epub May 20 (10.1021 / bi5003426). 47. D. H. Mathews, M. D. Disney, J. L. Childs, S. J. Schroeder, M. Zuker, D. H. Turner, Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc Natl Acad Sci U S A 101, 7287-7292 (2004); published online Epub May 11 (10.1073 / pnas.0401799101). 48. K. Darty, A. Denise, Y. Ponty, VARNA: Interactive drawing and editing of the RNA secondary structure. Bioinformatics 25, 1974 - 1975 (2009); published online Epub Aug 1 (10.1093 / bioinformatics / btp250). 49. P. Cordero, J. B. Lucks, R. Das, An RNA Mapping DataBase for curating RNA structure mapping experiments. Bioinformatics 28, 3006 - 3008 (2012); published online Epub Nov 15 (10.1093 / bioinformatics / bts554).

[0137] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that changes and modifications may be made thereto without departing from the spirit or scope of the appended claims in light of the teachings of the present invention.

[0138] Accordingly, the foregoing is merely illustrative of the principles of the present invention. Those skilled in the art can devise various arrangements, which are not explicitly described or illustrated herein, but which will embody the principles of the present invention and will be apparent to be within its spirit and scope. Further, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the present invention and the concepts contributed by the inventors to advance the art, and are to be construed as not being limited to such specifically recited examples and conditions. Also, all descriptions in this specification listing the principles, aspects, and embodiments of the present invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, such equivalents are intended to include both currently known equivalents and equivalents developed in the future, i.e., any elements developed to perform the same function regardless of structure. The scope of the present invention is, therefore, not intended to be limited to the embodiments illustrated and described herein. Rather, the scope and spirit of the present invention are embodied by the appended embodiments.

