JP2023514576A - Pan-genetic agents against respiratory viruses and methods of using them - Google Patents

Abstract

Methods of inhibiting respiratory viruses (ie, viruses associated with respiratory disease, eg, influenza A, influenza B, RSV, etc.) in a sample are provided. Aspects of the method include contacting a sample containing viral RNA (vRNA) having a target motif with an effective amount of an agent that specifically binds to the target motif and inhibits respiratory viruses. Also provided are methods of treating or preventing a respiratory viral infection in a subject. Also provided are compounds and pharmaceutical compositions comprising oligonucleotide sequences complementary to the target vRNA regions used in the subject methods. [Selection drawing] Fig. 1A

Description

CROSS-REFERENCE This application is a continuation-in-part of U.S. patent application Ser. Continuation-in-Part of Application No. 16/081,818, which is a 371 National Stage Application of PCT/US2017/20241 filed March 1, 2017 and filed March 2, 2016 No. 62/302,548 filed, all of which are hereby incorporated by reference in their entireties.

This application also claims the benefit of US Provisional Patent Application No. 62/992,659, filed March 20, 2020, which is hereby incorporated by reference in its entirety.

INTRODUCTION 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, are subtype restricted, and suffer from increased antiviral resistance to all drug class members.

The IAV genome consists of eight single-stranded negative-sense viral RNA (vRNA) segments that encode a minimum of 14 known viral proteins. vRNA forms the complete viral ribonucleoprotein (vRNP) together with the nucleoprotein (NP) and a heterotrimeric polymerase complex consisting of the PB2, PB1, and PA proteins. To be fully infected, IAV virions must incorporate at least one of the vRNPs of each segment. Each vRNP interacts with at least one other partner in a supramolecular complex likely maintained by intersegmental RNA-RNA and/or protein-RNA interactions postulated to induce the packaging process. to form

The individual is infected with influenza A virus alone and/or other respiratory viruses including, but not limited to, influenza B virus (IBV), coronavirus (Cov), respiratory syncytial virus (RSV), etc. can do. IBV is a segmented RNA virus whose genome consists of eight segments of linear negative-sense single-stranded RNA.

Respiratory syncytial virus (RSV), also called human respiratory syncytial virus (hRSV), is a negative-sense, single-stranded RNA virus. Patients infected with RSV are at increased risk of respiratory failure. It is a very common human respiratory viral pathogen.

Coronaviruses (CoVs) are enveloped RNA viruses that typically cause self-limiting respiratory tract infections in humans. However, in the last two decades, the 2003 Severe Respiratory Syndrome (SARS) outbreak, the 2012 Middle East Respiratory Syndrome (MERS), and most recently the 2019 CoV outbreak originating from Wuhan, China (SARS) There have been life-threatening conditions caused by new CoV strains such as those found in CoV-2). New treatments are desperately needed to contain current and future outbreaks.

Aspects of the present disclosure provide pan-genotypic compositions designed to disrupt RNA structural elements of respiratory disease-associated viruses, such as IAV, IBV, RSV, coronaviruses. Aspects of the present disclosure provide pan-genotypic compositions designed to disrupt an RNA structural element of IAV called packaging stem loop 2 (PSL2) within the 5′ packaging signal region of genome segment PB2. . Disruption of PSL2 structure dramatically inhibits IAV. PSL2 is conserved across all influenza A subtypes tested.

A method of inhibiting influenza A virus in a sample is provided. Aspects of the method include contacting a sample containing viral RNA (vRNA) with 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, vRNA is isolated from virions or cells. In some cases, the vRNA is in virions. In some cases, the vRNA is in infected cells. Also provided are methods of treating or preventing influenza A virus infection in a subject. Also provided is a method 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. including deciding whether Compounds and pharmaceutical compositions comprising oligonucleotide sequences complementary to the PB2 vRNA (or its complementary strand) regions used in the subject methods are also provided.

A method of inhibiting influenza B virus in a sample is provided. Aspects of the method include contacting a sample containing viral RNA (vRNA) having a motif with an effective amount of an agent that specifically binds to the RNA motif to inhibit influenza B virus. In some cases, vRNA is isolated from virions or cells. In some cases, the vRNA is in virions. In some cases, the vRNA is in infected cells. Also provided are methods of treating or preventing influenza B virus infection in a subject. Also provided is a method for screening a candidate agent for the ability to inhibit influenza B virus in a cell, the method comprising contacting a sample with a candidate agent and determining whether the candidate agent specifically binds to the RNA motif of the vRNA. including deciding whether Also provided are compounds and pharmaceutical compositions comprising oligonucleotide sequences complementary to the IBV vRNA regions used in the subject methods.

Those skilled in the art will appreciate that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

1A-1F depict wild-type PB2 (SEQ ID NO: 1) and packaging mutant vRNAs, PB2 m757 (SEQ ID NO: 2), m745 (SEQ ID NO: 3), 1918 Pandemic (H1N1) (SEQ ID NO: 4), highly pathogenic Avian (H5M=N1) (SEQ ID NO: 5) and 2009 pig (H1N1) (SEQ ID NO: 6) RNA secondary structures are shown. 1A-1F depict wild-type PB2 (SEQ ID NO: 1) and packaging mutant vRNAs, PB2 m757 (SEQ ID NO: 2), m745 (SEQ ID NO: 3), 1918 Pandemic (H1N1) (SEQ ID NO: 4), highly pathogenic Avian (H5M=N1) (SEQ ID NO: 5) and 2009 pig (H1N1) (SEQ ID NO: 6) RNA secondary structures are shown. 1A-1F depict wild-type PB2 (SEQ ID NO: 1) and packaging mutant vRNAs, PB2 m757 (SEQ ID NO: 2), m745 (SEQ ID NO: 3), 1918 Pandemic (H1N1) (SEQ ID NO: 4), highly pathogenic Avian (H5M=N1) (SEQ ID NO: 5) and 2009 pig (H1N1) (SEQ ID NO: 6) RNA secondary structures are shown. 1A-1F depict wild-type PB2 (SEQ ID NO: 1) and packaging mutant vRNAs, PB2 m757 (SEQ ID NO: 2), m745 (SEQ ID NO: 3), 1918 Pandemic (H1N1) (SEQ ID NO: 4), highly pathogenic Avian (H5M=N1) (SEQ ID NO: 5) and 2009 pig (H1N1) (SEQ ID NO: 6) RNA secondary structures are shown. Predicted RNA secondary structures conserved across coronavirus B viruses are shown. Panels A and B show the reactivity of full-length PB2 vRNA. Figures 3A-3E show wild-type reactivity (SEQ ID NO: 7), PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11) with packaging-deficient mutations. indicate collapse. Figures 3A-3E show wild-type reactivity (SEQ ID NO: 7), PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11) with packaging-deficient mutations. indicate collapse. Figures 3A-3E show wild-type reactivity (SEQ ID NO: 7), PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11) with packaging-deficient mutations. indicate collapse. Figures 3A-3E show wild-type reactivity (SEQ ID NO: 7), PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11) with packaging-deficient mutations. indicate collapse. Figures 3A-3E show wild-type reactivity (SEQ ID NO: 7), PB2m744b (SEQ ID NO: 8), PB2m745 (SEQ ID NO: 9), PB2m55c (SEQ ID NO: 10) and PB2m757 (SEQ ID NO: 11) with packaging-deficient mutations. indicate collapse. Shows the conservation of nucleotide sequences containing the PSL2 structure. Figures 5A-5D show two-dimensional mutation and map analysis of PSL2 RNA secondary structure (Figure 5C, SEQ ID NO: 12). Figures 5A-5D show two-dimensional mutation and map analysis of PSL2 RNA secondary structure (Figure 5C, SEQ ID NO: 12). Figures 5A-5D show two-dimensional mutation and map analysis of PSL2 RNA secondary structure (Figure 5C, SEQ ID NO: 12). Figures 5A-5D show two-dimensional mutation and map analysis of PSL2 RNA secondary structure (Figure 5C, SEQ ID NO: 12). Panels AB show the design of compensating mutations for the previously described PR8 PB2 mutant (Panel A, SEQ ID NO: 13). Panels AD show synonymous mutations of single highly conserved codons of PR8 PB2 vRNA (Panel A, SEQ ID NOs: 14-15; Panel B, SEQ ID NOs: 16-23, top to bottom). A table showing the nomenclature of the PB2 hull packaging mutants and the corresponding site of mutation is shown. Figures 9A-9C show the effect of synonymous mutations on the PSL2 structure, PB2m731 (SEQ ID NO:24), PB2m751 (SEQ ID NO:25), and PB2m748 (SEQ ID NO:26). Figures 9A-9C show the effect of synonymous mutations on the PSL2 structure, PB2m731 (SEQ ID NO:24), PB2m751 (SEQ ID NO:25), and PB2m748 (SEQ ID NO:26). Figures 9A-9C show the effect of synonymous mutations on the PSL2 structure, PB2m731 (SEQ ID NO:24), PB2m751 (SEQ ID NO:25), and PB2m748 (SEQ ID NO:26). Panels AI show the effect of compensating mutations in PR8 PB2 packaging-deficient mutants on viral packaging and titer. Panels AD show multidimensional chemical mapping of the PB2 packaging-deficient and compensating mutant partner (Panel B, SEQ ID NO:27). Panels a-t show two-dimensional mutational map rescue analysis. Design of primer sequences for two-dimensional mutation map rescue mutants. The sequences correspond from top to bottom to SEQ ID NOs:28-43. Panels AB show that packaging-deficient virus is attenuated in vivo. Panels AD show the antiviral activity of locked nucleic acids targeting the PSL2 RNA structure (Panel A, SEQ ID NO:44). Figures 16A-16B show analysis for LNA-RNA binding. Figures 16A-16B show analysis for LNA-RNA binding. Panels AB show the percent survival and percent weight loss of mice over time following intranasal administration of a single dose of exemplary compound LNA9. Panels AD, show susceptibility of influenza virus to oseltamivir after serial passage under drug pressure. Panels AC show susceptibility of influenza virus to exemplary compound LNA9, including virus after serial passage under drug pressure and drug-resistant virus. Antiviral effects of miRNA-directed LNAs designed to disrupt respiratory viral infections. Huh7 cells were pretreated with 25 nM miRNA-directed LNA 12-24 hours prior to infection with 0.3 MOI of fully replicating BSL3 SARS-CoV-2-nLuc reporter virus. The luciferase signal was read 48 hours after infection. Results are presented as log ₁₀ luciferase signal. Samples were run in duplicate, N=2 and controls in quadruplicate, N=4. Statistical analysis was performed by GraphPad Prism software and calculated using conventional one-way ANOVA with Dunnett's multiple comparison test between samples and scrambled LNA (Scr.LNA) control means. As a positive control, the positive control nucleoside analog EIDD-2801 (EIDD) was included. Huh7 cells were pretreated with 25 nM LNA combinations (12.5 nM each LNA = 25 nM total) 12-24 hours prior to infection with 0.3 MOI of fully replicating BSL3 SARS-CoV-2-nLuc reporter virus. bottom. The luciferase signal was read 48 hours after infection. Results are presented as log10 luciferase signal. Samples were run in duplicate, N=2 and controls in quadruplicate, N=4. Statistical analysis was performed by GraphPad Prism software and calculated using conventional one-way ANOVA with Dunnett's multiple comparison test between samples and DMSO control means. As a positive control, the positive control nucleoside analog EIDD-2801 (EIDD) was included. In vivo efficacy of LNA combinations against respiratory viruses. Human ACE2 transgenic mice were treated with a single intranasal administration of a combination of vehicle, small molecule A, or LNA 5 days prior to infection with a lethal inoculation of SARS-Cov-2. After infection, animals are monitored daily by clinical score, 1=asymptomatic, higher scores indicate worsening clinical status. ACE2-A549 cells were treated with 50 nM, 25 nM, or 5 nM CoV-2 plus strand targeted LNA 12-24 hours prior to infection with 0.3 MOI of fully replicating BSL3 SARS-CoV-2-nLuc reporter virus. , CoV-2 minus strand targeted LNAs, or miRNA-directed LNAs. The luciferase signal was read 48 hours after infection. Results are presented as log10 luciferase signal. Samples were run in 6 replicates, N=6. Statistical analysis was performed by GraphPad Prism software and calculated using conventional one-way ANOVA with Dunnett's multiple comparison test between samples and scrambled LNA (Scr.LNA) control means. As a positive control, the positive control nucleoside analog EIDD-2801 (EIDD) was included. (ab) Effect of intranasal LNA prophylaxis on survival of virus-infected mice. Kaplan-Meier survival plot. Mice (n=7 mice/group) were intranasally administered a single dose of LNA9, scrambled LNA, or vehicle (mock treatment), followed by lethal inoculation with wild-type PR8 virus, (a a) dosed with 20 μg LNA 3 days prior to infection (day −3) or 1 day prior to infection (day −1); and (b) treated with a single dose of 30 μg LNA9 or vehicle control 1 week prior. rice field. (c) LNA9 and newly designed LNA14 target sites mapped to the PSL2 structure. (d) Electrophoretic profiles of SHAPE analysis performed on untreated, scrambled LNA, LNA9, and LNA14 at a concentration of 100 nM in the presence of PR8 PB2 vRNA. Labeling with 1M7 SHAPE reagent is shown. (e) Kaplan-Meier survival plot of mice (n=7 mice/group) pretreated intranasally with a single dose of 30 μg LNA14 or vehicle control one week prior to lethal PR8 infection (day −7). . (fh) A single dose of 40 μg LNA14 or vehicle was administered I.V. two weeks prior to PR8 virus infection (day -14). N. dosed. (f) Kaplan-Meier survival plot. (g) Percent body weight of mice relative to day 0. (h) clinical score; (il) Mice (n=7) received a single intranasal dose of 40 μg LNA14 one week prior to 1 LD100 primary lethal PR8 virus infection (e). Sixty-five days after the first infection, surviving mice from (e) were challenged a second time with 10 LD100 along with age-matched naive controls (n=7/group). (i) Load study timeline. (j) Percent body weight of mice relative to day 0. (k) clinical score; (l) Kaplan-Meier survival curve. (m) Mice (n=10/group) were infected with a lethal dose of PR8 wild-type virus. Three days after infection, mice received a single dose of 40 μg of LNA14, LNA9, scrambled LNA, or vehicle control by intravenous injection. Kaplan-Meier survival plot.

Definitions Before describing the illustrative embodiments in greater detail, the following definitions are provided to illustrate and define the meaning and scope of terms used in the description.

Unless defined otherwise, 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.J. Y. (1991) provides those skilled in the art with a general meaning for many of the terms used herein. Nevertheless, certain terms are defined below for clarity and ease of reference.

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Please note. For example, the term "primer" refers to one or more primers, ie, single primers and multiple primers. It is further noted that the claims may be drafted to exclude any optional element. Therefore, this description should not be used for exclusive terms such as "solely" and "only" or for the use of "negative" limitations in connection with the recitation of claim elements. It is intended to function as an antecedent.

As used herein, the term "effective amount" refers to that amount of a substance (eg, drug of interest) that produces some desired local or systemic effect. An effective amount of a desired active agent will vary depending on a variety of factors, including, but not limited to, the weight and age of the subject, the condition being treated, the severity of the condition, the mode of administration, etc., and is readily determined, e.g. , can be determined empirically using data such as that provided in the experimental section below.

The term "sample" as used herein relates to a material or mixture of materials containing one or more constituents of interest, typically, but not necessarily, in fluid, i.e., aqueous form. Samples can be derived from a variety of sources, such as from a solid or fluid such as a biological sample or tissue isolated from an individual, e.g., plasma, serum, spinal fluid, semen, lymph, external to the skin. Sections, respiratory, intestinal, and urinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also in vitro cell culture components (conditioned media resulting from growth of cells in cell culture media, virus-infected (including, but not limited to, putative cells, recombinant cells, and cell components). A component in a sample is referred to herein as an "analyte." In many embodiments, the sample comprises at least about ¹⁰² , ^5x102 , ¹⁰³ , 5x103, ¹⁰⁴ , ^5x104 , ¹⁰⁵ , ^5x105 , ¹⁰⁶ , ^5x106 , ¹⁰⁷ , ^{5x107 , 108} , A composite sample containing 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² or more species of analyte.

Antibody fragments comprise portions of intact antibodies, eg, the antigen-binding or variable regions of intact antibodies. 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, nanobodies, and multispecific and multifunctional antibodies formed from antibody fragments. Papain digestion of an antibody produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc," a name that reflects its ability to crystallize readily. produce fragments. Pepsin treatment yields an F(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

The terms "polypeptide" and "protein", used interchangeably herein, refer to polymeric forms of amino acids of any length, including encoded and non-encoded amino acids, chemical or biochemical Amino acids modified or derivatized to , as well as polypeptides with modified peptide backbones. The term "fusion protein" or its grammatical equivalents means a protein composed of multiple polypeptide components, which are typically unjoined in their native state, but typically joined at their respective amino and carboxyl termini via bonds, eg, peptide bonds, to 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 fusion proteins with heterologous amino acid sequences, fusions with heterologous and homologous leader sequences with or without an N-terminal methionine residue, immunologically tagged proteins, detectable For example, fusion proteins with fluorescent proteins, β-galactosidase, luciferase, and the like as fusion partners, including but not limited to fusion proteins. Additionally, the term "protein" may further include post-translational modifications including, but not limited to glycosylation, phosphorylation, methylation, and acetylation.

Generally, a polypeptide can be of any length, e.g., greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 300 amino acids, usually up to about It can be of 500 or 1000 or more amino acids. A "peptide" is generally greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, usually up to about 50 amino acids. In some embodiments, peptides are 5-30 amino acids long.

The term "specific binding" refers to the ability of an agent to preferentially bind to a particular target (eg, PSL2) present in a homogeneous mixture of various analytes. In some cases, specific binding interactions typically discriminate between desirable and undesirable analytes in a sample by about 10-100 fold or more (eg, about 1000 fold or more). Specific binding can involve hybridization, polypeptide-nucleic acid interactions, or small molecule-nucleic acid interactions.

"Oligonucleotide" refers to ribose and/or deoxyribose nucleoside subunit polymers having from about 2 to about 200 contiguous subunits. The nucleoside subunits are phosphodiesters, phosphotriesters, alkyl phosphonates such as methyl phosphonates, P3'→N5' phosphoramidates, N3'→P5' phosphoramidates, N3'→P5' thiophosphoramidates , phosphorodiamidite, and phosphorothioate linkages. In certain cases, the intersubunit bonds have chiral atoms. Representative chiral intersubunit linkages include, but are not limited to, alkylphosphonates, phosphorodiamidites, and phosphorothioates. Furthermore, an "oligonucleotide" may have modifications known to those of skill in the art, such as sugars (e.g., 2' substitutions), bases (see definition of "nucleoside" below), and/or 3' and 5' ends. including chemical and biochemical modifications to, etc. In embodiments where the oligonucleotide portion includes multiple intersubunit linkages, each linkage may be formed using the same chemistry, or a mixture of linkage chemistries. In embodiments where the oligonucleotide portion comprises multiple intersubunit linkages, one or more of the linkages may be chiral. Bonds containing chiral atoms can be prepared as racemic mixtures or as separate enantiomers. The terms "oligonucleotide", "nucleic acid", "nucleic acid molecule", "nucleic acid fragment", "nucleic acid sequence or segment", or "polynucleotide" are used interchangeably and may also be used interchangeably to encode genes, cDNAs, , DNA, and RNA may be used interchangeably.

A "bicyclic nucleic acid" or "bridged nucleic acid" (BNA) is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, thereby forming a bicyclic ring system. Point. BNA monomers can contain 5-, 6-, or 7-membered bridge structures with a fixed 3'-end structure. Crosslinked nucleic acids include, but are not limited to, locked nucleic acids (LNA), ethylene bridged nucleic acids (ENA) and constrained ethyl (cEt). "Bridge" refers to a chain of atoms or valence bonds that connect two bridge heads, where a "bridge head" is a ring system (e.g., ribose ring system) is any backbone atom. In some embodiments, the bridge in BNA has 7-12 ring members and 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Unless otherwise specified, BNA is optionally substituted with one or more substituents including, but not limited to, alkyl, substituted alkyl, alkoxy, substituted alkoxy, hydroxy, amino, and halogen.

A "locked nucleic acid" (LNA) is a modified RNA nucleotide in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, thereby forming a bicyclic ring system. The bridge often "locks" the ribose of the 3'-end structure found in A-type duplexes. Locked nucleic acids are also encompassed by the term "bicyclic nucleic acid" or "bridged nucleic acid" (BNA). LNA nucleotides can be mixed, if desired, with any convenient nucleotides or nucleotide analogs such as DNA or RNA residues in oligonucleotides. LNAs hybridize to DNA or RNA according to Watson-Crick base pairing rules. Such oligomers can be chemically synthesized. In general, the locked ribose conformation enhances base stacking and backbone pre-organization and increases the hybridization properties (melting temperature) of oligonucleotides. In some cases, the locked nucleic acid is an α-l-locked nucleic acid (α- l-LNA).

"Ethylene-bridged nucleic acids" (ENA) refer to LNA-modified RNA nucleotides in which the ribose moiety is modified with an extra bridge containing two carbon atoms between the 2' oxygen and the 4' carbon (e.g. Morita et al. al., Bioorganic Medicinal Chemistry, 2003, 11(10), 2211-2226). Ethylene-bridged nucleic acids are also encompassed by the term "bicyclic nucleic acid" or "bridged nucleic acid" (BNA).

"Constrained Ethyl (cEt)" refers to LNA-modified RNA nucleotides in which the ribose moiety is modified with an extra bridge connecting the 2' oxygen and the 4' carbon, the carbon atom of the bridge containing a methyl group. In some cases, cEt is (S) constrained ethyl. In other cases, cEt is (R) constrained ethyl (see, eg, Pallan et al., Chem. Commun. (Camb)., 2012, 48(66), 8195-8197). Constrained ethyl nucleic acids are also encompassed by the term "bicyclic nucleic acid" or "bridged nucleic acid" (BNA).

As used herein, the terms "2'-modified" or "2'-substituted" refer to sugars that contain a substituent other than H or OH at the 2'-position. 2′-modified nucleotides include alkyl, allyl, amino, azido, fluoro, thio, O-alkyl, such as O-methyl, O-allyl, OCF ₃ , O—(CH ₂ ) ₂ —O—CH ₃ (eg, 2′-O-methoxyethyl (MOE)), O—(CH ₂ ) ₂ SCH ₃ ,)—(CH ₂ ) ₂ —ONR ₂ , and O—CH ₂ C(O)—NR ₂ 'including moieties with substituents, where each R is independently selected from H, alkyl, and substituted alkyl.

The present disclosure includes isolated or substantially purified nucleic acid molecules and compositions containing those molecules. An "isolated" or "purified" DNA or RNA molecule, in the context of this disclosure, is a DNA or RNA molecule that exists away from its natural environment and is therefore not a product of nature. An isolated DNA or RNA molecule can exist in purified form or can exist in a non-native environment such as, for example, a transgenic host cell. For example, an "isolated" or "purified" nucleic acid molecule or biologically active portion thereof is substantially free of other cellular material or culture medium when produced by recombinant techniques, or When chemically synthesized, it is substantially free of chemical precursors or other chemicals. In one embodiment, an "isolated" nucleic acid includes sequences that naturally flank it (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. do not have. For example, in various embodiments, an isolated nucleic acid molecule is less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, 0.5 kb, or may contain 0.1 kb of nucleotide sequence. Fragments and variants of the disclosed nucleotide sequences are also encompassed by this disclosure. By "fragment" or "portion" is meant a full-length or less than full-length nucleotide sequence. The siRNA of this disclosure can be produced by any method known in the art, including in vitro transcription, recombinant, or synthetic means. In one example, siRNA can be produced in vitro by using a recombinant enzyme such as T7 RNA polymerase and a DNA oligonucleotide template.

A "small interfering" or "short interfering RNA" or siRNA is an RNA duplex of nucleotides that is targeted to a gene of interest. "RNA duplex" refers to the structure formed by complementary pairing between two regions of an RNA molecule. The siRNA is "targeted" to a gene, and the nucleotide sequence of the duplex portion of the siRNA is complementary to the nucleotide sequence of the targeted gene. In some embodiments, the siRNA duplex length is less than 30 nucleotides. In some embodiments, the duplex is or 10 nucleotides long. In some embodiments, the length of the duplex is 19-25 nucleotides long. The RNA duplex portion of the siRNA can be part of a hairpin structure. In addition to the duplex portion, the hairpin structure may contain a loop portion located between the two sequences forming the duplex. Loops can vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides long. Hairpin structures may also contain 3' or 5' overhang portions. In some embodiments, the overhangs are 3' or 5' overhangs of 0, 1, 2, 3, 4 or 5 nucleotides in length.

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

Other definitions of terms may appear throughout the specification.

DETAILED DESCRIPTION Before describing the various embodiments, it is to be understood that the teachings of this disclosure are not limited to particular embodiments described, as such may, of course, vary. Moreover, since the scope of the present teachings is limited only by the appended claims, the terminology used herein is for the purpose of describing particular embodiments only and is intended to be limiting. It should also be understood that it is not a thing.

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. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as would be appreciated by those skilled in the art.

Unless defined otherwise, 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.

Citation of any publication is for its disclosure prior to the filing date of the application and should be construed as an admission that the claims are not entitled to antedate such publication by virtue of prior invention. isn't it. Further, the dates of publication provided may be different from the actual publication dates which can be independently confirmed.

It will be apparent to those of ordinary skill in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein can be modified into various other embodiments without departing from the scope or spirit of the present teachings. It has separate components and features that can be easily separated from or combined with any of the features. Any described method can be performed in the order of events described or in any other order that is logically possible.

All patents and publications, including all sequences disclosed within such patents and publications, referenced herein are expressly incorporated by reference.

Methods for Modulating Host MicroRNAs Host microRNAs can regulate a wide variety of physiological processes. In some cases, host microRNAs may modulate the fitness of influenza A, B, SARS, SARS-Cov-2, coronaviruses including MERS, and other respiratory viruses. In some cases, the miRNA is microRNA191 (miR191). In some cases, the miRNAs are , miR-10396, miR-4749, miR-6787, miR-4706, miR-3675, miR-6810, miR-6812, miR-663a, miR-381, miR-744, miR-4508, miR-4730, miR -6777, miR-10396, miR-4749, miR-6787, miR-4706, miR-3675, miR-6810, miR-3675, miR-6812, miR-6796; Not limited. In some cases, miRNAs can bind directly to influenza A virus RNA in virions or infected cells. In some cases, miRNAs can bind directly to influenza B virus RNA in virions or infected cells. In some cases, miRNAs can directly bind to coronavirus viral RNAs, including SARS-Cov, SARS-Cov2, MERS, in virions or infected cells. In some cases, miRNAs may directly bind to respiratory syncytial virus (RSV) RNA in virions or infected cells. In some cases, miRNAs may modulate host cell processes that otherwise modulate levels of influenza A virus RNA in virions or infected cells. In some cases, miRNAs may modulate host cell processes that otherwise modulate levels of influenza A virus RNA in virions or infected cells. In some cases, miRNAs may modulate host cellular processes that otherwise modulate levels of influenza B virus RNA in virions or infected cells. In some cases, miRNAs may modulate host cellular processes that otherwise regulate levels of coronavirus viral RNA, including SARS-Cov, SARS-Cov2, MERS viral RNA, in virions or infected cells. In some cases, miRNAs may modulate host cellular processes that otherwise modulate levels of respiratory syncytial virus (RSV) RNA in virions or infected cells. In some cases, conserved RNA secondary structure involves the presence of miR191 binding sites. In some cases, the subject agents or compositions are designed to sequester miR191 in CoV-transfected cells.

Methods for Inhibiting Influenza A Viruses As summarized above, aspects of the present disclosure provide an RNA structural element of IAV called packaging stem loop 2 (PSL2) within the 5′ packaging signal region of genome segment PB2. including pan-genotypic compositions designed to disrupt By "pan-genotypic" is meant that the composition is effective across a variety of different types of IAV in which the PSL2 structural elements are conserved. In some cases, the subject compositions can be referred to as broad spectrum. As used herein, the term "broad spectrum" refers to 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. It refers to the antiviral activity of a single moiety that is active against viruses. The two or more different viruses can be selected from different virus subgroups (eg, influenza A group 1 or influenza A group 2) or within the same group (eg, groups H1, H2, H5, H6, H8 and H9). 1 influenza A virus, or two or more of H3, H4, H7 and H10 group 2 influenza A viruses).

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

Aspects of the present disclosure include methods for inhibiting influenza A virus (IAV) in a sample. In some embodiments, the method comprises contacting a sample comprising 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. include. In some cases the sample is in vitro. In certain cases, the sample is in vivo. The vRNA in the sample can be contained in virions. In some cases, vRNA is contained in cells, such as cells infected with viral particles.

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

In some embodiments, the vRNA in the sample (eg, cells) comprises PB2 vRNA. As used herein, "PB2 vRNA" refers to viral RNA (eg, IAV RNA) that contains a conserved RNA structural element called packaging stem loop 2 (PSL2). Agents can bind to specific sites in the PSL2 motif and disrupt the overall structure of the vRNA, thereby inhibiting the virus (see, eg, Figure 14). In some cases, the agent inhibits the packaging capacity of vRNA, thereby inhibiting the virus. For example, Figure 1 shows the RNA secondary structure of wild-type PB2 and packaging mutant vRNA.

In some embodiments, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 5 or more, of the virus is removed by contacting the sample (eg, cells) with the agent. A 1 log _{10 or greater titer loss of virus is produced, such as a 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 log 10 or} even greater titer loss.

In some embodiments, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more of the virus are removed by contacting the sample (eg, cells) with an agent. , resulting in a 2 log ₁₀ or greater titer loss of virus, such as a 9 or greater, 10 log ₁₀ or even greater titer loss. In some embodiments, the agent is an oligonucleotide compound (eg, as described herein) comprising a sequence complementary to the PSL2 motif of vRNA, or a salt thereof.

In some cases of the method, binding (eg, via hybridization) of an oligonucleotide compound (eg, one of the above sequences) to the PB2 vRNA region yields the overall secondary RNA structure of the PB2 vRNA. collapse. In some cases, binding of the oligonucleotide compound inhibits the packaging ability of PB2 vRNA, thereby inhibiting the virus. In some cases, the subject compounds target at least a portion of the region defined by nucleotides 34-87 in the (-)sense notation of the 5'end coding region of 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'end coding region of PB2 vRNA. In some cases of the method, the method further comprises recruiting RNase to PSL2 to degrade vRNA.

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

Any convenient protocol for administering an agent to a subject may be used. The particular protocol employed may vary, eg, depending on the site of administration and whether the agent is, eg, an oligonucleotide, antibody, protein, peptide, or small molecule. For in vivo protocols, any convenient administration protocol may be used. drug identity and binding affinity, desired response, mode of administration, e.g., topical or systemic, intraocular, periocular, retrobulbar, intramuscular, intravenous, intraperitoneal, subcutaneous, subconjunctival, intranasal, topical, eye drops, i. v. s. c. , i. p. , oral, etc., depending on the half-life, number of cells or size of the graft bed or tissue, different protocols can be used. In some cases, cells are administered intranasally. In some cases, the drug is administered as an aerosol. In certain cases, the drug is administered by a nebulizer. In certain cases, the drug is administered through the assistance of respiratory assistance devices including, but not limited to, non-invasive positive pressure ventilation or mechanical ventilation. In certain cases, the drug is administered intravenously. In certain other cases, the drug is administered subcutaneously. In certain cases, the drug is administered by intramuscular injection.

Pharmaceutical compositions containing the subject agents are also provided. Any convenient excipients, carriers and the like can be utilized in the compositions. Pharmaceutically acceptable carriers used in the compositions may include sterile non-aqueous 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), and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases may also be present. Pharmaceutical compositions can also be lyophilized for subsequent reconstitution and use. Compositions may include a carrier as described herein. Examples of carriers that may be used include, but are not limited to, alum, microparticles, liposomes, and nanoparticles. Any convenient additive can be included in the subject compositions to enhance delivery of the subject active agents. Additives of interest include cell uptake enhancers, carrier proteins, lipids, dendrimeric carriers, carbohydrates, and the like.

In some cases, the pharmaceutical composition further comprises one or more additional active agents. Active agents of interest include the additional oligonucleotide compounds of this disclosure, and any convenient antiviral compound or drug of interest, including, but not limited to, amantadine, rimantadine, zanamivir, oseltamivir, peramivir, and the like.

Methods for Inhibiting Influenza B Virus Aspects of the present disclosure include pan-genotypic compositions designed to disrupt the RNA structural elements of IBV, which may include regions of genome segment HA.

Aspects of the present disclosure include methods for inhibiting influenza B virus (IBV) in a sample. In some embodiments, the method comprises contacting a sample comprising viral RNA (vRNA) having an IBV RNA motif with an effective amount of an agent that specifically binds to the IBV HA motif to inhibit influenza B virus. Including. In some cases the sample is in vitro. In certain cases, the sample is in vivo. The vRNA in the sample can be contained in virions. In some cases, vRNA is contained in cells, such as cells infected with viral particles.

Aspects of the present disclosure include methods for inhibiting influenza B virus (IBV) in cells. In some embodiments, the method comprises contacting a cell containing viral RNA (vRNA) having an IBV RNA motif with an effective amount of an agent that specifically binds to the IBV RNA motif to inhibit influenza B virus. Including. In some cases the cell is in vitro. In certain cases, the cells are in vivo.

In some embodiments, 1.5 or more, 2 or more, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 5 or more, of the virus is removed by contacting the sample (eg, cells) with the agent. A 1 log _{10 or greater titer loss of virus is produced, such as a 6 or greater, 7 or greater, 8 or greater, 9 or greater, 10 log 10 or} even greater titer loss.

In some embodiments, 2.5 or more, 3 or more, 3.5 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more of the virus are removed by contacting the sample (eg, cells) with an agent. , resulting in a 2 log ₁₀ or greater titer loss of virus, such as a 9 or greater, 10 log ₁₀ or even greater titer loss. In some embodiments, the agent is an oligonucleotide compound (eg, as described herein) comprising a sequence complementary to the IBV RNA motif of vRNA, or a salt thereof.

In some cases of the method, binding (eg, via hybridization) of an oligonucleotide compound (eg, one of the sequences described above) to the IBV vRNA region determines the overall secondary RNA structure of the IBV vRNA. collapse. In some cases, binding of the oligonucleotide compound inhibits the packaging ability of HA vRNA, thereby inhibiting the virus. In some cases of the method, the method further comprises recruiting RNase to the IBV vRNA to degrade the vRNA.

Aspects of the present disclosure include methods of treating or preventing influenza B 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 binds to the IBV HA RNA motif of viral RNA. Thus, in some cases, the subject is a virally infected subject. In certain cases, the subject is a subject at risk or suspected of contracting a virus.

Any convenient protocol for administering an agent to a subject may be used. The particular protocol employed may vary, eg, depending on the site of administration and whether the agent is, eg, an oligonucleotide, antibody, protein, peptide, or small molecule. For in vivo protocols, any convenient administration protocol may be used. drug identity and binding affinity, desired response, mode of administration, e.g., topical or systemic, intraocular, periocular, retrobulbar, intramuscular, intravenous, intraperitoneal, subcutaneous, subconjunctival, intranasal, topical, eye drops, i. v. s. c. , i. p. , oral, etc., depending on the half-life, number of cells or size of the graft bed or tissue, different protocols can be used. In some cases, cells are administered intranasally. In some cases, the drug is administered as an aerosol. In certain cases, the drug is administered by a nebulizer. In certain cases, the drug is administered through the assistance of respiratory assistance devices including, but not limited to, non-invasive positive pressure ventilation or mechanical ventilation. In certain cases, the drug is administered intravenously. In certain other cases, the drug is administered subcutaneously. In certain cases, the drug is administered by intramuscular injection.

Pharmaceutical compositions containing the subject agents are also provided. Any convenient excipients, carriers and the like can be utilized in the compositions. Pharmaceutically acceptable carriers used in the compositions may include sterile non-aqueous 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), and the like. Preservatives and other additives such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases may also be present. Pharmaceutical compositions can also be lyophilized for subsequent reconstitution and use. Compositions may include a carrier as described herein. Examples of carriers that may be used include, but are not limited to, alum, microparticles, liposomes, and nanoparticles. Any convenient additive can be included in the subject compositions to enhance delivery of the subject active agents. Additives of interest include cell uptake enhancers, carrier proteins, lipids, dendrimeric carriers, carbohydrates, and the like.

In some cases, the pharmaceutical composition further comprises one or more additional active agents. Active agents of interest include the additional oligonucleotide compounds of this disclosure, and any convenient antiviral compound or drug of interest, including, but not limited to, amantadine, rimantadine, zanamivir, oseltamivir, peramivir, and the like.

Methods for Inhibiting Respiratory Syncytial Virus (RSV) As summarized above, aspects of the present disclosure include agents and compositions designed to disrupt the RNA secondary structure of RSV. Disruption of the RSV RNA secondary structure can inhibit the RSV virus.

Methods for Inhibiting Coronavirus (CoV) As summarized above, aspects of the present disclosure include agents and compositions designed to disrupt the RNA secondary structure of coronavirus (CoV). Disruption of CoV RNA secondary structure can inhibit CoV viruses. Agents and compositions are effective across a variety of different types of coronaviruses (CoV), and RNA secondary structure is important in the viral life cycle. In some cases, the subject compositions can be referred to as broad spectrum. As used herein, the term "broad spectrum" refers to 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. It refers to the antiviral activity of a single moiety that is active against viruses. The two or more different viruses belong to different viral subgroups (e.g. human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), SARS-CoV (the etiological agent of severe acute respiratory syndrome (SARS)) , human coronavirus NL63 (HCoV-NL63, New Haven coronavirus), human coronavirus HKU1, MERS-CoV (“Middle East respiratory syndrome coronavirus” or MERS), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV -2) or COVID-19 (coronavirus disease 2019).

Disruption of CoV RNA secondary structure can dramatically inhibit CoV. FIG. 1G shows examples of predicted RNA secondary structures conserved across coronaviruses that can be targeted in the subject method. In some cases, the subject compositions are one selected from HCoV-299E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, and SARS-CoV-2 or COVID-19 It has broad-spectrum activity against CoV, such as activity against CoV described above. In some cases, the target CoV is SARS-CoV-2 or COVID-19. In some cases, the target CoV is SARS-CoV. In some cases, the target CoV is MERS-CoV. In some cases, the subject compositions disrupt the conserved RNA secondary structure of the target CoV without inducing caspases or interferons.

Aspects of the present disclosure include methods for inhibiting coronavirus (CoV) in a sample. In some embodiments, the method comprises contacting a sample comprising viral RNA (vRNA) with a CoV conserved RNA secondary structure with an effective amount of an agent that specifically binds to the secondary structure, Including inhibiting CoV. In some embodiments, the method includes treating a sample comprising viral RNA (vRNA) with a CoV conserved RNA secondary structure with an amount effective to inhibit interaction between host microRNA (miRNA) and CoV. Including contact with drugs. In some cases, the miRNA is microRNA191 (miR191). In some cases, the miRNAs are -3675, miR-6810, miR-3675, miR-6812, miR-6796. In some cases, conserved RNA secondary structure includes the presence of microRNA binding sites. In some cases, the microRNA is miR-191. In some cases, the microRNA can be any of the microRNAs listed above that have predicted binding sites in viral RNA. In some cases, the subject agents or compositions are designed to sequester miR191 in CoV-transfected cells. In some cases, cells are transfected with a CoV 5' terminal RNA segment conjugated to a luciferase report. In some cases the CoV is SARS-CoV-2 or COVID-19.

In some cases the sample is in vitro. In certain cases, the sample is in vivo. The vRNA in the sample can be contained in virions. In some cases, vRNA is contained in cells, such as cells infected with viruses.

Aspects of the present disclosure include methods for inhibiting coronavirus (CoV) in cells. Aspects of the present disclosure include methods for inhibiting coronavirus (CoV) in humans. In some embodiments, the method comprises contacting a cell containing viral RNA (vRNA) with a CoV conserved RNA secondary structure with an effective amount of an agent that specifically binds to the secondary structure, Including inhibiting CoV. In some cases the cell is in vitro. In certain cases, the cells are in vivo. In some embodiments, agents (e.g., as described herein) bind to specific sites of conserved RNA secondary structure motifs, disrupting the overall structure of vRNA, thereby disrupting the viral structure. can inhibit In some cases, the agent inhibits the packaging capacity of vRNA, thereby inhibiting the virus.

Agents Any convenient agent can be utilized as the agent of interest (eg, PSL2) in the subject methods and compositions. Agents of interest include ligands of PSL-2, PSL2-binding antibodies, scaffolding protein-binding agents of PSL2, oligonucleotides, small molecules, and peptides, or fragments, variants, or derivatives thereof, or any of the foregoing. Combinations include, but are not limited to.

Antibodies that may be used as agents in connection with the present disclosure include monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab) ₂ antibody fragments, Fv antibody fragments (e.g., _VH or _VL ) , single chain Fv antibody fragments, and dsFv antibody fragments. Furthermore, antibody molecules can be fully human antibodies, humanized antibodies, or chimeric antibodies. Antibodies that may be used in connection with the present disclosure may comprise any antibody variable region, mature or unprocessed, joined to any immunoglobulin constant region. Minor variations in the amino acid sequence of an antibody or immunoglobulin molecule, provided that the amino acid sequence variation maintains 75% or more, such as 80% or more, 90% or more, 95% or more, or 99% or more of the sequence, are encompassed by the present disclosure. In particular, conservative amino acid substitutions are contemplated. Conservative substitutions are those that take place within a family of amino acids related to their side chains. Whether an amino acid change results in a functional peptide can be determined by assaying the specific activity of the polypeptide derivative. In some embodiments, the agent is an antibody fragment (eg, as described herein).

In some embodiments, the agent is a scaffold polypeptide binding agent. A scaffold refers to the underlying peptide framework (eg, consensus sequence or structural motif) from which a polypeptide agent arises. The underlying scaffolding sequence includes fixed residues as well as variant residues that can confer different functions to the polypeptide agents, such as specific binding to target receptors. Such structural motifs can be structurally characterized and compared as a combination of specific secondary and tertiary structural elements, or alternatively as equivalent primary sequences of amino acid residues. Any convenient scaffold and scaffold polypeptide can be utilized as an agent in the subject methods. In some embodiments, such agents may be identified using recombinant screening methods such as phage display screening. Scaffold polypeptide binding agents of interest include, but are not limited to, synthetic small proteins and recombinant small proteins such as Affibodies.

In some cases, the agent is a small molecule that binds to PSL2. Small molecules of interest include small organic or small inorganic compounds having a molecular weight (MW) greater than 50 Da and less than about 2,500 Da, such as greater than 50 Daltons (Da) and less than about 1000 Da, or greater than 50 Da and less than about 500 Da. including but not limited to: "Small molecule" encompasses numerous biological and chemical classes including synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules, and includes synthetic, recombinant, or naturally occurring polypeptides and nucleic acids. . Small molecules of interest may contain functional groups necessary for structural interactions with proteins, particularly hydrogen bonding, and may contain at least an amine, carbonyl, hydroxyl, or carboxyl group, and at least two of the functional chemical groups can include Small molecules can comprise cyclical carbon or heterocyclic structures, and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Small molecules are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Oligonucleotide Compounds In some embodiments, the agent is an oligonucleotide or derivative thereof, or salt thereof (eg, a pharmaceutically acceptable salt). In some cases, an oligonucleotide is complementary to a particular segment of a target motif (eg, as described herein). Complementary oligonucleotides used in the subject methods are in some cases at least 5, such 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 oligonucleotides are 75 nucleotides or less in length, such as 50 nucleotides or less, 45 nucleotides or less, 40 nucleotides or less, or 35 nucleotides or less in length, wherein the length determines the efficiency of inhibition, Defined by specificity, including the absence of cross-reactivity, and the like. In some cases, the complementary oligonucleotides are 30 nucleotides or less in length, such as 25 nucleotides or less, 20 nucleotides or less, or 15 nucleotides or less in length, and the length determines the efficiency of inhibition, absence of cross-reactivity, It is defined by a specificity including The disclosure provides short oligonucleotides, eg, 7, 8-15, or 15-16 nucleotides in length, which can be potent and selective inhibitors of target functions.