[0139] Notwithstanding the appended claims, the disclosure described herein is also illustrated by the following clauses. Clause 1. An oligonucleotide compound, or a salt thereof, comprising an oligonucleotide sequence complementary to PB2 vRNA, wherein the region comprises nucleotides 34 - 87 in the (-) sense notation of the 5'-terminal coding region of PB2 vRNA. Clause 2. The compound according to Clause 1, comprising an oligonucleotide sequence comprising at least 8 nucleoside subunits complementary to a region of PB2 vRNA. Clause 3. The compound according to any one of Clauses 1 - 2, wherein the oligonucleotide is complementary to a region of the packaging stem - loop 2 (PSL2) motif of the PB2 vRNA region. Clause 4. The compound according to any one of Clauses 1 - 3, wherein the oligonucleotide comprises internucleoside linkages selected from phosphorothioate, phosphorodithioate, phosphoramidate, and thiophosphoramidate linkages. ​Item 5. The compound according to any one of Items 1 to 4, wherein all of the internucleoside linkages of the oligonucleotide are selected from phosphorothioate, phosphorodithioate, phosphoramidate, thiophosphoramidate, and phosphodiester linkages. Item 6. The compound according to any one of Items 1 to 5, wherein the oligonucleotide contains locked nucleic acid (LNA) nucleotides. Item 7. The oligonucleotide is 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 45), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 46), 5’ TAGCATACTTA 3’ (SEQ ID NO: 47), 5’ CCAAAAGA 3’ (SEQ ID NO: 48), 5’ CATACTTA 3’ (SEQ ID NO: 49), 5’ CAGACACGACCAAAA 3’ (SEQ ID NO: 50), 5’ TACTTACTGACAGCC 3’ (SEQ ID NO: 51), 5’ AGACACGACCAAAAG 3’ (SEQ ID NO: 52), 5’ ACCAAAAGAAT 3’ (SEQ ID NO: 53), 5’ TGGCCATCAAT 3’ (SEQ ID NO: 54), 5’ TAGCATACTTA 3’ (SEQ ID NO: 55), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 56), 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 57), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 58), 5’ GATGGCCATCAATTA 3’ (SEQ ID NO: 59), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 60), 5’ TCTAGCATACTTACT 3’ (SEQ ID NO: 61), 5’ GAATTCGGATGGCCA 3’ (SEQ ID NO: 62), 5’ GGCCATCAATTAGTG 3’ (SEQ ID NO: 63), 5’ TTCGGATGGCCATCA 3’ (SEQ ID NO: 64), 5’ AGCCAGACAGCGA 3’ (SEQ ID NO: 65), and a compound according to any one of items 1 - 6, comprising a sequence selected from 5’ GACAGCCAGACAGCA 3’ (SEQ ID NO: 66). The compound according to item 7, wherein all nucleotides of the oligonucleotide are locked nucleic acid (LNA) nucleotides. Item 9. The oligonucleotide is LNA1: 5’ AccAaaAGaaT 3’ (SEQ ID NO: 67), LNA2: 5’ TggCcATcaaT 3’ (SEQ ID NO: 68), LNA3: 5’ TagCAtActtA 3’ (SEQ ID NO: 69), LNA4: 5’ CCAAAAGA 3’ (SEQ ID NO: 70), LNA5: 5’ CATACTTA 3’ (SEQ ID NO: 71), LNA6: 5’ CagaCaCGaCCaaAA 3’ (SEQ ID NO: 72), LNA7: 5’ TAcTtaCTgaCagCC 3’ (SEQ ID NO: 73), LNA8: 5’ AGACacgaccaAAAG 3’ (SEQ ID NO: 74), LNA9: 5’ TACTtactgacaGCC 3’ (SEQ ID NO: 75), LNA9.2: 5’ TACttactgacAGCC 3’ (SEQ ID NO: 76), LNA10: 5’ ACCaaaagAAT 3’ (SEQ ID NO: 77), LNA11: 5’ TGGccatcAAT 3’ (SEQ ID NO: 78), LNA12: 5’ TAGcatacTTA 3’ (SEQ ID NO: 79), LNA13: 5’ CgacCAaaAGaattC 3’ (SEQ ID NO: 80), LNA14: 5’ CGACcaaaagaATTC 3’ (SEQ ID NO: 81), LNA15: 5’ GaTGgCcATcaAttA 3’ (SEQ ID NO: 82), LNA16: 5’ GATGgccatcaATTA 3’ (SEQ ID NO: 83), LNA17: 5’ TcTAgCaTActTacT 3’ (SEQ ID NO: 84), LNA18: 5’ TCTAgcatactTACT 3’ (SEQ ID NO: 85), LNA19: 5’ GAAttcggatgGCCA 3’ (SEQ ID NO: 86), LNA20: 5’ GGCCatcaattaGTG 3’ (SEQ ID NO: 87), LNA21: 5’ TTCGgatggccaTCA 3’ (SEQ ID NO: 88), LNA22: 5’ AGCCagacagCGA 3’ (SEQ ID NO: 89), LNA23: 5’ GACAgccagacaGCA 3’ (SEQ ID NO: 90), LNA9.G74C: 5’ TACTtactgacaGTC 3’ (SEQ ID NO: 91), and LNA9.T80C: 5’ TACTtaccgacaGCC 3’ (SEQ ID NO: 92), and contains a sequence selected from The compound according to item 7, wherein the capital letters indicate LNA nucleotides and the lowercase letters indicate DNA nucleotides. Item 10. The compound according to any one of items 1 to 9, wherein the oligonucleotide contains at least 5 deoxyribonucleotide units and can recruit RNase. Item 11. The compound according to any one of items 1 to 10, wherein the binding of the compound to the PB2 vRNA region disrupts the overall secondary RNA structure of PB2 vRNA. Item 12. The compound according to any one of items 1 to 11, wherein the compound is an oligonucleotide complex having enhanced cellular uptake. Item 13. The compound according to item 12, wherein the compound is an oligonucleotide-lipid complex. Item 14. The compound according to any one of items 1 to 12, wherein the compound is an oligonucleotide complex containing a cell-specific protein. Item 15. A method for inhibiting influenza A virus in cells, the method comprising contacting a sample containing viral RNA (vRNA) having a PSL2 motif with an agent in an effective