In some embodiments, the agent comprises 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) that are complementary to the target motif of the vRNA. , at least 14, at least 16, at least 18, or at least 20) or a salt thereof. In certain cases, the linkage of the oligonucleotide is a modified phosphate group, eg, one or more oxygens of the phosphate have been replaced with various substituents. Without being bound by any particular theory, such modifications can increase the resistance of oligonucleotides to nucleolytic degradation. Examples of modified phosphate groups include phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogenphosphonates, phosphoramidites, alkylphosphonates or arylphosphonates, and phosphotriesters, including Not limited. In some cases, the linkage of the oligonucleotide is a modified phosphate group and one or more of the non-bridging phosphonate oxygen atoms in the linkage are S, Se, BR ^a ₃ , alkyl, substituted alkyl, aryl, substituted substituted with a group selected from aryl, H, NR ^a ₂ or OR ^b , where R ^a is H, alkyl, substituted alkyl, aryl, substituted aryl and R ^b is H, alkyl, substituted alkyl , aryl, or substituted aryl. In certain cases, one or more bonds of the phosphorous atoms of the oligonucleotide are chiral, eg, asymmetric centers. Asymmetric phosphorus atoms can have either the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). In certain embodiments, binding of the oligonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or It contains one or more asymmetric phosphorus atoms with an enantiomeric excess of Sp configuration above it. In certain others, binding of the oligonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. It contains one or more asymmetric phosphorus atoms with an enantiomeric excess of Rp configurations greater than or equal to.

In certain embodiments, one or more of the linkages of the oligonucleotide are methylphosphonate, P3'→N5'phosphoramidate, N3'→P5'phosphoramidate, N3'→P5'thiophosphoramidate. date, phosphorodithioate and phosphorothioate linkages. In certain cases, one or more linkages of the oligonucleotide are phosphorothioate linkages. In certain cases, the phosphorus atoms in one or more of the phosphorothioate linkages are chiral. In some cases, the chiral phosphorus atom in one or more phosphorothioate linkages has the Rp configuration. In some cases, the chiral phosphorus atom in one or more phosphorothioate linkages has the Sp configuration. In certain embodiments, binding of the oligonucleotide is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or It contains one or more phosphorothioate linkages with an enantiomeric excess of Sp configuration above it. In certain other embodiments, oligonucleotide binding is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% , or one or more phosphorothioates with an enantiomeric excess of the Rp configuration.

In certain cases, the oligonucleotide sequences are bridging nucleic acids (eg, as described herein). In certain cases, the oligonucleotide sequences are 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 more, such as comprising one or more bridging nucleic acid nucleotides of

In certain cases, the oligonucleotide sequence is a locked nucleic acid. In certain cases, the oligonucleotide sequences are 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 more, such as containing one or more locked nucleic acid nucleotides of

In certain cases, the oligonucleotide sequence is an ethylene-bridged nucleic acid (ENA). In certain cases, the oligonucleotide sequences are 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 more, such as contains one or more ENA nucleotides of

In certain cases, the oligonucleotide sequence is a constrained ethyl (cEt) nucleic acid. In certain cases, the oligonucleotide sequences are 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 more, such as containing one or more cEt nucleotides of In certain cases, the oligonucleotide sequences are 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 more, such as containing one or more (S) constrained ethyl nucleic acids of In certain cases, the oligonucleotide sequences are 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 more, such as containing one or more (R)-constrained ethyl nucleic acids of In certain cases, the oligonucleotide sequence contains one or more ribose modifications. In some cases, an oligonucleotide sequence includes one or more 2'-modified ribose sugars (also referred to herein as 2'-modified nucleotides). In certain cases, the oligonucleotide sequences are 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 more, such as contains one or more 2' modified nucleotides of 2′-modified nucleotides include alkyl, allyl, amino, azido, fluoro, thio, O-alkyl, such as O-methyl, O-allyl, OCF ₃ , O—(CH ₂ ) ₂ —O—CH ₃ (eg, 2′-O-methoxyethyl (MOE)), O—(CH ₂ ) ₂ SCH ₃ ,)—(CH ₂ ) ₂ —ONR ₂ , and O—CH ₂ C(O)—NR ₂ Including but not limited to moieties with 'substituents, each R is independently selected from H, alkyl, and substituted alkyl. In certain cases, the oligonucleotide sequences are 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 more, such as contains one or more 2′-O-methoxyethyl (MOE) modifications of

In some embodiments, the agent comprises at least 5 deoxyribonucleotide units (e.g., units complementary to a target motif, e.g., a 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 is capable of recruiting RNase. In some cases, oligonucleotides recruit RNases to catalyze the degradation of target vRNA into smaller components. Any convenient methods and moieties for recruiting RNases can be incorporated into the subject agents (eg, oligonucleotides). In some cases, the oligonucleotide agent further comprises a sequence that recruits the RNase of interest. Unless otherwise indicated, oligonucleotide sequences presented herein include DNA sequences, RNA sequences (e.g., U may optionally be substituted for T), mixed RNA/DNA sequences, and analogs thereof. wherein one or more nucleotides of the sequence are BNA analogues, LNA analogues, ENA analogues, cEt analogues, 2′ modified analogues, and/or one or more internucleoside linkages are, for example, phosphorothioate , modified nucleotides such as analogs substituted with non-naturally occurring linkages such as phosphorodithioate, phosphoramidate or thiophosphoramidate linkages. In embodiments in which one or more of the bonds of the oligonucleotide comprise a chiral phosphorus atom, e.g., an asymmetric center, the chiral phosphorus atom is in the "R" configuration (referred to herein as Rp), or the "S configuration (referred to herein as Sp).

In some embodiments, the active agent is a compound comprising an oligonucleotide sequence comprising at least eight 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 no more than 20 (eg, no more than 15) nucleoside subunits complementary to a region of PB2 vRNA.

A specific region of the endogenous strand PSL2 sequence is selected to be complemented by the oligonucleotide agent. The selection of specific sequences for oligonucleotides may use empirical methods, and based on structural analysis (eg, as described herein), several candidate sequences are likely to target IAV in vitro or in animal models. are assayed for inhibition of A combination of oligonucleotides and sequences may also be used, with several regions of target PSL2 selected for antisense complementation.

In some embodiments, the oligonucleotide is
5'ACCAAAAGAAT3' (SEQ ID NO: 45),
5'TGGCCATCAAT3' (SEQ ID NO: 46),
5'TAGCATACTTA3' (SEQ ID NO: 47),
5'CCAAAGA3' (SEQ ID NO: 48),
5'CATACTTA3' (SEQ ID NO: 49),
5′CAGACACGACCAAAA3′ (SEQ ID NO: 50),
5'TACTTACTGACAGCC3' (SEQ ID NO: 51),
5′AGACACGACCAAAG3′ (SEQ ID NO: 52),
5'ACCAAAAGAAT3' (SEQ ID NO: 53),
5'TGGCCATCAAT3' (SEQ ID NO: 54),
5'TAGCATACTTA3' (SEQ ID NO: 55),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 56),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 57),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 58),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 59),
5'TCTAGCATACTTACT3' (SEQ ID NO: 60),
5'TCTAGCATACTTACT3' (SEQ ID NO: 61),
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′GGCCATCAATTAGTG3′ (SEQ ID NO: 63),
5′TTCGGATGGCCATCA3′ (SEQ ID NO: 64),
5'AGCCAGACAGCGA3' (SEQ ID NO:65) and 5'GACAGCCAGACAGCA3' (SEQ ID NO:66).

In certain embodiments, the oligonucleotide comprises the sequence: 5'ACCAAAAGAAT3' (SEQ ID NO:45). In certain embodiments, the oligonucleotide comprises the sequence: 5'TGGCCATCAAT3' (SEQ ID NO:46). In certain embodiments, the oligonucleotide comprises the sequence: 5'TAGCATACTTA3' (SEQ ID NO:47). In certain embodiments, the oligonucleotide comprises the sequence: 5'CCAAAAGA3' (SEQ ID NO:48). In certain embodiments, the oligonucleotide comprises the sequence: 5'CATACTTA3' (SEQ ID NO:49). In certain embodiments, the oligonucleotide comprises the sequence: 5'CAGACACGACCAAAA3' (SEQ ID NO:50). In certain embodiments, the oligonucleotide comprises the sequence: 5'TACTTACTGACAGCC3' (SEQ ID NO:51). In certain embodiments, the oligonucleotide comprises the sequence: 5'AGACACGACCAAAAG3' (SEQ ID NO:52). In certain embodiments, the oligonucleotide comprises the sequence: 5'ACCAAAAGAAT3' (SEQ ID NO:53). In certain embodiments, the oligonucleotide comprises the sequence: 5'TGGCCATCAAT3' (SEQ ID NO:54). In certain embodiments, the oligonucleotide comprises the sequence: 5'TAGCATACTTA3' (SEQ ID NO:55). In certain embodiments, the oligonucleotide comprises the sequence: 5'CGACCAAAAGAATTC3' (SEQ ID NO:56). In certain embodiments, the oligonucleotide comprises the sequence: 5'CGACCAAAAGAATTC3' (SEQ ID NO:57). In certain embodiments, the oligonucleotide comprises the sequence: 5'GATGGCCATCAATTA3' (SEQ ID NO:58). In certain embodiments, the oligonucleotide comprises the sequence: 5'GATGGCCATCAATTA3' (SEQ ID NO:59). In certain embodiments, the oligonucleotide comprises the sequence: 5'TCTAGCATACTTACT3' (SEQ ID NO:60). In certain embodiments, the oligonucleotide comprises the sequence: 5'TCTAGCATACTTACT3' (SEQ ID NO:61). In certain embodiments, the oligonucleotide comprises the sequence: 5'GAATTCGGATGGCCA3' (SEQ ID NO:62). In certain embodiments, the oligonucleotide comprises the sequence: 5'GGCCATCAATTAGTG3' (SEQ ID NO:63). In certain embodiments, the oligonucleotide comprises the sequence: 5'TTCGGATGGCCATCA3' (SEQ ID NO:64). In certain embodiments, the oligonucleotide comprises the sequence: 5'AGCCAGACAGCGA3' (SEQ ID NO:65). In certain embodiments, the oligonucleotide comprises the sequence: 5'GACAGCCAGACAGCA3' (SEQ ID NO: 66).

In some embodiments, the oligonucleotide is
5'CGACCAAAGAATT3' (SEQ ID NO:98) and 5'GACCAAAAGAATTCGG3' (SEQ ID NO:99).

In certain embodiments, the oligonucleotide comprises the sequence: 5'CGACCAAAAGAATT3' (SEQ ID NO:98). In certain embodiments, the oligonucleotide comprises the sequence: 5'GACCAAAAGAATTCGG3' (SEQ ID NO:99).

In some embodiments, the oligonucleotide is
5′AGCATACTTACTGACA3′ (SEQ ID NO: 100),
5'CATACTTACTGACA3' (SEQ ID NO: 101),
5'ATACTTACTGACAG3' (SEQ ID NO: 102),
5'CATACTTACTGACAGC3' (SEQ ID NO: 103),
5'AGACAGCGACCAAAAG3' (SEQ ID NO: 104), and 5'ACAGCGACCAAAAG (SEQ ID NO: 105).

In certain embodiments, the oligonucleotide comprises the sequence: 5'AGCATACTTACTGACA3' (SEQ ID NO: 100). In certain embodiments, the oligonucleotide comprises the sequence: 5'CATACTTACTGACA3' (SEQ ID NO: 101). In certain embodiments, the oligonucleotide comprises the sequence: 5'ATACTTACTGACAG3' (SEQ ID NO: 102). In certain embodiments, the oligonucleotide comprises the sequence 5'CATACTTACTGACAGC3' (SEQ ID NO: 103). In certain embodiments, the oligonucleotide comprises the sequence: 5'AGACAGCGACCAAAAG3' (SEQ ID NO: 104). In certain embodiments, the oligonucleotide comprises the sequence: 5'ACAGCGACCAAAAG (SEQ ID NO: 105).

In some embodiments, the oligonucleotide is
5′CAGCCAGACAGCGAC3′ (SEQ ID NO: 106),
5′CAGCCAGACAGCGA3′ (SEQ ID NO: 107),
5'ACAGCCAGACAGCG3' (SEQ ID NO: 108), and 5'GACAGCCAGACAGCG3' (SEQ ID NO: 109).

In certain embodiments, the oligonucleotide comprises the sequence: 5'CAGCCAGACAGCGAC3' (SEQ ID NO: 106). In certain embodiments, the oligonucleotide comprises the sequence: 5'CAGCCAGACAGCGA3' (SEQ ID NO: 107). In certain embodiments, the oligonucleotide comprises the sequence: 5'ACAGCCAGACAGCGA3' (SEQ ID NO: 108). In certain embodiments, the oligonucleotide comprises the sequence: 5'GACAGCCAGACAGCG3' (SEQ ID NO: 109).

In some embodiments, the oligonucleotide is
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′AGCCAGACAGCGA3′ (SEQ ID NO: 65),
5'CATCAATTAGTGTCG3' (SEQ ID NO: 110),
5′CCATCAATTAGTGTCG3′ (SEQ ID NO: 111),
5′GCCATCAATTAGTGTG3′ (SEQ ID NO: 112),
5′AAGAATTCGGATGGC3′ (SEQ ID NO: 113),
5'CAGACAGCGACCAA3' (SEQ ID NO:114), and 5'TGACAGCCAGACAGC3' (SEQ ID NO:115).

In certain embodiments, the oligonucleotide comprises the sequence: 5'GAATTCGGATGGCCA3' (SEQ ID NO:62). In certain embodiments, the oligonucleotide comprises the sequence: 5'AGCCAGACAGCGA3' (SEQ ID NO:65). In certain embodiments, the oligonucleotide comprises the sequence: 5'CATCAATTAGTGTCG3' (SEQ ID NO: 110). In certain embodiments, the oligonucleotide comprises the sequence: 5'CCATCAATTAGTGTCG3' (SEQ ID NO: 111). In certain embodiments, the oligonucleotide comprises the sequence: 5'GCCATCAATTAGTGTG3' (SEQ ID NO: 112). In certain embodiments, the oligonucleotide comprises the sequence: 5'AAGAATTCGGATGGC3' (SEQ ID NO: 113). In certain embodiments, the oligonucleotide comprises the sequence: 5'CAGACAGCGACCAA3' (SEQ ID NO: 114). In certain embodiments, the oligonucleotide comprises the sequence: 5'TGACAGCCAGACAGC3' (SEQ ID NO: 115). In some cases, binding (eg, via hybridization) of an oligonucleotide compound (eg, one of the above sequences) to the PB2 vRNA region disrupts the overall secondary RNA structure of the PB2 vRNA. In certain cases, binding of oligonucleotide compounds to regions of PB2 vRNA inhibits the packaging ability of PB2 vRNA, thereby inhibiting the virus.

In some embodiments, the oligonucleotide comprises a sequence selected from (COV2).
5′GGTGACATGGTACCACATATATCACGTC3′ (SEQ ID NO: 192)
5′GACGTGATATATGTGGTACCATGTCACC3′ (SEQ ID NO: 193)
5′GACGTGATATATGTGGG3′ (SEQ ID NO: 194)
5'CGTGATATAGTGTGGTA3' (SEQ ID NO: 195)
5'GATATGTGGTACCAT3' (SEQ ID NO: 196)
5'TGGTACCATGTCAC3' (SEQ ID NO: 197)
5' GTCACTAAGAAAATCTGCTGCTGAGG 3' (SEQ ID NO: 198)
5'CCTCAGCAGCAGATTTCTTAGTGAC3' (SEQ ID NO: 199)
5' CCTCAGCAGCAGATTT 3' (SEQ ID NO: 200)
5'TCAGCAGCAGATTTC3' (SEQ ID NO: 201)
5' CAGCAGCAGATTTC 3' (SEQ ID NO: 202)
5'CAGATTTCTTAGTGAC3' (SEQ ID NO: 203)
5'TGCTTACGGTTTCGTCCGTGTTGCA3' (SEQ ID NO: 204)
5'TGCAACACGGACGAAAACCGTAAGCA3' (SEQ ID NO: 205)
5'TGCAACACGGACGAAA3' (SEQ ID NO: 206)
5′GCAACACGGACGAAAC3′ (SEQ ID NO: 207)
5'ACACGGACGAAACCG3' (SEQ ID NO: 208)
5′GGACGAACCCGTAA3′ (SEQ ID NO: 209)
5′ACGAAACCGTAAGCA3′ (SEQ ID NO: 210)
5'ACGAAACCGTAAGCA3' (SEQ ID NO: 211)
5′GCAACACGGACGAAAC3′ (SEQ ID NO: 212)
5'GCAACACGGACGAAA3' (SEQ ID NO: 213)
5′GCAACACGGACGAAAC3′ (SEQ ID NO: 214)
5′GCAACACGGACGAAAC3′ (SEQ ID NO: 215)
5′GCAACACGGACGAAAC3′ (SEQ ID NO: 216)
5′GCAACACGGACGAA3′ (SEQ ID NO: 217)
5' ACACGGACGAAACCG 3' (SEQ ID NO: 218)
5′ACACGGACGAAACCG3′ (SEQ ID NO: 219)
5' AACACGGACGAAACCG 3' (SEQ ID NO: 220)
5' AACACGGACGAAACCG 3' (SEQ ID NO: 221)
5'ACGAAACCGTAAGCA3' (SEQ ID NO: 222)
5′ACGAAACCGTAAGCA3′ (SEQ ID NO: 223)
5'ATAGGTCCAGACATGTTCC3' (SEQ ID NO: 224)
5′GGAACATGTCTGGACCTAT3′ (SEQ ID NO: 225)
5′AACATGTCTGGACCTA3′ (SEQ ID NO: 226)
5'ACATGTCTGGACCTAT3' (SEQ ID NO: 227)
5'TATGACTATGTCATATTCA3' (SEQ ID NO: 228)
5'AGTGAATATGACATAGTCATATT3' (SEQ ID NO: 229)
5'TGAATATGACATAGT3' (SEQ ID NO: 230)
5'AATATGACATAGTC3' (SEQ ID NO: 231)
5′ GATCTCTTGTAGATCTGTTCTCTAAACGAACTTTAAATCTGTG 3′ (SEQ ID NO: 232)
5'CACAGATTTTAAAGTTCGTTTAGAGAACAGATCTACAAGAGATC3' (SEQ ID NO: 233)
5'CACAGATTTTAAAGTT3' (SEQ ID NO: 234)
5'CACAGATTTTAAAGTT3' (SEQ ID NO: 235)
5'CAGATTTTAAAGTTCG3' (SEQ ID NO: 236)
5'GATTTTAAAGTTCGT3' (SEQ ID NO: 237)
5'TAAAGTTCGTTTAGA3' (SEQ ID NO: 238)
5' AAAGTTCGTTTAGA 3' (SEQ ID NO: 239)
5'TCGTTTAGAGAACAGAT3' (SEQ ID NO: 240)
5'AGAGAACAGATCTACA3' (SEQ ID NO: 241)
5'AGATCTACAAGAGA3' (SEQ ID NO: 242)
5'ATCTACAAGAGATC3' (SEQ ID NO: 243)
5'AGATTTTAAAGTTCGT3' (SEQ ID NO: 244)
5'GATTTTAAAGTTCGT3' (SEQ ID NO: 245)
5'GATTTTAAAGTTCGT3' (SEQ ID NO: 246)
5'AGATTTTAAAGTTCG3' (SEQ ID NO: 247)
5'TCGTTTAGAGAACAGAT3' (SEQ ID NO: 248)
5' TTCGTTTAGAGAACAG 3' (SEQ ID NO: 249)
5' TTCGTTTAGAGAACAG 3' (SEQ ID NO: 250)
5' GAGAAAACACACGTCCAACTCAGTTTGCCTG 3' (SEQ ID NO: 251)
5′CAGGCAAAACTGAGTTGGACGTGTGTTTTCTC3′ (SEQ ID NO: 252)
5'CAGGCAAAACTGAGTTG3' (SEQ ID NO: 253)
5'CAGGCAAAACTGAGTTG3' (SEQ ID NO: 254)
5'CAGGCAAAACTGAGT3' (SEQ ID NO: 255)
5′CAAAACTGAGTTGGACG3′ (SEQ ID NO: 256)
5'AAAACTGAGTTGGAC3' (SEQ ID NO: 257)
5'AAAACTGAGTTGGACGT3' (SEQ ID NO: 258)
5′GAGTTGGACGTGTGT3′ (SEQ ID NO: 259)
5'TTGGACGTGTGTTTTC3' (SEQ ID NO: 260)
5′GGACGTGTGTTTTCTC3′ (SEQ ID NO: 261)
5′GGTTTCGTCCGGGTGTGACCG3′ (SEQ ID NO: 262)
5'TTTCGGTCACACCCGGACGAAACCTAGAT3' (SEQ ID NO: 263)
5'TTTCGGTCACACCCGG3' (SEQ ID NO: 264)
5'CGGTCACACCCGGACG3' (SEQ ID NO: 265)
5'TCACACCCGGACGAAA3' (SEQ ID NO: 266)
5'CACCCGGACGAAAC3' (SEQ ID NO: 267)
5'CACCCGGACGAAACC3' (SEQ ID NO: 268)
5'CACCCGGACGAAACCT3' (SEQ ID NO: 269)
5'CCGGACGAAACCTA3' (SEQ ID NO: 270)
5'CGGTCACACCCGGACGA3' (SEQ ID NO: 21)
5'CGGTCACACCCGGACG3' (SEQ ID NO: 272)
5'CGGTCACACCCGGACG3' (SEQ ID NO: 273)
5'CGGTCACACCCGGACG3' (SEQ ID NO: 274)
5' GTCACACCCGGACG 3' (SEQ ID NO: 275)
5' GTCACACCCGGACG 3' (SEQ ID NO: 276)
5'CACCCGGACGAAACC3' (SEQ ID NO: 277)
5'CACCCGGACGAAACC3' (SEQ ID NO: 278)
5'CACCCGGACGAAAC3' (SEQ ID NO: 279)
5'CACACCCGGACGAAAC3' (SEQ ID NO: 280)
5'CACACCCGGACGAAA3' (SEQ ID NO: 281)
5'CACACCCGGACGAA3' (SEQ ID NO: 282)
5'CGAGGCCACGCGGAGTACGATCGA3' (SEQ ID NO: 283)
5′ ACTCGATCGTACTCGCGCGTGGCCTCGGTGAA 3′ (SEQ ID NO: 284)
5'TCGATCGTACTCCGC3' (SEQ ID NO: 285)
5'TCGATCGTACTCCG3' (SEQ ID NO: 286)
5'ATCGTACTCGCGTG3' (SEQ ID NO: 287)
5'ACTCGATCGTACTC3' (SEQ ID NO: 288)
5'CGTGGCCTCGGTGAA3' (SEQ ID NO: 289)
5'GTGGCCTCGGTGAA3' (SEQ ID NO: 290)
5' TAGTTAACTTTAATCTCCACATAGCAATCTTTAATC 3' (SEQ ID NO: 291)
5' GATTAAAGATTGCTATGTGAGATTAAAGTTAACTA 3' (SEQ ID NO: 292)
5' GATTAAAGATTGCTAT 3' (SEQ ID NO: 293)
5'TAAAGATTGCTATGTG3' (SEQ ID NO: 294)
5' AAGATTGCTATGTGAG 3' (SEQ ID NO: 295)
5'GCTATGTGAGTTAAAG3' (SEQ ID NO: 296)
5'TATGTGAGTTAAAGTT3' (SEQ ID NO: 297)
5'TGTGAGTTAAAGTTAA3' (SEQ ID NO: 298)
5′ CGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAG 3′ (SEQ ID NO: 299)
5′CTGCGTAGAAGCCTTTTGGCCAATGTTTGTTCCTTGAGGAAGTTGTAGCACG3′ (SEQ ID NO: 300)
5'TCGTAGAAGCCTTTTG3' (SEQ ID NO: 301)
5'CGTAGAAGCCTTTTGGC3' (SEQ ID NO: 302)
5'TAGAAGCCTTTTGGCA3' (SEQ ID NO: 303)
5' AAGCCTTTTGGCAATG 3' (SEQ ID NO: 304)
5'TTGGCAATGTTGTTCC3' (SEQ ID NO: 305)
5′GCAATGTTGTTCCT3′ (SEQ ID NO: 306)
5′CTTGAGGAAGTTGT3′ (SEQ ID NO: 307)
5'AGGAAGTTGTAGCACG3' (SEQ ID NO: 308)
5′GCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAG3′ (SEQ ID NO: 309)
5′CTACTAAAATTAATTTTTACACATTAGGGCTCTTCCATAGGC3′ (SEQ ID NO: 310)
5'CTACTAAAATTAATT3' (SEQ ID NO: 311)
5' TACTAAAATTAATTT 3' (SEQ ID NO: 312)
5'ACTAAAATTAATTT3' (SEQ ID NO: 313)
5'AATTAATTTTACACAT3' (SEQ ID NO: 314)
5'ATTTTACACATTAGGG3' (SEQ ID NO: 315)
5'TACACATTAGGGGCTC3' (SEQ ID NO: 316)
5′ACATTAGGGCTCTTC3′ (SEQ ID NO:317)
5'TAGGGCTCTTCCCATA3' (SEQ ID NO: 318)
5′GCTCTTCCATATAGG3′ (SEQ ID NO:319)
5'AGGTAAGATGGAGAGCCTT3' (SEQ ID NO: 320)
5'AAGGCTCTCCATCTTACCTTTCG3' (SEQ ID NO: 321)
5' AAGGCTCTCCATCTTA 3' (SEQ ID NO: 322)
5'AAGGCTCTCCATCT3' (SEQ ID NO: 323)
5′GCTCTCCATCTTACCT3′ (SEQ ID NO:324)
5'TCCATCTTACCTTTCG3' (SEQ ID NO: 325)
5'TGTGTAACATTAGGGAGG3' (SEQ ID NO: 326)
5'CCTCCCTAATGTTACACA3' (SEQ ID NO: 327)
5'CCTCCCTAATGTTACA3' (SEQ ID NO: 328)
5'CTCCCTAATGTTACAG3' (SEQ ID NO: 329)
5'CTCCCTAATGTTACAG3' (SEQ ID NO: 330)
5' TCATGTGGTAGTGTTGGTTTTA 3' (SEQ ID NO: 331)
5'TAAACCAACACTACCACATGA3' (SEQ ID NO: 332)
5'TAAACCAACACTACC3' (SEQ ID NO: 333)
5'AAACCAACACTACCAC3' (SEQ ID NO: 334)
5'AAACCAACACTACCAC3' (SEQ ID NO: 335)
5'ACCAACACTACCACAT3' (SEQ ID NO: 336)
5'CAACACTACCACATGA3' (SEQ ID NO: 336)

In some embodiments, the oligonucleotide comprises a sequence selected from: (IAV PSL2(+)
5'TGTCAGTAAGTATG3' (SEQ ID NO: 337)
5'CTGGCTGTCAGTAAGT3' (SEQ ID NO: 338)
5'TCGCTGTCTGGCTG3' (SEQ ID NO: 339)
5'CTTTTGGTCGCTGTCT3' (SEQ ID NO: 340)
5'CTTTTGGGTCGCTGT3' (SEQ ID NO: 341)
5'TTGGTCGCTGTCTGGC3' (SEQ ID NO: 342)
5'TTGGTCGCTGTCTG3' (SEQ ID NO: 343)
5'GAATTCTTTTGGTCG3' (SEQ ID NO: 344)
5′GAATTCTTTTGGTCG3′ (SEQ ID NO:345)
5'AATTCTTTTGGTCGC3' (SEQ ID NO: 346)
5'CGAATTCTTTTGGTCG3' (SEQ ID NO: 347)
5'ACACTAATTGATGGC3' (SEQ ID NO: 348)
5'AATTGATGGCCAT3' (SEQ ID NO: 349)

In some embodiments, the oligonucleotide comprises a sequence selected from: (IBV(-)
5'CCACAAAATGAAGGCA3' (SEQ ID NO: 350)
5'CACAAAATGAAGGCA3' (SEQ ID NO: 351)
5'CACAAAATGAAGGC3' (SEQ ID NO: 352)
5′CAATAATTGTACTA3′ (SEQ ID NO:353)
5′CAATAATTGTACTA3′ (SEQ ID NO:354)
5′CAATAATTGTACTA3′ (SEQ ID NO:355)
5'CAATAATTGTACTAC3' (SEQ ID NO: 356)
5'CAATAATTGTACTAC3' (SEQ ID NO: 357)
5'TGTACTACTCATGGTA3' (SEQ ID NO: 358)
5'TGTACTACTCATGGTA3' (SEQ ID NO: 359)
5'TGTACTACTCATGGTA3' (SEQ ID NO: 360)
5'GTACTACTCATGGT3' (SEQ ID NO: 361)
5'TTGTACTACTCATGG3' (SEQ ID NO: 362)
5'CATGGTAGTAACATC3' (SEQ ID NO: 363)
5′CTCATGGTAGTAACATC3′ (SEQ ID NO:364)
5′CTCATGGTAGTAACAT3′ (SEQ ID NO:365)
5′CTCATGGTAGTAACAT3′ (SEQ ID NO:366)
5' ACTCATGGTAGTAACA 3' (SEQ ID NO: 367)
5' ACTCATGGTAGTAACA 3' (SEQ ID NO: 368)
5'ACTCATGGTAGTAAC3' (SEQ ID NO: 369)
5' ACTCATGGTAGTAA 3' (SEQ ID NO: 370)
5' CAATGCAGATCGAAT 3' (SEQ ID NO: 371)
5' CAATGCAGATCGAAT 3' (SEQ ID NO: 372)
5′GATTGCCTTCACGAAA3′ (SEQ ID NO:373)
5GATTGCCTTCACGAAA3' (SEQ ID NO:374)

In some embodiments, the oligonucleotide comprises a sequence selected from: (IBV(+)
5′GCCTTCATTTTGTG3′ (SEQ ID NO:375)
5'ATTGCCTTCATTTTGT3' (SEQ ID NO: 376)
5'ATTGCCTTCATTTTGT3' (SEQ ID NO: 377)
5'TAGTACAATTATTGCC3' (SEQ ID NO: 378)
5'AGTACAATTATTGCC3' (SEQ ID NO: 379)
5′GTACAATTATTGCCTT3′ (SEQ ID NO: 380)
5'ACCATGAGTAGTACA3' (SEQ ID NO: 381)
5'CATGAGTAGTACAAT3' (SEQ ID NO: 382)
5' GATGTTACTACCATGAG 3' (SEQ ID NO: 383)
5' ATGTTACTACCATGAG 3' (SEQ ID NO: 384)
5'CATTGGATGTTACTAC3' (SEQ ID NO: 385)
5' ATTGGATGTTACTACC 3' (SEQ ID NO: 386)
5'ATTGGATGTTACTAC3' (SEQ ID NO: 387)
5'TTTCGTGAAGGCAATC3' (SEQ ID NO: 388)
5'TCGTGAAGGCAATC3' (SEQ ID NO: 389)

In some embodiments, the oligonucleotide comprises a sequence selected from (miRNA targeting sequences).
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 390)
5′ GTTTCGTCCGTGTT3′ (SEQ ID NO:391)
5′GCTGTCGCCCGTGTC3′ (SEQ ID NO:392)
5′GCTGTCGCCCGTGTC3′ (SEQ ID NO:393)
5′GCTGTCGCCCGTGTC3′ (SEQ ID NO:394)
5′GCTGTCGCCCGTGT3′ (SEQ ID NO:395)
5'TTCGTCCGTG3' (SEQ ID NO: 396)
5'TTCGTCCGTG3' (SEQ ID NO: 397)
5'TTCGTCCGTG3' (SEQ ID NO: 398)
5'CCCACCCAC3' (SEQ ID NO: 399)
5'CCCACCCAC3' (SEQ ID NO: 400)
5'CCCACCCAC3' (SEQ ID NO: 401)
5'TTTCGTCCGT3' (SEQ ID NO: 402)
5'TTTCGTCCGT3' (SEQ ID NO: 403)
5'TTTCGTCCGT3' (SEQ ID NO: 404)
5'CCCCACCCAC3' (SEQ ID NO: 405)
5'CCCCACCCAC3' (SEQ ID NO: 406)
5'CCCCACCCAC3' (SEQ ID NO: 407)
5'TTTCGTCCGTGT3' (SEQ ID NO: 408)
5'TTTCGTCCGTGT3' (SEQ ID NO: 409)
5'TTTCGTCCGTGT3' (SEQ ID NO: 410)
5'TTCCATCCATGT3' (SEQ ID NO: 411)
5'TTCCATCCATGT3' (SEQ ID NO: 412)
5'TTCCATCCATGT3' (SEQ ID NO: 413)
5'TTCCATCCATGT3' (SEQ ID NO: 414)
5'GTTTCGTCCGTGTT3' (SEQ ID NO: 415)
5′ GTTTCGTCCGTGTT3′ (SEQ ID NO: 416)
5′ GTTTCGTCCGTGTT3′ (SEQ ID NO: 417)
5'GTTTCGGCCATGTG3' (SEQ ID NO: 418)
5'CGTCCGTGTT3' (SEQ ID NO: 419)
5'CGTCCGTGTT3' (SEQ ID NO: 420)
5'CGTCCGTGTT3' (SEQ ID NO: 421)
5'CGTCCGTGTT3' (SEQ ID NO: 422)
5'CCTCCGTGTC3' (SEQ ID NO: 423)
5'CCTCCGTGTC3' (SEQ ID NO: 424)
5'CCTCCGTGTC3' (SEQ ID NO: 425)
5'TCCGTGTTGC3' (SEQ ID NO: 426)
5'TCCGTGTTGC3' (SEQ ID NO: 427)
5'TCCGTGTTGC3' (SEQ ID NO: 428)
5'TCCGTCTTGC3' (SEQ ID NO: 429)
5′TCCGTCTTGC3 (SEQ ID NO: 430)
5'TCCGTTTG3' (SEQ ID NO: 431)
5'TTTCGTCCGTGTT3' (SEQ ID NO: 432)
5'TTTCGTCCGTGTT3' (SEQ ID NO: 433)
5'TTTCCTCTATGTT3' (SEQ ID NO: 434)
5'TTTCCTCTATGTT3' (SEQ ID NO: 435)
5'GTTTCGTCCGGT3' (SEQ ID NO: 436)
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 437)
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 438)
5'AAGTACGTCCTACTT3' (SEQ ID NO: 439)
5'GGTTTCGTCC3' (SEQ ID NO: 440)
5'GGTTTCGTCC3' (SEQ ID NO: 441)
5'TGGGTTTCGTCCGTT3' (SEQ ID NO: 442)
5'TTTTGGGATTCCGT3' (SEQ ID NO: 443)
5′GCTTTTGGGATTCCGT3′ (SEQ ID NO: 444)
5′GCTTTTGGGATTCCGT3′ (SEQ ID NO: 445)
5'AGCTGCTTTTGGGATT3' (SEQ ID NO: 446)
5′GAGCTGCTTTTGGGAT3′ (SEQ ID NO: 447)
5'CTGCTTTTGGGATTCC3' (SEQ ID NO: 448)
5'TGCTTTTGGGATTC3' (SEQ ID NO: 449)
5′AGCTGCTTTTGGGATT3′ (SEQ ID NO: 450)
5'AGCTGCTTTTGGGA3' (SEQ ID NO: 451)
5'CAGCTGCTTTTGGGAT3' (SEQ ID NO: 452)
5′AGCTGCTTTTGGGATT3′ (SEQ ID NO: 453)
5′AGCTGCTTTTGGGA3′ (SEQ ID NO: 454)
5′AGCTGCTTTTGGGA3′ (SEQ ID NO: 455)
5′AGCTGCTTTTGGGATT3′ (SEQ ID NO: 456)
5'TGCTTTTGGGATTC3' (SEQ ID NO: 457)
5′GCGGCGCCCCGCCT3′ (SEQ ID NO: 458)
5′GCGGCGCCCCGCCT3′ (SEQ ID NO: 459)
5'CGGTCCCGCGGCGCCCCGCCT3' (SEQ ID NO: 460)
5'TTTGGGCTTCCTCGCT3' (SEQ ID NO: 461)
5'TTTGGGCTTCCTCG3' (SEQ ID NO: 462)
5'TTTGGGCTTCCTCG3' (SEQ ID NO: 463)
5'TGGGCTTCCTCGCT3' (SEQ ID NO: 464)
5'ATATACTTTGGGCTTC3' (SEQ ID NO: 465)
5'ATATACTTTGGGCTTC3' (SEQ ID NO: 466)
5'ATATACTTTGGGCT3' (SEQ ID NO: 467)
5'ATATACTTTGGGCTT3' (SEQ ID NO: 468)
5'TAGCCCTAGGCCCCGCA3' (SEQ ID NO: 469)
5'TAGCCCTAGGCCCCGCA3' (SEQ ID NO: 470)
5'TAGCCCTAGGCCCCG3' (SEQ ID NO: 471)
5'TGCTGTTAGCCCTA3' (SEQ ID NO: 472)
5'TGCTGTTAGCCCTA3' (SEQ ID NO: 473)
5'GTGTTAGCCCTAGCC3' (SEQ ID NO: 474)
5′GCGCCCAGCCCCGC3′ (SEQ ID NO: 475)
5′GCGCCCAGCCCCGC3′ (SEQ ID NO: 476)
5'CGCGCGCCCAGCCC3' (SEQ ID NO: 477)
5'CGCGCGCCCAGCCCCGC3' (SEQ ID NO: 478)
5'TGGCATGGAATGGG3' (SEQ ID NO: 479)
5'ATGGAATGGGCTCCGC3' (SEQ ID NO: 480)
5'ATGGAATGGGCTCC3' (SEQ ID NO: 481)
5'AATGGGCTCCGCCAG3' (SEQ ID NO: 482)
5'AATGGGCTCCGCCAG3' (SEQ ID NO: 483)
5'AATGGGCTCCGCCA3' (SEQ ID NO: 484)
5'AATGGGCTCCGCCA3' (SEQ ID NO: 485)
5'CTTACCCGAGGCGGTC3' (SEQ ID NO: 486)
5'TTACCCGAGGCGGT3' (SEQ ID NO: 487)
5'ACCGTACCTTACCCGA3' (SEQ ID NO: 488)
5'CGTACCTTACCCGA3' (SEQ ID NO: 489)
5'CTCCGAGCCCCGCC3' (SEQ ID NO: 490)
5'CCCGGCTCCGAGCCCCGCC3' (SEQ ID NO: 491)
5'TGGCCTGTCCCCGCA3' (SEQ ID NO: 492)
5'TGGCCTGTCCCCGCCA3' (SEQ ID NO: 493)
5'GATGCCCTGGCCTG3' (SEQ ID NO: 494)
5'GATGCCCTGGCCTG3' (SEQ ID NO: 495)
5'GCAGCCAGCTCTAC3' (SEQ ID NO: 496)
5'GCAGCCAGCTCTAC3' (SEQ ID NO: 497)
5'AGCCAGCTCTACCC3' (SEQ ID NO: 498)
5'AGCTCTACCCCCGCCA3' (SEQ ID NO: 499)
5'AGCTCTACCCCCGCCA3' (SEQ ID NO: 500)
5'AAAGCAGCGCCCCACTT3' (SEQ ID NO: 501)
5'ACTTCCTCCCCGCT3' (SEQ ID NO: 502)
5'CTACAGAAGCCCCATA3' (SEQ ID NO: 503)
5'CTACAGAAGCCCCATA3' (SEQ ID NO: 504)
5′GAAATCTCTACAGAAG3′ (SEQ ID NO: 505)
5′GAAATCTCTACAGAAG3′ (SEQ ID NO: 506)
5'TGATCCCTGTCCCCAT3' (SEQ ID NO: 507)
5'ATGCTGATCCCCTGTC3' (SEQ ID NO: 508)
5′GCCATGCTGATCCCTGTCCCCCAT3′ (SEQ ID NO: 509)
5'CCATCTCACCCCAT3' (SEQ ID NO: 510)
5'GCTGCTCCTCCCCAT3' (SEQ ID NO: 511)
5'TCTCCAACCCCCACAA3' (SEQ ID NO: 512)
5'CTCCAACCCCCACAA3' (SEQ ID NO: 513)
5'CAGCCAGCTCTCCAA3' (SEQ ID NO: 514)
5'AGCCAGCTCTCCAA3' (SEQ ID NO: 515)

In some embodiments, the oligonucleotide comprises a sequence selected from (minus strand targeting LNA).
5' ATCTGTTCTCTAAAACGA 3' (SEQ ID NO: 516)
5' ATCTGTTCTCTAAAACG 3' (SEQ ID NO: 517)
5'AGATCTGTTCTCTAAA3' (SEQ ID NO: 518)
5'TCTCTAAACGAACTTT3' (SEQ ID NO: 519)
5'TCTCTAAACGAACTTT3' (SEQ ID NO: 520)
5'CTCTAAACGAACTTTA3' (SEQ ID NO: 521)
5'ACTTTAAATCTGT3' (SEQ ID NO: 522)
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 523)
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 524)
5′GGTTTCGTCCGTGTT3′ (SEQ ID NO: 525)
5'TGCTTACGGTTTCGTCC3' (SEQ ID NO: 526)
5'GTTTCGTCCGTGTTGC3' (SEQ ID NO: 527)
5'TAGGTTTCGTCCCGG3' (SEQ ID NO: 528)
5'TCGTCCGGGTGTGA3' (SEQ ID NO: 529)
5'TTCGTCCGGGTGTGA3' (SEQ ID NO: 530)
5'TTTCGTCCGGGTGTGA3' (SEQ ID NO: 531)
5'AGGTTTCGTCCGGGT3' (SEQ ID NO: 532)
5'AGGTTTCGTCCGGG3' (SEQ ID NO: 533)
5'AGGTTTCGTCCGGGT3' (SEQ ID NO: 534)
5'TTTCGTCCGGGTGTG3' (SEQ ID NO: 535)
5'AGGCCACGCGGAGTA3' (SEQ ID NO: 536)
5′CACGCGGAGTACGATC3′ (SEQ ID NO: 537)

An oligonucleotide sequence may contain any convenient number of DNA, RNA BNA, LNA, ENA, cEt, 2' modified nucleotides, or other chemically modified nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences are mixed RNA/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed BNA/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed BNA/RNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences contain only BNA nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences are mixed LNA/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed LNA/RNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences contain only LNA nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences are mixed ENA/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed ENA/RNA sequences. In some cases of the oligonucleotide sequences described herein, the sequence contains only ENA nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences are mixed cEt/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed cEt/RNA sequences. In some cases of the oligonucleotide sequences described herein, the sequence contains only cEt nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences are mixed 2' modified nucleotide/DNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences are mixed 2' modified nucleotide/RNA sequences. In some cases of the oligonucleotide sequences described herein, the sequences contain only 2' modified nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences contain only DNA nucleotides. In some cases of the oligonucleotide sequences described herein, the sequences contain only RNA nucleotides.