amount that specifically binds to the PSL2 motif and inhibits influenza A virus. Item 16. The method according to item 15, wherein the agent is an oligonucleotide compound comprising at least 8 nucleoside subunits complementary to the PSL2 motif of vRNA, or a salt thereof. Item 17. The method according to item 15 or 16, wherein the agent is the oligonucleotide compound according to any one of items 1 to 14. Item 18. The method according to any one of items 15 to 17, wherein the vRNA in the sample is PB2 vRNA. Item 19. The method according to any one of items 15 to 18, wherein contacting the sample with the agent results in a loss of at least 2 log 10 in the titer of the virus. Item 20. The method according to any one of items 15 to 19, wherein the agent disrupts the overall structure of the PSL2 motif of vRNA. Item 21. The method according to any one of items 15 to 19, wherein the vRNA is isolated from virions or cells. Item 22. The method according to any one of items 15 to 19, wherein the vRNA is contained in virions or infected cells. Item 23. The method according to any one of items 15 to 22, wherein the sample is in vitro. Item 24. The method according to any one of items 15 to 19 or 22, wherein the sample is in vivo. Item 25. A method for 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 agent in an effective amount that specifically binds to the PSL2 motif of viral RNA (vRNA). Item 26. The method according to item 25, wherein the vRNA is PB2 vRNA. Item 27. The method according to any one of claims 25 to 26, wherein the agent is a compound comprising an oligonucleotide sequence comprising at least 8 nucleoside subunits complementary to a region of PB2 vRNA. Item 28. The method according to any one of Items 25 to 27, wherein the agent is the oligonucleotide compound according to any one of Items 1 to 14. Item 29. The method according to any one of Items 25 to 28, wherein the subject is at risk of influenza A virus infection, and administration of the oligonucleotide compound protects the subject from infection for at least one week or more (for example, two weeks or more, three weeks or more, one month or more, two months or more, three months or more, etc.). Item 30. The method according to Item 29, wherein the administration includes administration once a week, once every two weeks, or once a month of an effective dose of the oligonucleotide compound. Item 31. The administration results in a reduction in the titer of the virus in the sample of the subject by at least 2 log 10 The method according to any one of Items 25 to 30. Item 32. The method according to any one of Items 25 to 30, wherein the agent is an oligonucleotide complex having enhanced cellular uptake. Item 33. The method according to any one of Items 25 to 31, wherein the agent is an oligonucleotide complex containing a cell-specific protein. Item 34. The method according to any one of Items 25 to 31, wherein the pharmaceutical composition contains an enhancer of cellular uptake. Item 35. The method according to any one of Items 25 to 31, wherein the pharmaceutical composition further contains an additional agent selected from a second oligonucleotide agent and an antiviral agent. Item 36. The method according to any one of Items 25 to 31, wherein the agent is siRNA, shRNA, antisense RNA, or antisense DNA. Item 37. The method according to any one of Items 25 to 31, wherein the subject is at risk of influenza A virus infection and the method prevents infection. Item 38. The method according to any one of Items 25 to 31, wherein the subject is diagnosed as having or suspected of having influenza A virus infection and the method treats the infection. Item 39. A method for screening a candidate agent for the ability to inhibit influenza A virus in cells, comprising Contacting a sample containing viral RNA (vRNA) comprising a PSL2 motif with a candidate agent; Determining whether the candidate agent specifically binds to the PSL2 motif, and a method, wherein an agent that specifically binds to the PSL2 motif inhibits influenza A virus in a cell. Item 40. The method according to item 39, wherein the candidate agent is selected from small molecules, nucleic acids, and polypeptides. Item 41. The method according to item 40, wherein the determining step includes detecting a cell parameter, and a change in the parameter in the cell compared to that in a cell not contacted with the candidate agent indicates that the candidate agent specifically binds to the PSL2 motif. Item 42. The method according to any one of items 39 to 41, wherein an agent that specifically binds to the PSL2 motif treats a subject having influenza A virus infection.

Claims

[Claim 1] The invention as depicted in the drawings.

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