In certain cases, the subject oligonucleotides have one of the following arrangements of types of nucleotides in a sequence (eg, SEQ ID NOs:45-66, or one of SEQ ID NOs:98-115).
A.
ABA,
A-B-A-B-A,
A-B-A-B-A-B-A,
A-B-A-B-A-B-A-B-A,
B.
BAB,
B-A-B-A-B,
B-A-B-A-B-A-B, or B-A-B-A-B-A-B-A-A,
wherein each A is independently a sequence of modified nucleotides and each B is a sequence of DNA nucleotides. In certain cases, A is a sequence of modified nucleotides and the ribose moiety is modified to include modifications selected from BNA, LNA, ENA, cEt, and 2' modified nucleotides. In certain cases, each A is a sequence of 1-50 nucleotides, such as 1-40, 1-30, 1-20, 1-10, or 1-5. In certain cases, each B is a sequence of 1-50 nucleotides, such as 1-40, 1-30, 1-20, 1-10, or 1-5.

In certain cases, the subject oligonucleotides have one of the following arrangements of types of nucleotides in a sequence (eg, SEQ ID NOs:45-66, or one of SEQ ID NOs:98-115).
(A) _n1 ,
(A) _n2 −(B) _n3 −(A) _n2 ,
(A) n4 - (B) _n5 - (A) _n4 - (B) _n5 - (A) _n4 - (B) _n5 - (A) _n4 or (A) _n4 - (B) _n5 - (A) _n4 -(B) _n5 -(A) _n4 -(B) _n5 -(A) _n4 -(B) _n5 -(A) _n4 ,
wherein A is a modified nucleotide, B is a DNA nucleotide, n1 is 8 or more, n2 is 3-4, n3 is 6-8, n4 is , 1 to 2, and n5 is 1 to 3. In certain cases, A is a modified nucleotide and the ribose moiety is modified to include modifications selected from BNA, LNA, ENA, cEt, and 2' modified nucleotides.

In certain cases, the subject oligonucleotides have one of the following arrangements of types of nucleotides in a sequence (eg, SEQ ID NOs:45-66, or one of SEQ ID NOs:98-115).
A, where A is a sequence of 8 or more LNA nucleotides;
ABA, where B is a sequence of 6-8 DNA nucleotides and each A is a sequence of 3-4 LNA nucleotides;
ABA, where B is a sequence of 7-8 DNA nucleotides and each A is a sequence of 4 LNA nucleotides;
LDLDL, where each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 2 DNA nucleotides;
LDLDLDL, where each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-2 DNA nucleotides;
LDLDLDL, where each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-3 DNA nucleotides;
LDLDLLDLDL, where each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-3 DNA nucleotides , and LDLDLDL, where each L is a sequence of 1-2 LNA nucleotides and each D is a sequence of 1-2 DNA nucleotides is.

The subject oligonucleotide sequences may further comprise one or more modified internucleoside linkages such as phosphorothioate, phosphorodithioate, phosphoramidate, and/or thiophosphoramidate linkages. Non-naturally occurring internucleoside linkages may be included at any convenient position in the sequence of the subject oligonucleotides. In embodiments where the internucleoside linkage comprises a chiral phosphorus atom, e.g., an asymmetric center, the chiral phosphorus atom is in the "R" configuration (referred to herein as Rp) or the "S" configuration (referred to herein as Sp). A subject oligonucleotide sequence containing a chiral phosphorus atom can be prepared as a racemic mixture or as separate enantiomers.

In certain embodiments, the oligonucleotide has one of the following sequences, where uppercase letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides (ie, deoxyribonucleotide units).
LNA1: 5'AaaAGaaT3' (SEQ ID NO: 67),
LNA2: 5'TggCcATcaaT3' (SEQ ID NO: 68),
LNA3: 5'TagCAtActtA3' (SEQ ID NO: 69),
LNA4: 5'CCAAAGA3' (SEQ ID NO: 70),
LNA5: 5'CATACTTA3' (SEQ ID NO: 71),
LNA6: 5'CagaCaCGaCCaaAA3' (SEQ ID NO: 72),
LNA7: 5'TAcTtaCTgaCagCC3' (SEQ ID NO: 73),
LNA8: 5'AGACacgaccaAAAG3' (SEQ ID NO: 74),
LNA9: 5'TACTtactgacaGCC3' (SEQ ID NO: 75),
LNA9.2: 5'TACttactgacAGCC3' (SEQ ID NO: 76),
LNA10: 5'ACCaaaagAAT3' (SEQ ID NO: 77),
LNA11: 5'TGGccatcAAT3' (SEQ ID NO: 78),
LNA12: 5'TAGcatacTTA3' (SEQ ID NO: 79),
LNA13: 5'CgacCAaaAGaattC3' (SEQ ID NO: 80),
LNA14: 5'CGACcaaaagaATTC3' (SEQ ID NO: 81),
LNA15: 5′GaTGgCcATcaAttA3′ (SEQ ID NO: 82),
LNA16: 5'GATGgccatcaATTA3' (SEQ ID NO: 83),
LNA17: 5'TcTAgCaTActTacT3' (SEQ ID NO: 84),
LNA18: 5'TCTAgcatactTACT3' (SEQ ID NO: 85),
LNA19: 5′GAAttcggatgGCCA3′ (SEQ ID NO: 86),
LNA20: 5'GGCCatcaattaGTG3' (SEQ ID NO: 87),
LNA21: 5'TTCGgatggccaTCA3' (SEQ ID NO: 88),
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO:89), and LNA23: 5'GACAgccagacaGCA3' (SEQ ID NO:90).

For any of sequences LNA1-LNA23 (SEQ ID NO:67)-(SEQ ID NO:90), one or more of the LNA nucleotides are selected from BNA nucleotides, ENA nucleotides, cEt nucleotides, and 2' modified nucleotides It will be appreciated that modified nucleotides may be substituted. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with BNA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with ENA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with cEt nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be substituted with 2' modified nucleotides (eg, substituted with MOE and other substituents described herein).

In certain embodiments, the oligonucleotide has one of the following sequences, where uppercase letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides (ie, deoxyribonucleotide units).
LNA19: 5′GAAttcggatgGCCA3′ (SEQ ID NO: 86),
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO: 89),
LNA22.2: 5'CAGCcagacagCGAC3' (SEQ ID NO: 116),
LNA22.3: 5'CAGccagacagCGAC3' (SEQ ID NO: 117),
LNA22.5: 5'CAGccagacaGCGA3' (SEQ ID NO: 118),
LNA22.6: 5'CAGccagacagCGA3' (SEQ ID NO: 119),
LNA22.7: 5'CAGCcagacagCGA3' (SEQ ID NO: 120),
LNA22.8: 5'ACAgccagacagCGA3' (SEQ ID NO: 121),
LNA22.9: 5'ACAGccagacaGCGA3' (SEQ ID NO: 122),
LNA22.10: 5'ACAgccagacaGCGA3' (SEQ ID NO: 123),
LNA22.11: 5'GACAgccagacaGCG3' (SEQ ID NO: 124),
LNA22.13: 5'GACagccagacaGCG3' (SEQ ID NO: 125), and LNA22.14: 5'GACAgccagacAGCG (SEQ ID NO: 126).

one or more of the LNA nucleotides for any of the sequences LNA19, or LNA22-LNA22.14 ((SEQ ID NO:86), (SEQ ID NO:89), and (SEQ ID NO:116)-(SEQ ID NO:126)) can be replaced with modified nucleotides selected from BNA nucleotides, ENA nucleotides, cEt nucleotides and 2' modified nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with BNA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with ENA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with cEt nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be substituted with 2' modified nucleotides (eg, substituted with MOE and other substituents described herein).

In certain embodiments, the oligonucleotide has one of the following sequences, where uppercase letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides (ie, deoxyribonucleotide units).
LNA24: 5'CATcaattagtgTCG3' (SEQ ID NO: 127),
LNA25: 5'CCAtcaattagTCG3' (SEQ ID NO: 128),
LNA26: 5′GCCatcaattagtGTG3′ (SEQ ID NO: 129),
LNA27: 5'AAGAattcggaTGGC3' (SEQ ID NO: 130),
LNA28: 5'CAGacagcgacCAA3' (SEQ ID NO: 131), and LNA29: 5'TGAcagccagacAGC3' (SEQ ID NO: 132).

For any of sequence LNA24, or LNA29 (SEQ ID NO: 127)-(SEQ ID NO: 132), one or more of the LNA nucleotides are selected from BNA nucleotides, ENA nucleotides, cEt nucleotides, and 2' modified nucleotides. It will be understood that any modified nucleotide may be substituted. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with BNA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with ENA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with cEt nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be substituted with 2' modified nucleotides (eg, substituted with MOE and other substituents described herein).

In certain embodiments, the oligonucleotide has the sequence LNA14, or derivative thereof, as shown in Table 1, wherein the "+" before the letter is an LNA nucleotide or (e.g., ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence LNA9, or a derivative thereof, as shown in Table 2, wherein the "+" before the letter is an LNA nucleotide or (e.g., ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence LNA8a, or derivative thereof, as shown in Table 3, wherein the "+" before the letter is an LNA nucleotide or (e.g., ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence IAV-Pos, or a derivative thereof, as shown in Table 4, wherein the "+" before the letter is an LNA nucleotide or (eg, described), and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence IBV, or a derivative thereof, as shown in Table 5, where the "+" before the letter is an LNA nucleotide or (e.g., ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence IBV-Pos, or a derivative thereof, as shown in Table 6, where the "+" before the letter is an LNA nucleotide or (eg, described), and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence Neg, or a derivative thereof, as shown in Table 7, where the "+" before the letter is an LNA nucleotide or (e.g., as described herein ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence miR, or derivative thereof, as shown in Table 8, wherein the "+" before the letter is an LNA nucleotide or (e.g., ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has the sequence Cov, or a derivative thereof, as shown in Table 8, where the "+" before the letter is an LNA nucleotide or (e.g., as described herein ) indicates other chemically modified nucleotides, and other letters indicate DNA nucleotides (ie, deoxyribonucleotide units).

In certain embodiments, the oligonucleotide has one of the following sequences, where uppercase letters indicate LNA nucleotides and lowercase letters indicate DNA nucleotides (ie, deoxyribonucleotide units).
a) LNA14: 5'CGACcaaaagaATTC3' (SEQ ID NO: 81),
b) LNA14.5: 5'CGACcaaaagaATT3' (SEQ ID NO: 135),
c) LNA14.8: 5'CGACcaaaagaaTTC3' (SEQ ID NO: 137),
d) LNA14.28: 5'GACcaaagaatTCGG3' (SEQ ID NO: 148),
e) LNA14.30: 5'GACCaaaagaattCGG3' (SEQ ID NO: 149),
f) LNA9: 5'TACTtactgacaGCC3' (SEQ ID NO: 75),
g) LNA9.1: 5'AGCAtacttactGACA3' (SEQ ID NO: 159),
h) LNA9.2a: 5'CATacttactgACA3' (SEQ ID NO: 160),
i) LNA9.8: 5'ATActactgACAG (SEQ ID NO: 164),
j) LNA9.12: 5'CATActtactgacAGC (SEQ ID NO: 167),
k) LNA8a: 5′AGAcagcgaccaaAAG (SEQ ID NO: 188),
l) LNA8a. 1:5'AGACagcgaccaAAAG (SEQ ID NO: 189), and m) LNA8a. 2: 5'ACAGcgaccaAAAG (SEQ ID NO: 190).

In certain embodiments, the oligonucleotide has a sequence selected from (a)-(e). In certain cases, the oligonucleotide has a sequence selected from (f)-(j). In certain other cases, the oligonucleotide has a sequence selected from (k) through (m).

For any of sequences (a)-(m) or a sequence selected from any one of Tables 1-9, one or more of the LNA nucleotides are BNA nucleotides, ENA nucleotides, cEt It will be appreciated that modified nucleotides selected from nucleotides and 2' modified nucleotides may be substituted. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with BNA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with ENA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with cEt nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be substituted with 2' modified nucleotides (eg, substituted with MOE and other substituents described herein).

It will also be appreciated that for any of the above oligonucleotide sequences, one or more of the DNA nucleotides may be modified to provide the corresponding BNA, LNA, ENA, cEt, or 2' modified nucleotides. be. In certain cases, 1, 2, 3, 4 or more of the DNA nucleotides may be replaced with BNA nucleotides. In certain cases, one, two, three, four or more of the DNA nucleotides may be modified to provide the corresponding LNA nucleotides. In certain cases, one, two, three, four or more of the DNA nucleotides may be modified to provide the corresponding ENA nucleotides. In certain cases, one, two, three, four or more of the DNA nucleotides may be modified to provide the corresponding cEt nucleotides. In certain cases, one, two, three, four or more of the DNA nucleotides may be modified to provide the corresponding 2' modified nucleotides (eg, as described herein).

Sequence variants of the above oligonucleotide sequences are also encompassed by this disclosure. One, two, three, four or more of the nucleotides in any of the sequences described herein are mutated to provide desirable properties such as enhanced inhibitory activity, conjugation to modifiers, etc. It is understood to obtain

In some cases, any one of the sequences described herein (eg, one of SEQ ID NOs:45-907) is more Contained in a long array. In certain cases, the subject oligonucleotides are 75 nucleotides or less in length, such as 50 nucleotides or less, 40 nucleotides or less, or 35 nucleotides or less in length. In certain cases, the subject oligonucleotides are 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 in length. , 30 nucleotides or less in length, such as 13 nucleotides or less, 12 nucleotides or less, 11 nucleotides or less, or 10 nucleotides or less.

In some cases, the subject oligonucleotide compounds comprise sequences having deletions relative to one of the sequences set forth herein (eg, one of SEQ ID NOs:45-907). For example, sequences in which 1, 2, or 3 nucleotides are deleted from the 5' and/or 3' ends of one of, for example, SEQ ID NOs:45-96. In some cases, the deleted sequence has one nucleotide missing from the 5' end of one of SEQ ID NOs:45-907. In some cases, the deleted sequence has one nucleotide missing from the 3' end of one of SEQ ID NOs:45-191. In some cases, the deletion sequence is two nucleotides missing from the 5' end of one of SEQ ID NOs:45-907. In some cases, the deletion sequence is two nucleotides missing from the 3' end of one of SEQ ID NOs:45-907.

In certain cases, the oligonucleotide comprises a sequence that has at least 70% homology with any one of (eg, defined herein) sequences (SEQ ID NO:45) to (SEQ ID NO:907) . In certain cases, the oligonucleotide comprises any one of sequences (SEQ ID NO:45) to (SEQ ID NO:907) and 70, such as 75% or more, 80% or more, 85% or more, 90% or more, or more. Includes sequences with 10% or more homology. In certain cases, the oligonucleotide comprises a sequence with 70-80% homology to any one of sequences (SEQ ID NO:45)-(SEQ ID NO:907). In certain cases, the oligonucleotide comprises a sequence with 80%-90% homology to any one of sequences (SEQ ID NO:45)-(SEQ ID NO:907). In certain cases, the oligonucleotide comprises a sequence that has 90% to 99% homology with any one of sequences (SEQ ID NO:45)-(SEQ ID NO:907).

In certain cases, the oligonucleotide sequences may contain mutations designed to cover single nucleotide polymorphisms (SNPs) in the target PSL2 sequence. In some cases, the oligonucleotide is a modified version of LNA9 with a single mutation site that protects against the PLS2 target sequence containing nucleotide changes with the LNA9 target sequence. It is understood that the SNP mutations of interest can be applied to any of the sequences described herein. Mutant sequences of interest are:
5'TACTTACTGACAGTC3' (SEQ ID NO: 91),
5'TACTTACCGACAGCC3' (SEQ ID NO:92), and 5'GGATTTCGGATGGCCA3' (SEQ ID NO:93).

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'TACTtactgacaGTC3' (SEQ ID NO: 94),
LNA9. T80C: 5'TACTtaccgacaGCC3' (SEQ ID NO: 95), and LNA19. U56C: 5'GGATttcggatggCCA3' (SEQ ID NO: 96).

for any of the sequences (SEQ ID NO:94)-(SEQ ID NO:96), or one or more of the LNA nucleotides are replaced with modified nucleotides selected from ENA nucleotides, cEt nucleotides, and 2′ modified nucleotides It will be understood to obtain In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with ENA nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be replaced with cEt nucleotides. In certain cases, 1, 2, 3, 4 or more of the LNA nucleotides may be substituted with 2' modified nucleotides (eg, substituted with MOE and other substituents described herein).

In certain cases, the oligonucleotide has a maximum length corresponding to a particular region of the PSL2 structure (eg, a subregion corresponding to nucleotides 34-87). In certain cases, the length of the oligonucleotide is 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, 11 nucleotides or less, 10 nucleotides or less, 9 nucleotides or less, 8 nucleotides or less, 7 nucleotides or less, or less, or the like. 20 nucleotides or less.

Oligonucleotides may 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 the native phosphodiester structure to increase intracellular stability and binding affinity. Several such modifications have been described in the literature that alter the chemistry of the backbone, sugars or heterocyclic bases.

Among the useful variations in backbone chemistry are phosphorothioates, phosphorodithioates in which both non-bridging oxygens are replaced with sulfur, phosphoramidites, alkylphosphotriesters and boranophosphates. Achiral phosphate derivatives include 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate, 3′-CH ₂ -5′-O-phosphonate, 3′-NH-5 '-O-phosphoramidates and thiophosphoramidates are included. Peptide nucleic acids replace the entire ribose phosphodiester backbone with peptide bonds. Sugar modifications are also used to enhance stability and affinity. The α-anomer of deoxyribose can be used, with the bases reversed relative to the natural β-anomer. In certain cases, the 2'-OH of the ribose sugar can be changed, eg, as described herein. The 2'-OH of ribose sugars may be changed to form 2'-O-methyl or 2'-O-allyl sugars, which provide resistance to degradation without compromising affinity. In certain cases, modification of the 2'-OH of the ribose sugar can ameliorate toxicity. Modification of heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxythymidine and 5-methyl-2'-deoxycytidine and 5-bromo-2'-deoxycytidine for deoxycytidine. 5-Propynyl-2'-deoxyuridine and 5-propynyl-2'-deoxycytidine have been shown to increase affinity and biological activity when substituted for deoxythymidine and deoxycytidine, respectively. ing.

Oligonucleotide agents may be derivatized with any convenient modifier, such as by conjugation of the modifier to the 5' and/or 3' ends of the oligonucleotide sequence. In some cases, modifiers are moieties (eg, lipids) that enhance cellular uptake. Any convenient lipid can be conjugated to the subject oligonucleotides. In some cases, the modifier is a fatty acid attached to the 5' or 3' end via an optional linker. Lipid groups can be aliphatic hydrocarbons or fatty acids, including but not limited to derivatives of hydrocarbons and fatty acids, examples being myristic acid (tetradecanoic acid), palmitic acid (hexadecanoic acid), and stearic acid (octadecane acids) and their corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane, and octadecane, saturated linear compounds with 14-20 carbons. Examples of other suitable lipid groups that may be used are sterols such as cholesterol, and substituted fatty acids and hydrocarbons, especially polyfluorinated forms of these groups. The range of lipid groups includes amines, amides, esters, and derivatives such as carbamate derivatives.

In some cases, the modifier is an additional nucleic acid sequence that has a desired activity (eg, recruitment of RNases as described herein). In certain cases, the modifier has a specific binding activity that provides delivery of the oligonucleotide to a particular target, such as a cell-specific protein. In some cases, the modifying agent is an antibody of interest that specifically binds to a cell-specific target of interest. In certain cases, an antibody modifier specifically binds to a hemagglutinin (HA) target.

Oligonucleotide active agents may be utilized in any convenient form. In some cases, the oligonucleotide active agent is single stranded. In some cases, the oligonucleotide active agent is double-stranded. In some cases, the oligonucleotide active agent is siRNA. In some cases, the oligonucleotide active agent is shRNA. In some cases, the oligonucleotide active agent is ssRNA. In some cases, one or more nucleotides of the ssRNA can be replaced with LNA nucleotides. In some cases, the oligonucleotide active agent is ssDNA. In some cases, one or more nucleotides of the ssDNA can be replaced with LNA nucleotides.

Methods of Treatment The individual is infected with influenza A virus alone and/or other respiratory viruses including, but not limited to, influenza B virus (IBV), coronavirus (Cov), respiratory syncytial virus (RSV). can do.

The present invention is particularly well suited for the prevention and/or treatment of respiratory infections and to serve as a just-in-time vaccine. Additionally, the present invention can be co-administered with other vaccines to provide immediate protection during the period needed for the other vaccine to be effective. Additionally, the present invention may be administered to vaccinated individuals at risk of exposure to vaccine-resistant variants. In addition, the present invention can be administered to vaccinated individuals who are asymptomatic but still capable of transmitting the virus.

Oligonucleotides according to the invention may be used alone or in combination with other oligonucleotides to prevent and/or treat respiratory viruses described herein.

Aspects of the present disclosure include methods of treating or preventing influenza A virus infection, influenza B, coronavirus, respiratory syncytial virus (RSV) in a subject. The subject oligonucleotide compounds are used as a new class of antiviral therapeutics that can efficiently disrupt packaging and completely prevent otherwise fatal disease in vivo. As demonstrated in the Examples section, intranasal dosing of exemplary oligonucleotide compounds in vivo produced potent antiviral effects and prevented lethal IAV infection in mice.

Aspects of the method include administering a therapeutically effective amount of a subject compound to a subject in need thereof to treat the subject for infection or to prevent infection in the subject. By "therapeutically effective amount" is meant a concentration of the compound sufficient to elicit the desired biological effect (eg, treatment or prevention of a condition or disease, influenza A virus infection). By "treatment" is meant that at least an amelioration of symptoms associated with the condition afflicting the host is achieved; amelioration broadly refers to parameters associated with the condition being treated, e.g. used to refer to at least a reduction in sluggishness. Thus, treatment also includes that the pathological condition, or at least the symptoms associated therewith, is completely inhibited, e.g., prevented from occurring so that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. or suspended, e.g., terminated. Thus, treatment is (i) prophylactic, i.e., reducing the risk of developing clinical symptoms, including not developing clinical symptoms, e.g., preventing progression of the disease to an adverse condition, (ii) inhibiting, i.e. preventing the onset or further onset of clinical symptoms, e.g., reducing or completely inhibiting active disease (e.g., infections); and/or (iii) alleviating, i.e., clinical symptoms Including causing regression. In the context of influenza A virus infection, the term "treating" includes reducing the number of viral cells in a patient sample, inhibiting viral cell replication, and one or more treatments associated with the infection. including any or all of ameliorating symptoms.

The subject to be treated can be one in need of treatment and the host to be treated is a host amenable to treatment using the subject compounds. In some embodiments, the subject is suspected of having an influenza A virus infection. In certain embodiments, the subject is diagnosed with an influenza A virus infection. Thus, in some cases, the subject is a virally infected subject.

In certain cases, the subject is a subject at risk or suspected of contracting a virus. In some embodiments, the vRNA is PB2 vRNA. The subject methods can be used to prevent infection with influenza A virus in a subject. By "prophylaxis" is meant that a subject at risk of influenza A virus infection does not become infected despite being exposed to the virus under conditions that would normally lead to infection. In some cases, administration of the subject active agents (e.g., oligonucleotide compounds) relieves the subject from infection for one week or more, such as two weeks or more, three weeks or more, one month or more, two months or more, three months or more. Protect. Multiple doses of the subject compounds may be administered according to the subject methods to provide long-term protection against the subject forms of infection. Timing and dosages can be readily determined using conventional methods.

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

Accordingly, a wide variety of subjects may be amenable to treatment using the subject compounds and pharmaceutical compositions disclosed herein. As used herein, the terms "subject" and "host" are used interchangeably. Generally, such subjects are "mammals," and humans are the subject of interest. Other subjects include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, etc.), rodents (e.g., mice, guinea pigs, and rats, e.g., diseased animals). models), and non-human primates (eg, chimpanzees and monkeys).

The amount of the subject compound administered is sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle, using any convenient method. can be determined by Unit dosage form specifications of the invention will depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

Aspects of the present disclosure include methods of treating or preventing coronavirus (CoV) infections. The subject oligonucleotide compounds are used as a new class of antiviral therapeutics that can efficiently disrupt the RNA secondary structure of CoV and thus treat or prevent CoV infection in a subject. In some cases, the subject oligonucleotide compounds can inhibit host miRNA interaction with CoV. In some cases, the subject oligonucleotide compounds are novel antimicrobials that can efficiently disrupt CoV RNA secondary structure, disrupt packaging, and completely prevent otherwise fatal disease in vivo. Used as an antiviral agent. In certain cases, the CoV is selected from HCoV-229E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, and SARS-CoV-2. In certain cases the CoV is SARS-CoV-2.

Aspects of the method include administering a therapeutically effective amount of a subject compound to a subject in need thereof to treat the subject for infection or to prevent infection in the subject. A "therapeutically effective amount" is a sufficient concentration of a compound to elicit a desired biological effect (e.g., treatment or prevention of a condition or disease, influenza A, influenza B, coronavirus infection, RSV infection). means By "treatment" is meant that at least an amelioration of symptoms associated with the condition afflicting the host is achieved; amelioration broadly refers to parameters associated with the condition being treated, e.g. used to refer to at least a reduction in sluggishness. Thus, treatment also includes developing a pathological condition, or at least symptoms associated therewith, that are partially or completely inhibited, e.g., such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. is prevented or stopped, e.g., terminated. Thus, treatment is (i) prophylactic, i.e., reducing the risk of developing clinical symptoms, including not developing clinical symptoms, e.g., preventing progression of the disease to an adverse condition, (ii) inhibit, i.e. prevent the onset or further onset of clinical symptoms, e.g. reduce or completely inhibit an active disease (e.g. an infection); (iii) alleviate, i.e. cause regression of clinical symptoms (iv) prevent the need for critical conditions requiring hospitalization or intensive care unit (ICU); (v) shorten the duration of hospitalization or ICU stay; (vi) supplemental oxygen; preventing or reducing the need for assisted ventilation, including but not limited to non-invasive positive pressure ventilation (CPAP and/or BPAP), mechanical ventilation; (vii) preventing the development of multiple bacterial infections; and (viii) preventing mortality. In the context of influenza A, influenza B, and coronavirus (CoV) infections, the term "treating" includes reducing the number of viruses in the body, reducing the number of virus-infected cells in a patient, Inhibiting replication of intracellular and/or extracellular viruses and virus-infected cells, and ameliorating one or more symptoms associated with the infection.

The subject to be treated can be one in need of treatment and the host to be treated is a host amenable to treatment using the subject compounds. In some embodiments, the subject is suspected of having a coronavirus infection. In certain embodiments, the subject is diagnosed with a coronavirus infection. Thus, in some cases, the subject is a virally infected subject.

In certain cases, the subject is a subject at risk or suspected of contracting a virus. The subject methods can be used to prevent infection with coronavirus in a subject. “Prophylaxis” means that a subject at risk of coronavirus infection does not become infected despite being exposed to the virus under conditions that would normally lead to infection, or means that the intensity is attenuated. In some cases, administration of the subject active agents (e.g., oligonucleotide compounds) relieves the subject from infection for one week or more, such as two weeks or more, three weeks or more, one month or more, two months or more, three months or more. Immediate protection. Multiple doses of the subject compounds may be administered according to the subject methods to provide long-term protection against the subject forms of infection. Timing and dosages can be readily determined using conventional methods.

In some cases, the subject treatment methods include determining or diagnosing whether the subject has a coronavirus infection. The determining step may be performed using any convenient method. In some cases, the determining step includes obtaining a biological sample from the subject and assaying the sample for the presence of intracellular and/or extracellular virus and virus-infected cells. A sample can be a cell sample. The determining step may include identifying intracellular and/or extracellular viruses containing specific mutations and virus-infected cells.

In some embodiments, the effective dosage of the subject compounds is about 50 ng/ml to about 50 μg/ml (eg, about 50 ng/ml to about 40 μg/ml, about 30 ng/ml to about 20 μg/ml, about 50 ng/ml /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 /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).

In some embodiments, the effective amount of the subject compounds is from about 10 pg to about 100 mg, such as from about 10 pg to about 50 pg, from about 50 pg to about 150 pg, from about 150 pg to about 250 pg, from about 250 pg to about 500 pg, from 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 Amounts ranging from 1 mg to about 100 mg, or from about 50 mg to about 100 mg. The amount can be a single dose amount or can be a total daily amount. The total daily dose can range from 10 pg to 100 mg, or can range from 100 mg to about 500 mg, or can range from 500 mg to about 1000 mg.

In some embodiments, a single dose of the subject compound is administered. In other embodiments, multiple doses of the subject compounds are administered. When multiple doses are administered over a period of time, the subject compounds are administered twice daily (qid), daily (qd), every other day (qod), every 3 days, thrice weekly (tiw), Or it can be administered twice weekly (biw). For example, the compounds can be administered qid, qd, qod, tiw, or biw over a period of 1 day to about 2 years or more. For example, the compound is administered at any of the frequencies described above for 1 week, 2 weeks, 1 month, 2 months, 6 months, 1 year, or 2 years, or more, depending on various factors. can be

Any of a variety of methods may be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual treated with the subject method can be assayed for the presence and/or level of viral cells. Assessing the efficacy of a therapeutic method for a subject can include evaluating the subject before, during and/or after treatment using any convenient method. Aspects of the subject method further comprise assessing the subject's therapeutic response to the treatment.

In some embodiments, the method comprises assessing the subject's condition and assessing one or more symptoms of the subject associated with the disease or condition of interest to be treated (e.g., as described herein). Including diagnosing or evaluating. In some embodiments, the method obtains a biological sample from a subject, e.g., for the presence of viral cells or components thereof associated with a disease or condition of interest (e.g., as described herein). , including assaying the sample. A sample can be a cell sample. The evaluating 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 evaluating step includes identification and/or quantification of viral cells. In certain cases, evaluating a subject includes diagnosing whether the subject has or has symptoms of a viral infection.

Screening Methods Aspects of the present disclosure also include screening assays configured to identify agents for use in the methods of the invention, eg, as outlined above. Aspects of the present disclosure include methods for screening candidate agents for their ability to inhibit influenza A virus in cells. In some cases, the method comprises contacting a sample comprising viral RNA (vRNA) that includes a PSL2 motif with a candidate agent and determining whether the candidate agent specifically binds to the PSL2 motif. . In some cases, agents that specifically bind to PSL2 motifs treat subjects with influenza A virus infection. In some cases, the method includes contacting a sample comprising viral RNA (vRNA) comprising a segment of influenza B genomic RNA with a candidate agent and determining whether the candidate agent specifically binds to the vRNA. Including. In some cases, agents that specifically bind to the HA motif treat subjects with influenza B virus infection. Assessing or determining is at least predicting 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 desired. means

Candidate agents are selected from small molecules, oligonucleotides, antibodies, and polypeptides. In some cases, the determining step comprises detecting a cellular parameter, wherein a change in the parameter within the cell relative to cells not contacted with the candidate agent indicates that the candidate agent specifically binds to the PSL2 motif. indicates that In some cases, the subject screening method is a method of RNA structure mapping, such as SHAPE analysis (selective 2'-hydroxyl acylation analyzed by primer extension). In certain cases, candidate agents are oligonucleotides.

Drug screening can be performed using in vitro models, genetically modified cells or animals, or purified PSL2 protein. Drug screening can identify ligands that compete with, modulate, or mimic the action of the lead drug. Drug screens identify agents that bind to specific sites in 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 of protein binding, and the like. 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.

The term "agent" as used herein describes any molecule, eg, an oligonucleotide, protein, or pharmaceutical, that has the ability to bind to PSL2 and inhibit IAV. Generally, multiple assay mixtures are run in parallel at different drug concentrations to obtain differential responses to the various concentrations. Typically one of these concentrations serves as a negative control, ie at zero concentration or below the level of detection.

Aspects of the present disclosure also include screening assays configured to identify agents for use in the methods of the invention, eg, as outlined above. Aspects of the present disclosure include methods for screening candidate agents for their ability to inhibit coronaviruses in cells. In some cases, the method comprises contacting a sample comprising viral RNA (vRNA) comprising a CoV conserved RNA secondary structure with a candidate agent, and wherein the candidate agent is associated with the conserved RNA secondary structure motif. Including determining whether it specifically binds. In some cases, agents that specifically bind to conserved RNA secondary structure motifs treat subjects with coronavirus infections. Assessing or determining is at least predicting 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 desired. means

Candidate agents encompass numerous chemical classes, including oligonucleotides, antibodies, polypeptides, and organic molecules, eg, small organic compounds having molecular weights greater than 50 and less than about 2,500 daltons. Candidate agents contain functional groups necessary for structural interactions, particularly hydrogen bonding, with proteins, typically at least an amine, carbonyl, hydroxyl, or carboxyl group, preferably at least one of the functional chemical groups. Including two. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries. Known pharmacological agents can undergo direct or random chemical modifications, such as acylation, alkylation, esterification, amidation, etc., to produce structural analogs. In certain embodiments, the interest is a compound that crosses the blood-brain barrier.

When the screening assay is a binding assay, one or more of the molecules may be conjugated to a member of a signal-producing system, such as a label, which directly or indirectly provides a detectable signal. can be done. 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 antidigoxin. For specific binding members, the complementary member is usually labeled with a molecule that provides for detection according to known procedures.

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

The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the invention, and are intended to limit the scope of what the inventors regard as their invention. and is not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (eg amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. standard abbreviations such as bp, base pairs; kb, kilobases; pl, picoliters; s or sec, seconds; min, minutes; h or hr, hours; aa, amino acids; pair; nt, nucleotide; i. m. , intramuscularly; i. p. , intraperitoneally; s. c. , subcutaneous, etc. may be used.

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

Plasmid construction and cloning: 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/An Hoi/1/ A plasmid containing the wild-type PB2 segment from 2013 (H7N9) was used. For the generation of PR8 packaging mutant vRNA, the Stratagene QuickChange XL site-directed mutagenesis kit (Stratagene) was utilized for mutagenesis of the pDZ plasmid containing the PB2 gene of PR8. The sequence of each mutant construct was confirmed by automated sequencing.

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 produce recombinant PR8 virus, 293T/MDCK co-cultures of 10 ⁶ cells utilize a bidirectional dual POL I/II promoter system for co-synthesis of genomic vRNA and mRNA. One of each of the eight segments contained within the plasmid was transfected with 1 ug of Lipofectamine 3000 (Invitrogen). Cells were harvested 24 hours after transfection and inoculated into the allantoic cavity of 10-day-old chicken embryos (Charles River, research grade specific pathogen-free eggs). Rescue of recombinant virus was assessed by hemagglutination activity. Each newly rescued virus was further plaque titered and the mutation confirmed by sequencing of the mutated gene. Plaque assays were performed on confluent MDCK cells as previously described (Szretter et al., 2006). Hemagglutination (HA) assays were performed in 96-well round-bottom plates using 50ul of virus diluent and 50ul of 0.5% turkey red blood cell suspension in phosphate-buffered saline (PBS). , was performed at room temperature.

Viral growth kinetics: The growth kinetics of PR8 virus was determined by inoculating 10-day-old chicken eggs with 100 plaque-forming units (PFU) of virus. 72 hours after inoculation, virus titers in allantoic fluid were determined by titration of plaques on MDCK cells.

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

qPCR analysis of packaged vRNA: Approximately 200 ng of extracted vRNA was analyzed using a universal 3′ primer (5′-AGGGCTCTTCGGCCAGCRAAAGCAGG) (SEQ ID NO: 97) and Superscript III reverse transcriptase (RT) (Invitrogen). reverse transcribed. The RT product was diluted 10,000-fold and used as template for quantitative PCR (qPCR). Separate PCRs were then performed with segment-specific primers as previously described (Marsh et al., 2007). A 10 ul reaction mix contains 1 ul of diluted RT product, 0.5 uM primer concentration, and SYBR GreenER dye, 200 uM deoxynucleoside triphosphates, heat-labile UDG, optimized SYBR Green Select Buffer, and AmpliTaq DNA. SYBR Select Master Mix with polymerase UP enzyme (Applied Biosystems) was included. Relative vRNA concentrations were determined by cycle threshold analysis, total vRNA amounts were normalized by equalization of levels of HA vRNA, and percentages of incorporation were then calculated relative to the levels of wt vRNA packaging. Viral packaging results represent the mean level of vRNA incorporation±s.d. from two independent virus purifications, vRNA levels were quantified in triplicate, n=6.

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

Design and Preparation of Locked Nucleic Acids (LNAs): Oligonucleotides containing locked nucleic acids (LNAs) were custom synthesized from Exiqon. Uppercase letters indicate LNAs. Lower case letters indicate typical (unlocked) DNA nucleotides. All oligonucleotides contained phosphorothioate internucleoside linkages. LNAs were designed to be complementary to various sequences contained in the PSL2 structure of segment PB2. LNAs 8a and 9 are designed to contain stretches of 6-8 DNA nucleotides for RNAse-H recruitment. The sequences of all LNAs are shown below.
LNA1: 5'AaaAGaaT3' (SEQ ID NO: 67)
LNA2: 5'TggCcATcaaT3' (SEQ ID NO: 68)
LNA3: 5'TagCAtActtA3' (SEQ ID NO: 69)
LNA4: 5'CCAAAGA3' (SEQ ID NO: 70)
LNA5: 5'CATACTTA3' (SEQ ID NO: 71)
LNA6: 5'CagaCaCGaCCaaAA3' (SEQ ID NO: 72)
LNA7: 5'TAcTtaCTgaCagCC3' (SEQ ID NO: 73)
LNA8a: 5′AGAcagcgaccaaAAG3′-having RNase-H activity (SEQ ID NO: 188)
LNA9: has 5'TACTtactgacaGCC3'-RNase-H activity (SEQ ID NO: 75)
LNA9.2: 5'TACttactgacAGCC3' (SEQ ID NO: 76)
LNA10: 5'ACCaaaagAAT3' (SEQ ID NO: 77)
LNA11: 5'TGGccatcAAT3' (SEQ ID NO: 78)
LNA12: 5'TAGcatacTTA3' (SEQ ID NO: 79)
LNA13: 5'CgacCAaaAGaattC3' (SEQ ID NO: 80)
LNA14: 5'CGACcaaaagaATTC3' (SEQ ID NO: 81)
LNA15: 5'GaTGgCcATcaAttA3' (SEQ ID NO: 82)
LNA16: 5'GATGgccatcaATTA3' (SEQ ID NO: 83)
LNA17: 5'TcTAgCaTActTacT3' (SEQ ID NO: 84)
LNA18: 5'TCTAgcatactTACT3' (SEQ ID NO: 85)
LNA19: 5'GAAttcggatgGCCA3' (SEQ ID NO: 86)
LNA20: 5'GGCCatcaattaGTG3' (SEQ ID NO: 87)
LNA21: 5'TTCGgatggccaTCA3' (SEQ ID NO: 88)
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO: 89)
LNA23: 5'GACAgccagacaGCA3' (SEQ ID NO: 90)

The following oligonucleotides were designed to cover single nucleotide polymorphisms (SNPs) in the PSL2 sequence. The exemplary sequence below is a modified version of LNA9 with a single mutation site that protects the PLS2 sequence from some avian and bat strains containing nucleotide changes with the LNA9 target sequence. It should be understood that similar designs can be applied to any of the sequences described herein.
LNA9. G74C: 5'TACTtactgacaGTC3' (SEQ ID NO: 94)
LNA9. T80C: 5'TACTtaccgacaGCC3' (SEQ ID NO: 95)
LNA19. U56C: 5'GGATttcggatggCCA3' (SEQ ID NO: 96)

Antiviral Assay: LNA was reconstituted at 100 μM in RNAse-free water, aliquoted and stored at −20° C. prior to single use. Cells were transfected with LNAs at final concentrations of 1 μM, 100 nM, 10 nM, and 1 nM using Lipofectamine 3000 (Life Technology) according to the manufacturer's protocol. For prophylactic antiviral assays, 10 ⁶ MDCK cells were seeded in 6-well plates 24 hours prior to transfection with the indicated LNAs. Cells were then infected with 0.01 MOI of PR8 (H1N1) or HK68 (H3N2) virus 4 hours, 2 hours, or 1 hour after transfection. After infection, MDCK cells were infected with PR8 or HK68 as described for therapeutic antiviral evaluation. LNAs were then transfected 4 hours, 2 hours, or 1 hour after infection. Forty-eight hours after infection, supernatants were collected and virus titers were determined by plaque assay.

Cellular SARS-CoV-2 replication assay: For cell replication assays, LNA ASOs were reconstituted at 100 μM stock solution in RNase-free water, aliquoted and stored at −20° C. prior to single use. . One day prior to transfection, Huh-7, Vero E6, or ACE-A549 cells were seeded in 96-well clear-bottom plates to 60-70% confluency when treated with LNA ASO or scrambled LNA. bottom. Cells were transfected with LNA ASOs at final concentrations of 25 nM or 100 nM using Lipofectamine 3000® (Life Technologies) according to the manufacturer's protocol. Cells were then infected with a SARS-CoV-2 reporter virus expressing nanoluciferase (SARS-CoV-2 nLUC) at an MOI of 0.3 for 1 hour, after which the virus was removed and fresh medium was added. bottom. Recombinant SARS-CoV-2 nLUC is a bona fide fully replicating virus with ORF7 deleted and replaced with nLUC. Measurement of nLUC expression is therefore a surrogate marker of viral replication that allows for screening of antiviral compounds.

In vitro transcription of vRNA: For each wild-type isolate (PR8, 1918, VN1203, NY470, NY312, CA09, and A/Anhui/1/2013 H7N9) and the PR8 packaging mutant clone, segment-specific primers were used under the T7 promoter. was used to amplify the PB2 cDNA from the plasmid. Amplified cDNA was gel purified using the Invitrogen DNA gel kit. vRNA was then generated by in vitro transcription using T7-MEGAscript. vRNA for SHAPE was purified by MEGAclear (Thermofisher, cat# AM1908) and verified for purity and length by capillary electrophoresis.

sf-SHAPE analysis of vRNA: PB2 vRNA was folded in 100 mM HEPES (pH=8) (100 mM NaCl, 2.5 mM MgCl, 65°C for 1 min, room temperature for 5 min, chilled at 37°C for 20 min). ~30 minutes). As previously described (Mortimer and Weeks, 2009), 2 min acylation and reverse transcription (RT) primer extension with NMIA (Wilkinson et al., 2006) at 45°C for 1 min, 52°C for 25 min, Performed at 65° C. for 5 minutes. 6FAM was used for all labeled primers. Exceptions to these protocols were as follows. (i) post-acylation RNA purification was performed using RNA C&C columns (Zymo Research) rather than ethanol precipitation and (ii) the mixture was left at room temperature for 2-5 minutes before and after adding SHAPE primer buffer. , significantly enhances RT transcription yield, (iii) DNA purification is performed using Sephadex G-50 size exclusion resin in a 96-well format, followed by concentration by vacuum centrifugation to more significantly remove primers, and (iv) 2 pmol of RNA was used in the ddGTP RNA sequencing reaction.

An 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 sec. The GeneScan program was used to acquire data for each sample consisting of purified DNA resuspended in 9.75 ul of Hi-Di formamide to which 0.25 ul of ROX500 internal size standard (ABI Catalog 602912) was added. The PeakScanner parameters were set to the following parameters: smoothing=none, window size=25, size call=local southern, baseline window=51, peak threshold=15. Fragments 250 and 340 were computationally excluded from the ROX500 standard (Akbari et al., 2008). Data from PeakScanner were then processed into SHAPE data using FAST (Fast Analysis of SHAPE Traces), a custom program (Pang et al., 2011). FAST uses the ddGTP ladder as an external sizing standard and local Southern methods to automatically correct for signal differences due to handling errors, adjust for signal decay, and convert fragment lengths to nucleotide positions (Pang et al. , 2011 and Pang et al., 2012).

RNAstructure parameters: Slope and intercept parameters of 2.6 and −0.8 kcal/mol were initially tried as suggested (Deigan et al., 2009) and close to 0.0 kcal/mol (eg about −0.3) A smaller intercept was found to produce a less optimal structure (within a maximum energy difference of 10%). This small parameter difference can be attributed to the precise fitting achieved between the experimental and reference datasets by the automated FAST algorithm. In the current implementation, FAST is integrated into RNAstructure, which requires MFC (Microsoft Foundation Classes). RNA structures were drawn and colored using RNAViz2 (De Rijk et al., 2003) and finished in Adobe Illustrator.

Construct design, RNA synthesis, and chemical modification for mutation and map experiments: Double-stranded DNA templates were prepared by PCR assembly of DNA oligomers designed by automated MATLAB®, as previously described. (NA_Thermo, available at "https:" followed by "//github." followed by "com/DasLab/NA_thermo") (Kladwang and Cordero et al., 2011). The construct for mutation and map (M ² ) contains all single mutations to their Watson-Crick counterparts. Compensating 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, mutation and map ( ^M2 ), 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 is heated and cooled to remove secondary structural heterogeneity, then properly folded and treated with SHAPE reagent (5 mg/mL 1-methyl-7-nitroisatoic anhydride (1M7 ))) (Mortimer and Weeks, 2007). The modification reaction was quenched and RNA was recovered by poly(dT) magnetic beads (Ambion) and FAM-labeled Tail2-A20 primer. RNA was washed twice with 70% ethanol (EtOH) and resuspended in _ddH2O . Subsequently, it was reverse transcribed into cDNA and subjected to heated NaOH treatment to remove RNA. The final cDNA library was recovered by magnetic bead separation, rinsed, eluted in Hi-Di formamide (Applied Biosystems) using a ROX-350 ladder, and loaded onto a capillary electrophoresis sequencer (ABI3100). Data processing, structural modeling, and data deposition: CE data were analyzed using version 2.0 of the HiTRACE software package (both MATLAB toolbox and web server available (Yoon et al., 2011; Kim et al., 2013)). Trace alignment, baseline subtraction, sequence assignment, profile fitting, attenuation correction, and normalization were accomplished as previously described (Kim et al., 2009; Kladwang et al., 2014). Sequence assignments were done manually with validation from the sequencing ladder. Data-driven secondary structure models were obtained using the RNAstructure package version 5.4 folding program (Mathews et al., 2004) with pseudo-energy gradients and intercept parameters of 2.6 kcal/mol and −0.8 kcal/mol. . Two-dimensional Z-score matrices of the ^M2 dataset and helixwise bootstrapping confidence values were calculated as previously described (Tian et al., 2014; Kladwang and VanLang et al., 2011). Z-score matrices were used as base-pair-wise pseudo-free energies with slopes and intercepts of 1.0 kcal/mol and 0 kcal/mol. Secondary structure images were generated by VARNA (Darty et al., 2009). All chemical mapping datasets, including one-dimensional mapping, mutations and maps, and mutations/rescue, have been deposited in the RNA Mapping Database (“http:” followed by “//rmdb.stanford.” followed by “edu”). (Cordero et al., 2012).

SHAPE analysis of LNA-targeted vRNA: DNA template for PR8 segment PB2 was prepared by PCR assembly of DNA oligomers, 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. Once 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 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.

Example 1:
SHAPE characterization of the IAV segment PB2 packaging signal identifies a conserved structure.
Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) and computational modeling were applied to the 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' folds in solution (Pang, 2011) and exists in a flexible single-stranded state. It was investigated using an electrophilic SHAPE reagent that reacts preferentially with nucleotides (Wilkinson et al., 2006) (Fig. 1). This analysis revealed that much of the 2341 nt vRNA was mostly unstructured (Fig. 2). This is consistent with a recent bioinformatics study that found more likely secondary structure conservation of (+)sense than (−)sense RNA for all segments containing PB2 (Priore et al., 2012). ; Moss et al., 2011). These previous studies did not analyze the terminal coding region (TCR), instead stopping 80 nucleotides short of the end of the PB2 5'TCR. SHAPE-induced modeling showed stable RNA secondary structures, most notably a stem-loop motif (termed herein packaging stem loop 2 (PSL2)) containing nucleotides 34-87 ((-) sense notation). Several sections of this region were suggested, including FIG. 1A). This segment contained a set of nucleotides previously involved in PB2 packaging (see Figures 1A-1B, circled nucleotides), through mutational analysis via an unidentified mechanism (Gao et al., 2012; Marsh et al., 2008; Liang et al., 2008; Gog et al., 2007). Supporting the hypothesis that these previous mutations acted through disruption of the PSL2 structure, SHAPE analysis of the mutants resulted in different conformations that all abrogated the wild-type PSL2 structure (Fig. 1C, Fig. 1C). 3). The 60-nucleotide region encompassing PSL2 exhibits nearly 100% sequence conservation at the single-nucleotide level between seasonal and pandemic strains of different subtypes and species origin (Fig. 4), maintaining an intact PSL2 structure. suggesting the existence of strict biological requirements for Different downstream sequences within PB2 vRNA can alter the secondary structure of PSL2, resulting in full-length wild-type PB2 isolated from various IAV strains and subtypes, including the highly pathogenic avian H5N1 and pandemic 1918H1N1 strains. The structural conservation of PSL2 was explored by performing SHAPE analysis on vRNA. Despite the presence of two different nucleotides within the stem-loop and significant flanking sequence divergence, the PSL2 stem-loop structure was restored in SHAPE-induced modeling of PB2 RNA across these diverse species and subtypes ( 1D-1F).

Figures 1A-1F show SHAPE chemical mapping performed on full-length (-) sense wild-type PB2 vRNA. Color indicates SHAPE reactivity proportional to the probability that the nucleotide is single-stranded. All structures have been truncated to highlight the 5' end sequence structure. Energy = ΔG free energy value of the determined structure generated by the RNAstructure modeling algorithm using the SHAPE pseudo free energy parameter. (FIG. 1A) Wild-type PB2 RNA secondary structure from strain A/Puerto Rico/8/1934 'PR8' (H1N1). Color-coded circles correspond to nucleotide sites where synonymous mutations have been reported to affect PB2 packaging (Gao et al., 2012; Marsh et al., 2011). (Fig. 1B) Packaging efficiency of synonymous mutants in (Fig. 1a) as determined by qPCR. Results were run in triplicate. Error bars = ±SD. Boxes below indicate variant names and corresponding mutational changes. Nucleotide numbering is shown in the genomic (-) sense orientation. (FIG. 1C) SHAPE-determined structures of PB2 packaging-deficient mutant vRNAs, m757 (G44C) and m745 (A80U). Black boxes = sites of synonymous mutations, (Fig. 1D-F) SHAPE-determined structures of wild-type PB2 from pandemic and highly pathogenic strains, including different subtypes: (Fig. 1D) 1918 Pandemic (A/Brevig Mission /1/1918 (H1N1)), (Fig. 1E) highly pathogenic birds (A/Vietnam/1203/2004 (H5N1)), (Fig. 1F) 2009 pandemic "pig" (A/California/04/2009 (H1N1) ).

FIG. 2, panels AB show SHAPE reactivity of full-length PB2 vRNA. (FIG. 2, Panel A) Average SHAPE reactivity as a function of nucleotide position (window bin size=100 nt) of full-length (-) sense PB2 vRNA from IAV strain A/Puerto Rico/8/1934 (H1N1). The PSL2 region (highlighted in blue, nt 34-86) has a high density of codons conserved in the third position and has one of the lowest SHAPE reactivities within vRNA. 'includes the packaging signal domain. The region after PSL2 is relatively unstructured but still contains another potential site for the presence of an RNA structure at nucleotides 1400-1500. Interestingly, this second internal region was also predicted by a 2011 bioinformatics study to contain structural elements (ref. 19). (Fig. 2, panel B) Enlarged view of the PSL2 region from (Fig. 2a). Window bin size = 10 nt.

Figures 3A-3E show that packaging-deficient mutations disrupt wild-type SHAPE reactivity. Left: Mutant SHAPE reactivity plotted as change versus WT. Nucleotide numbering starts from the 5' end of the (-) sense vRNA. Orange bars indicate sites of mutation. Energy values represent the ΔG free energy of predicted structures generated by the RNAstructure modeling algorithm using the SHAPE pseudo free energy parameter. Right: SHAPE-determined structure of full-length (-) sense mutant PB2 vRNA from strain PR8 (H1N1). The image was cut to highlight the 5' terminal region. (Fig. 3A) Wild type. Packaging-deficient mutant: (Fig. 3B) m744b (AG83, 85UA). (Fig. 3C) m745 (A80U). (Fig. 3D) m55c (CU35, 36UC). (Fig. 3E) m757 (G44C).

Figure 4 shows the conservation of nucleotide sequences containing the PSL2 structure. Graphical representation of nucleic acid sequence alignments across various 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 symbol within each position indicates the relative frequency of each nucleotide at that position. Black boxes = PSL2 regions. Sequences included in the alignment: Highly Pathogenic A/Brevig Mission/1/1918 (H1N1), Pandemic "Swine Flu" A/California/04/2009 (H1N1), Modern 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 direction. Sequence alignment of the terminal 5' region of PB2 corresponding to the above sequences. Shaded blue boxes encompass PSL2 RNA secondary structure elements. Black dots designate the nucleotide sites that differ.

Mutation and Map Strategy Validates PSL2 Structure and Predicts Novel Packaging Mutants SHAPE analysis of PSL2 RNA structure was further tested to reveal additional beneficial mutations needed for in vivo studies, multidimensional A chemical mapping (Kladwang and Das, 2010) method was applied to the PSL2 segment. First, by mutation and map (M ² ) measurements, chemical reactivity upon systematic mutation of each stem residue, including changes at nucleotides previously found to be important for PB2 packaging. Collapse of the pattern was confirmed (see FIG. 5A, annotated regions) (Marsh et al., 2008; Gog et al., 2007). Automated computational analysis based on these ^M2 data reliably recovered the SHAPE-induced PSL2 structure (Figs. 1C, 3, 5B-5D), further validating the structural model. Second, as a predictive test, compensatory mutations were designed to restore the base pairs of the wild-type stem-loop structure disrupted by the initial packaging-deficient mutation (Figure 6, panels AB). These mutation rescuers indeed restore the PSL2 SHAPE pattern, provide base pair resolution for in vitro validation of modeled structures, and generate sequence variants to test the role of PSL2 structure in vivo. suggested.

Figures 5A-5D show two-dimensional mutation and map (M2) analysis of PSL2 RNA secondary structure. (FIG. 5A) Systematic single-nucleotide mutations and resulting chemical accessibility mapping reveal interactions in the three-dimensional structure of RNA. Chemical accessibility plotted in gray scale (black = highest SHAPE reactivity) across 88 single mutations at single nucleotide resolution of the PSL2 element from PR8 strain PB2. Reactive peaks (from left to right) correspond to nucleotides from the 5' to 3' end of PB2 RNA. Nucleotide sites corresponding to known packaging mutations (reported by Marsh et al., 2008) are indicated in blue on the right. Red arrows indicate prominent packaging-deficient mutation sites predicted by M2 analysis. (Fig. 5B) Mutations isolated by Z-score analysis (number of standard deviations from the mean at each residue) and powerful features of map data. A Z-score was calculated for each nucleotide reactivity by subtracting the average reactivity of this nucleotide across all variants and dividing by the standard deviation (output_Zscore_from_rdat in HiTRACE). Squares indicate secondary structure models induced by mutations and map data. Dark signals highlight evidence of structured nucleotide pairing. (Fig. 5C) RNA secondary structure of the 5' packaging signal region (nt 30-93) derived from incorporating Z-scores into the RNAstructure modeling algorithm: bootstrap confidence estimates given as green precentage values. Bootstrap values provide a numerically accurate measure of structural reliability. Low bootstrap confidence values suggest the existence of alternative structural models. (Fig. 5D) Bootstrap support values for each base pair shown as grayscale shading.

FIG. 6 shows the design of compensating mutations for the PR8 PB2 mutants described above. (Fig. 6, Panel A) Mapping the aforementioned synonymous variants (m757, m745, m55c) to the PSL2 structure. (FIG. 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 mutation and map chemistry analyses. bottom. Black boxed nucleotides indicate sites of compensatory mutations. (-) indicates sense vRNA orientation. For mutations where non-synonymous changes were required to restore structure, alterations in the encoded protein sequence are indicated.

To test whether the PSL2 stem-loop structure observed in solution is related to viral packaging in the cellular environment, the same nine synonymous mutations reported by Gog et al., 2007 and Marsh et al., 2008 (Fig. 1A-B, Figure 7 panel A), as well as four new synonymous mutations characterized by ^M2 analysis (Figure 7, panel B) were cloned into the pDZ plasmid containing the PR8 PB2 gene (Marsh et al., 2008 Liang et al., 2008; Gog et al., 2007) (Fig. 8). The packaging efficiency of the nine known mutants currently in the PR8 background was comparable to that originally described in WSN33 virus ¹⁵ (Fig. 7, panel C). Of these, mutants m55c, m757, m745, and m744b were predicted to exhibit the most pronounced impairment based on their position within the stem region of PSL2 (Fig. 1C, Figs. 3A-3F, Fig. 7). In contrast, published mutations that do not affect PB2 packaging that map to the unstructured apical loop or fall outside of PSL2 and did not alter its structural integrity (e.g., m731) (Fig. 9A) (Marsh et al., 2008). Three novel synonymous mutants (m74-1, m74-2, and m68) identified by ^M2 analysis to have significant effects in addition to in vitro PSL2 structure (Fig. 5A) have significant effects on PB2 packaging. while mutation sites that showed negligible changes in SHAPE reactivity compared to the wild-type PSL2 structure, resulted in wild-type-like packaging efficiency levels (e.g., m56) ( FIG. 7, panel D).

FIG. 7 shows highly conserved single codon synonymous mutations of PR8 PB2 vRNA. (FIG. 7, panel A) A previously published synonymous mutation involved in PB2 packaging. The top row is the parental PR8 vRNA sequence ((+)-sense orientation) and the mutated single nucleotide is bolded in red in the bottom row. The numbering and nomenclature of the mutations introduced are based on reports by Marsh et al., 2008 and Gog et al., 2007. Regions highlighted in yellow indicate sequences containing the PSL2 structure. (Fig. 7, panel B) Design of primer sequences for cloning synonymous mutations identified from M2 analysis (see Supplementary Fig. 4a) into the pDZ plasmid. Sequences are in the (+) sense orientation. Highlighted nucleotides = mutation sites. (Fig. 7, Panels C-D) (Fig. 7, Panel C) packaging efficiency representing the ratio of mutant PB2 packaging to parental wild-type PB2 of previously published synonymous mutants, and (Fig. 7, Panel D). ) M2 analysis identified synonymous variants. Results from two independent experiments resulted in assays performed in triplicate (n=6). Error bars indicate mean±SD.

Figure 8 shows the nomenclature of the PB2 packaging mutants and the corresponding sites of mutation. 1) previously published synonymous mutations involved in PB2 packaging (indicated in blue), based on reports of Marsh et al., 2008 and Gog et al., 2007, and 2) PSL2 structural design single mutants (indicated in black). Mutational nomenclature chart showing the name and site of mutations from ) and double compensating mutants (indicated in red). The numbering and nomenclature of the introduced mutations are based on the genomic, (-) sense vRNA. Examples where mutations result in protein coding changes are indicated by synonymous (SYN) or non-synonymous (non-SYN) regions.

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

Consistent with the proposed hierarchical role of PB2 in IAV packaging, compensatory mutations not only affected viral packaging of segment PB2 (Fig. 10, panels A-C; Fig. 6, panels A-B), but also previously Other segments reported to be affected by deleterious mutations were also rescued (Muramoto et al., 2006; Gao et al., 2012; Marsh et al., 2008) (Figure 10, panels DF). In addition to restoring PB2 packaging, the compensatory mutations resulted in a complete or near-complete rescue of the viral titer loss caused by the deletion mutation (Fig. 10, panels G-I). Some non-synonymous compensating mutations may restore PB2 packaging better than others (m745-comp and m55c-comp compared to m757-comp) (Fig. 10, panels A-C). Reflected incomplete restoration of PB2 protein function via exogenous addition. Such exogenous addition was necessary because several non-synonymous mutations affected both PSL2 structure and protein sequence. The sharpest examination of PSL2 structure came from packaging experiments that did not require supplemental wild-type PB2 protein addition. Based on computational enumeration and multidimensional mutation rescue experiments (Tian et al., 2014), a single mutation rescue pair was found (m52/m65, FIG. 11, panels AB, FIG. 12, panel s, FIG. 13). Making each mutation alone (m52 and m65) yielded packaging efficiencies of PB2 integration below 4% and titer losses greater than 4 log ₁₀ , previously reported for packaging-deficient viruses (i.e., 2 log ₁₀ )) (Fig. 11, panels CD, Fig. 7, panel c, Fig. 10). Although the sequence was altered, the compensating mutations restored both packaging efficiency and viral titer to wild-type levels when introduced together into the double-mutated m52/65-comp strain that restored the PSL2 structure. .

FIG. 10, panels AI show the effect of compensating mutations in PR8 PB2 packaging-deficient mutants on viral packaging and titer. (FIG. 10, panels A-C) Packaging efficiency of packaging-deficient and compensating mutant PB2 vRNA. For compensatory mutations requiring non-synonymous changes, wild-type PB2 protein expression plasmids were co-transfected during virus rescue. pWT = expression plasmid encoding wild-type PR8 PB2 protein. Values given as percentage of PB2 vRNA packaging compared to wt parental PR8 virus. Results from two independent experiments resulted in assays performed in triplicate (n=6). (Figure panels DF) Packaging efficiency of packaging-deficient and compensatory mutant viruses and their effect on packaging of other interacting segments, PB1, PA, NP, and MX. Assays were performed in triplicate (n=6). (FIG. 10, panels G-I) Viral titers by plaque assay. Results are PFU/mL and assays were run in triplicate.

FIG. 11 shows that multidimensional chemical mapping reveals novel PB2 packaging defects and compensating mutation partners. (Fig. 11, panel A) Rescue individual and compensating double mutations (mutation maps) to test base-pairing from the 1D data-driven model and identify predicted successful synonymous PSL2-deficient and compensating mutant pairs. Electropherogram results from systematic single-nucleotide mutation mapping with rescue) analysis. Chemical accessibility plotted in gray scale (black = highest SHAPE reactivity) across 88 single mutations at single nucleotide resolution of the PSL2 element from PR8 strain PB2. Reactive peaks (from left to right) correspond to nucleotides from the 5' to 3' end of PB2 RNA. See Figure 12 for a complete list of mutation rescue pairs. (FIG. 11, panel B) Mutational design of single mutants m52 (G52U) and m65 (C65A) on the PSL2 structure, and double m52/65 rescue pair. (Fig. 11, panel C) Packaging efficiency of synonymous single and double mutant mutation and rescue pairs. Values given as percentage of PB2 vRNA packaging compared to wt parental PR8 virus. Results from two independent experiments resulted in assays performed in triplicate (n=6). (FIG. 11, panel D) Viral titers in PFU/mL, triplicate results. Error bars indicate mean±SD.

Figure 12 panels a-t show two-dimensional mutational map rescue (M2R) analysis. Mutation/rescue results validate PSL2 RNA secondary structure. Electropherogram of SHAPE analysis using compensating double mutations to test base-pairing from a 1D data-derived model and identify successful PSL2 deletion and compensating mutant pairs. Chemical accessibility plotted in gray scale (black = highest SHAPE reactivity) across 88 single mutations at single nucleotide resolution of the PSL2 element from PR8 strain PB2. For each tested pairing, the wild-type, single mutant 1, single mutant 2, and compensating double mutant "quartets" are grouped for comparison. (FIG. 12, panels a-r) Non-synonymous mutation and rescue pairs. All electropherograms not boxed are pairs in which no disruption and/or rescue was observed. Blue boxes indicate normal deletions and rescue mutations. (Fig. 12, panel) Double synonymous mutation and rescue pair. Green box = successful synonymous defect and rescue pair. (Fig. 12, panel t) Packaging efficiency of non-synonymous mutation and rescue pairs. Values given as percentage of PB2 vRNA packaging compared to wt parental PR8 virus. Results from two independent experiments resulted in assays performed in triplicate (n=6). Error bars represent ±SD.

FIG. 13 shows the design of primer sequences for two-dimensional mutation map rescue (M2R) mutants. SEQ ID NOs (28-43) from top to bottom. Primer sequences used for QuickChange mutant cloning of M2R mutants into pDZ plasmid. Sequences are in the (+) sense orientation. The left region shows synonymous (Syn.) or non-synonymous (non-syn.) changes. Highlighted nucleotides = mutation sites. Boxed mutant primer sets indicate double synonymous mutant partners, m52 and m65.

To test the relevance of PSL2 structure in an in vivo model, 6-8 week old BALB/C mice were intranasally challenged with 1000 PFU of wild-type PR8 virus or strains carrying mutations predicted to disrupt or restore PSL2 structure. inoculated internally. Mice infected with the PSL2-disrupting mutant m745 mutant (20% packaging efficiency) or the severely packaging-deficient single mutant virus, m52 (<4% packaging efficiency), showed no weight loss or survival compared to PBS controls. In either, they showed reduced or absent clinical signs of disease, respectively (Figure 14, panels AB). Remarkably, including compensatory mutations that restore PSL2 structure rescued viral virulence. Animals infected with m52/65-comp and m745-comp showed comparable mortality profiles and survival curves to mice infected with wild-type PR8 (Figure 14, panels AB). Consistent with APLAC guidelines, all mice were humanely sacrificed when >20% weight loss was reached.

FIG. 14 panels AB show that packaging-deficient virus is attenuated in vivo. Percent weight loss and percent survival of mice infected with single PSL2-disrupting and compensating PSL2-restoring double mutant viruses. Six to eight week old BALB/C female mice were given 1000 PFU of PR8 wild type (wt) virus, packaging defective single mutant viruses m52 and m745, compensating double mutant viruses m52/65 and m745-comp. , or PBS controls were infected intranasally. Mice were monitored daily for percent weight loss and percent survival relative to day 0. Results are given as the mean of two independent experiments with 6 mice per condition. (FIG. 14, panel A) Percent weight loss. (FIG. 14, panel B) Kaplan-Meir survival plots of the individual cohorts shown in (FIG. 14, panel A).

Example 2:
Therapeutic design and targeting of PSL2 structures inhibit IAV infection in vitro and in vivo.
To explore the therapeutic potential of targeting PSL2-mediated viral packaging, nine locked nucleic acids (LNAs) containing phosphorothioate internucleoside linkages (Vester and Wengel, 2004) were constructed in the overall RNA secondary structure of the elements. were designed against key residues predicted to disrupt , thereby inhibiting virus production (Figure 15, panel A). Two of the designed LNAs, LNA8a and LNA9, are identical in sequence to LNA6 and LNA7, respectively, but have 6-7 unmodified (unlocked) sequences optimized for RNase-H activation. DNA nucleotides (see, eg, LNA6/8-RNaseH and LNA7/9-RNaseH, respectively). First, to assess the effect of LNA binding on PSL2 RNA secondary structure, we performed toeprinting and SHAPE chemical mapping on PB2 vRNA in the presence of LNA. In support of the antiviral assay results, sequences encoded by LNA6-9 showed the greatest ability to bind and disrupt the wild-type PSL2 structure (Fig. 16).

FIG. 15, panels AD, show that locked nucleic acids targeting PSL2 RNA structures exhibit potent antiviral activity in vitro and in vivo. (FIG. 15, panel A) Location of complementary locking nucleic acids (LNA) designed against various regions of the PSL2 structure. (FIG. 15, Panel B) To screen LNAs for antiviral activity, MDCK cells were infected with either PR8 (H1N1) virus or A/Hong Kong/8/68 (H3N2) virus at 0.01 MOI. , pretreated with 100 nM of each indicated LNA for 1 hour by Lipofectamine transfection. Forty-eight hours after infection, supernatants were collected and virus titers were determined by plaque assay. Results from two independent experiments resulted in assays performed in triplicate (n=6). (FIG. 15, panel C) Time course of pre-treatment (RX) versus post-infection treatment with LNA9 at titers (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 or 4 hours prior to 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, after which the supernatant was changed and cells were treated with LNA9 either 2 or 4 hours post-infection. Supernatants were collected 48 hours later and virus titers were determined by plaque assay in triplicate. FIG. 15, panel d, shows the effect of intranasal LNA treatment on survival of virus-infected mice. Mice were intranasally administered 20 ug of LNA9, scrambled LNA, or PBS (uninfected control) 12 hours prior to infection with PR8 virus. All mice received two additional treatments at 8 hpi and 36 hpi (n=7 mice per condition).

As shown in Figure 15, panels AD, the sequence label "LNA8" refers to, for example, the sequence LNA8a (SEQ ID NO: 188) described herein. Further, in FIG. 15, panels AD, FIG. 16A and FIG. 16B, LNA6/8 refers to, for example, sequences LNA6 (SEQ ID NO:72) and LNA8a (SEQ ID NO:155) described herein.

Figures 16A-16B show SHAPE analysis for LNA-RNA binding. (FIG. 16A) Electrophoretic profiles of SHAPE analysis performed on LNAs 1, 2, 4, 5, 6/8a, 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, unlabeled reaction without SHAPE reagent 1M7. Right: with labeling reagent. (FIG. 16B) Electrophoresis profiles of SHAPE run on LNA-vRNA combinations at titrating concentrations of LNA. For each LNA, the left set of columns is without labeling reagent.

In a first pilot experiment to determine the antiviral potential of LNA-mediated targeting of PSL2 across two different IAV subtypes, MDCK cells were then treated with 0.01 MOI of wild-type PR8 (H1N1) virus or tissue. Prior to infection with any of the culture-adapted A/Hong Kong/8/68 (HK68) (H3N2) viruses, they were pretreated with 100 nM of each LNA and delivered by Lipofectamine™ transfection for 1 hour. Forty-eight hours after infection, supernatants were collected and virus production was measured by plaque assay (Figure 15, panel B). LNAs directed only against the upper loop of PSL2 (LNA1, LNA4) had little or no effect on virus titer. Similarly, LNAs 3 and 5, which target the 3' base of PSL2, also did not inhibit virus production. In contrast, nucleotide coverage of both the upper loop and the middle bulge by LNA6 resulted in greater than 2 log ₁₀ loss of potency for PR8 (Figure 15, panels AB). LNA8a, an RNase-H activated copy of LNA6, produced up to 3 logs greater antiviral activity against both viruses. Most strikingly, LNA9, an RNase-H activated copy of LNA7, had the most potent antiviral capacity, reducing virus production by nearly 5 and 4 logs relative to PR8 and HK68, respectively.

After identifying the best candidate LNAs, the treatment time course and concentration parameters of LNA9's antiviral activity were further investigated. MDCK cells were treated with 1 dose of 10-fold diluted LNA9 either 2 or 4 hours prior to infection, or alternatively, 2 or 4 hours after infection with 0.01 MOI of wild-type PR8 virus. processed with Cells pretreated with LNA had the most potent antiviral response (>4 logs) and showed strong viral inhibition (>2 logs) even at the lowest dilution (1 nM) (Figure 15, panel C). Antiviral activity tended to decrease with increasing post-infection treatment time, but more than 3 logs of viral titer suppression was achieved even at the most recent study time point.

Example 3:
In Vivo Efficacy Experiments: Prolonged Single Dose Prophylaxis Balb/C female mice (5 mice/group) were treated 3 days prior to infection (day −3) or 1 day prior to infection with a lethal dose of wild-type PR8 virus. One day before (day -1), they were pretreated intranasally with a single dose of 20ug LNA9. Mice were monitored daily for weight loss, clinical scores, and survival. FIG. 17, panel A shows percent mouse survival over time. FIG. 17, panel B shows the percent weight loss over time after dosing.

A single dose of 20 ug LNA9 3 days prior to infection completely protected mice from lethal influenza disease. Untreated control mice were humanely sacrificed when they lost more than 25% body weight in an average of 5.5 days. In contrast, the pretreatment group showed minimal weight loss, few clinical signs of disease, and full recovery to pre-infection weight.

This result demonstrates that a single inhalable dose of LNA9 administered several days prior to infection can provide sustained protection against fatal disease, indicating that the subject compounds are effective against influenza outbreaks and It suggests that it could be used in prophylactic treatment during pandemics.

Example 4:
Susceptibility of Influenza Viruses to Oseltamivir and LNA9 After Serial Passage in the Presence of Drugs: A Drug Selection Experiment Oseltamivir (Tamiflu) is the most widely used and stockpiled neuraminidase inhibitor (NAI) on the market. Like all NAIs, oseltamivir requires conformational rearrangements of the viral neuraminidase (NA) protein to be drug compatible. Any mutation in the NA protein that affects this rearrangement will reduce the binding affinity of oseltamivir and thus reduce drug efficacy. In particular, the H274Y mutation (also known as the H275Y mutation depending on nomenclature) is most commonly associated with oseltamivir resistance. Rapid selection of the H274Y mutation in immunocompromised patients may lead to clinical failure of the last-resort NAI drug peramivir, which has previously been believed to select multidrug-resistant viruses in immunocompromised hosts. suggesting that it may be more common than previously thought. This, together with the recent spread of oseltamivir-resistant and NAI-resistant viruses in circulation, points to the need to re-evaluate the general use of NAI. The development of new classes of antiviral drugs is essential to reduce the potential adverse effects of current and future influenza pandemics on human health.

The sequence region in segment PB2, which contains the PSL2 stem loop, is highly conserved across IAV subtypes, strains, and isolates from a wide range of host species, likely reflecting stringent biological requirements for its conservation. have a nature. SHAPE analysis of this region confirmed the maintenance of PSL2 structure among seasonal and pandemic viruses of different subtypes and host origins, strongly highlighting the potential of this structural element as a novel pan-genotypic therapeutic target. (Thus, LNA9 has broad-spectrum potential against IAV isolates). In addition, since the subject LNAs are directed against highly conserved viral genomic RNA targets that apparently have strong constraints on their mutational capacity, the subject LNAs targeting PSL2 are significantly more efficient than NAIs. are expected to have higher barriers to the development of resistance.

Susceptibility of influenza virus to LNA9 versus oseltamivir after serial passage under drug pressure was investigated. Oseltamivir had an onset _IC50 of 41 nM against PR8 at passage 1 of drug treatment as determined by plaque reduction assay. With increasing amounts of drug, the _IC50 for oseltamivir jumped to 50 uM, 1000× after only 6 virus passages. See FIG. 18, panels AD. In contrast, the _IC50 remained stable at 18-16 pM after 10 virus passages in the presence of LNA9. Figure 19, panels A and B.

LNA9 can also be used to treat drug-resistant viruses. Drug-resistant mutants of A/WSN/33(H1N1) virus were generated using the reverse gene virus rescue system to mutate the NA gene to contain the H274Y resistance mutation. Against this virus, oseltamivir has an _IC50 of 53uM. Importantly, LNA9 maintained potency and efficacy against WSN H274Y virus with picomolar activity. FIG. 19, panel C. This result is strong evidence for therapeutic treatment of NAI-resistant viruses with PSL2-targeted LNAs. This result also highlights the activity of LNA9 against various IAV isolates.

Example 5:
Antiviral activity of miR-targeted LNAs: Determine the in vitro antiviral efficacy of LNAs designed against microRNAs postulated to mediate respiratory virus replication. Individual LNAs designed to target microRNAs were transfected into Huh7 cells, which were subsequently infected with a fully replicated SARS-CoV-2-nLuc virus with a nanoluciferase reporter in ORF7. Forty-eight hours after infection, the effect on replication was measured by luciferase activity compared to control cells treated with the negative control scrambled LNA (Scr.LNA) or the positive control nucleoside analogue EIDD (Figure 20). . LNAs targeting the frameshift element (FSE) region of the SARS-CoV-2 RNA genome had minimal effect on viral replication, whereas our antivirus designed to sequester miR-191 did not. Multiple log ₁₀ reductions were observed for miR LNAs. The degree of inhibition with anti-miRLNA was greater than the EIDD positive control. Moreover, this degree of inhibition was observed at 25 nmol LNA, while the EIDD positive control was used at 5 micromolar. Other microRNA-targeted LNAs with anti-respiratory virus activity have been identified as well, including: LNA-602.1, LNA-602.6, LNA-6769-5P. 3, LNA-6769-5P. 6, LNA-942.3P. 4, LNA-376c. 3, LNA-4433b. 1, LNA-191.1, LNA-191.2, LNA-191.3, LNA-191.4, LNA-191.8, LNA-191.8, LNA-19110, LNA-191.11, LNA- 191.12, LNA-191.13, LNA-663.1, LNA-663.3, LNA-381.2, LNA-744.2, LNA-744.4, LNA-744.7, LNA-4508. 1, LNA-4508.4, LNA-4730.2, LNA-4730.6, LNA-6777.4, LNA-10396.1, LNA-10396.2, LNA-4749.2, LNA-4749.4, LNA-4706.3, LNA-4706.4, LNA-3675.2, LNA-3675.3, LNA6810.1, LNA-6810.2, LNA-6810.3, LNA-6812.1, LNA-6796. 1, LNA-6796.3, which have potent antiviral activity against SARS-Cov-2, all with approximately 1 log10 or greater inhibition of viral replication. These LNAs are listed in Table 8 above.

Example 6:
Determining the In Vitro Antiviral Effects of LNA Combinations Against Respiratory Viruses: Combining LNAs Targeting Conserved SARS-CoV-2 RNA Secondary Structures with LNAs Designed to Sequester miR-191 lead to profound inhibition of viral replication. Individual LNAs were combined to demonstrate anti-respiratory virus activity using the assay described in Example 5 above (Figure 21).

Example 7:
In vivo effects of LNA combinations against respiratory viruses. Human ACE2 transgenic mice were treated with a single intranasal administration of a combination of vehicle, small molecule A, or LNA 5 days prior to infection with a lethal inoculation of SARS-Cov-2. After infection, animals are monitored daily by clinical score, 1=asymptomatic, higher scores indicate worsening clinical status (FIG. 21). Combinations of LNAs inhibit respiratory virus infection in vivo

Example 8:
Antiviral activity of single microRNA-targeted LNAs or LNAs targeting either the minus or plus strand of respiratory viruses. Using the assays described in Example 5, individual LNAs were shown to have anti-respiratory virus activity (Figure 22). Other LNAs with anti-respiratory virus activity have been identified as well, including but not limited to: Neg3.1, Neg8.2, Neg8.4, Neg10.1, Neg10.1, Neg12.2, Neg18.2, Neg18.3, Neg18.4, Cov8.5, Cov10.1, Cov11.3, Cov12. 8, Cov13.9, Cov14.3, Cov16.3, Cov18.1, Cov1.1, Cov1.2, Cov1.4, Cov2.1, Cov2.2, Cov3.2, Cov3.2-2, Cov3. 2-3, Cov3.2-4, Cov3.2-5, Cov3.2-6, Cov3.2-7, Cov4.1, Cov4.2, Cov6.4, Cov6.5, Cov6.6, Cov6. 7, Cov6.8, Cov6.9, Cov6.7-1, Cov6.7-2, Cov6.8, Cov6.9, Cov6.7-1, Cov6.7-2, Cov7.2, Cov7.7, Cov7.9, Cov8.1, Cov8.2, Cov8.3, Cov8.4, Cov8.5, Cov8.6, Cov8.2-1, Cov10.1, Cov10.4, Cov11.1, Cov11.2, Cov11.3, Cov12.4, Cov12.5, Cov12.7, Cov12.8, Cov13.1, Cov13.4, Cov13.5, Cov13.6, Cov13.8, Cov13.9, Cov14.1, Cov14. 2, Cov14.3, Cov14.4, Cov16.1, Cov16.2, Cov16.3, Cov18.1, Cov18.5, IBV-LNA0.2, IBV-LNA4.4, IBV-LNA3.4, IBV- LNA2.5, IBV-LNA1.5, which have potent antiviral activity, all with about 1 log10 or greater inhibition of viral replication. These LNAs are listed in Tables 7 and 9 above.

Example 9:
LNAs that inhibit respiratory viruses can be used as just-in-time vaccines, as shown in FIG. (a-b) Effect of intranasal LNA prophylaxis on survival of virus-infected mice. Kaplan-Meier survival plot. Mice (n=7 mice/group) were intranasally administered a single dose of LNA9, scrambled LNA, or vehicle (mock treatment), followed by lethal inoculation with wild-type PR8 virus, (a a) dosed with 20 μg LNA 3 days prior to infection (day −3) or 1 day prior to infection (day −1); and (b) treated with a single dose of 30 μg LNA9 or vehicle control 1 week prior. rice field. (c) LNA9 and newly designed LNA14 target sites mapped to the PSL2 structure. (d) Electrophoretic profiles of SHAPE analysis performed on untreated, scrambled LNA, LNA9, and LNA14 at a concentration of 100 nM in the presence of PR8 PB2 vRNA. Labeling with 1M7 SHAPE reagent is shown. (e) Kaplan-Meier survival plot of mice (n=7 mice/group) pretreated intranasally with a single dose of 30 μg LNA14 or vehicle control one week prior to lethal PR8 infection (day −7). . (fh) A single dose of 40 μg LNA14 or vehicle was administered I.V. two weeks prior to PR8 virus infection (day -14). N. dosed. (f) Kaplan-Meier survival plot. (g) Percent body weight of mice relative to day 0. (h) clinical score; (il) Mice (n=7) received a single intranasal dose of 40 μg LNA14 one week prior to 1 LD100 primary lethal PR8 virus infection (e). Sixty-five days after the first infection, surviving mice from (e) were challenged a second time with 10 LD100 along with age-matched naive controls (n=7/group). (i) Load study timeline. (j) Percent body weight of mice relative to day 0. (k) clinical score; (l) Kaplan-Meier survival curve. (m) Mice (n=10/group) were infected with a lethal dose of PR8 wild-type virus. Three days after infection, mice received a single dose of 40 μg of LNA14, LNA9, scrambled LNA, or vehicle control by intravenous injection. Kaplan-Meier survival plot.

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

Accordingly, the foregoing merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements that embody the principles of the invention and that are within its spirit and scope, although not expressly described or shown herein. deaf. Moreover, all examples and conditional language provided herein are primarily intended to assist the reader in understanding the principles of the invention and the concepts that the inventors contribute to advancing the art. is intended and should be construed as not limited to such specifically described examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents include both now known equivalents and future developed equivalents, regardless of structure, i.e., any element developed to perform the same function regardless of structure. is intended. Accordingly, the scope of the invention is not intended to be limited to the embodiments shown and described herein. Rather, the scope and spirit of this invention is embodied by the accompanying embodiments.

Notwithstanding the claims appended hereto, the disclosure set forth herein is also described by the following paragraphs.
Section 1. An oligonucleotide compound comprising an oligonucleotide sequence complementary to a PB2 vRNA region, wherein the region comprises nucleotides 34-87 in the (−)sense notation of the 5′ end coding region of the PB2 vRNA, or a salt thereof Nucleotide compound.
Section 2. 2. The compound of paragraph 1, comprising an oligonucleotide sequence comprising at least eight nucleoside subunits complementary to a region of PB2 vRNA.
Item 3. 3. The compound of paragraph 1 or 2, wherein the oligonucleotide is complementary to a region of the packaging stem loop 2 (PSL2) motif of a region of PB2 vRNA.
Section 4. 4. The compound of any one of clauses 1-3, wherein the oligonucleotide comprises internucleoside linkages selected from phosphorothioate, phosphorodithioate, phosphoramidate, and thiophosphoramidate linkages.
Item 5. 5. The compound of any one of clauses 1-4, wherein all of the internucleoside linkages of the oligonucleotide are selected from phosphorothioate, phosphorodithioate, phosphoramidate, thiophosphoramidate, and phosphodiester linkages. .
Item 6. 6. The compound of paragraph 4 or 5, wherein the internucleoside linkages of the oligonucleotide are chiral.
Item 7. Clause 6. The compound of any one of clauses 1-5, wherein the oligonucleotide comprises bridging nucleic acid (BNA) nucleotides.
Item 8. 6. The compound of any one of clauses 1-5, wherein the oligonucleotide comprises locked nucleic acid (LNA) nucleotides.
Item 9. Clause 6. The compound of any one of clauses 1-5, wherein the oligonucleotide comprises ethylene-bridged nucleic acid (ENA) nucleotides.
Item 10. Clause 6. The compound of any one of clauses 1-5, wherein the oligonucleotide comprises constrained ethyl nucleic acid (cEt) nucleotides.
Item 11. Clause 6. The compound of any one of clauses 1-5, wherein the oligonucleotide comprises 2' modified nucleotides.
Item 12. the oligonucleotide
5'ACCAAAAGAAT3' (SEQ ID NO: 45),
5'TGGCCATCAAT3' (SEQ ID NO: 46),
5'TAGCATACTTA3' (SEQ ID NO: 47),
5'CCAAAGA3' (SEQ ID NO: 48),
5'CATACTTA3' (SEQ ID NO: 49),
5′CAGACACGACCAAAA3′ (SEQ ID NO: 50),
5'TACTTACTGACAGCC3' (SEQ ID NO: 51),
5′AGACACGACCAAAG3′ (SEQ ID NO: 52),
5'ACCAAAAGAAT3' (SEQ ID NO: 53),
5'TGGCCATCAAT3' (SEQ ID NO: 54),
5'TAGCATACTTA3' (SEQ ID NO: 55),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 56),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 57),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 58),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 59),
5'TCTAGCATACTTACT3' (SEQ ID NO: 60),
5'TCTAGCATACTTACT3' (SEQ ID NO: 61),
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′GGCCATCAATTAGTG3′ (SEQ ID NO: 63),
5′TTCGGATGGCCATCA3′ (SEQ ID NO: 64),
5′AGCCAGACAGCGA3′ (SEQ ID NO: 65),
5′GACAGCCAGACAGCA3′ (SEQ ID NO: 66),
5'CGACCAAAAGAATT3' (SEQ ID NO: 98),
5'GACCAAAAGAATTCGG3' (SEQ ID NO: 99),
5′AGCATACTTACTGACA3′ (SEQ ID NO: 100),
5'CATACTTACTGACA3' (SEQ ID NO: 101),
5'ATACTTACTGACAG3' (SEQ ID NO: 102),
5'CATACTTACTGACAGC3' (SEQ ID NO: 103),
5′AGACAGCGACCAAAAG3′ (SEQ ID NO: 104),
5′ACAGCGACCAAAAG (SEQ ID NO: 105),
5′CAGCCAGACAGCGAC3′ (SEQ ID NO: 106),
5′CAGCCAGACAGCGA3′ (SEQ ID NO: 107),
5′ACAGCCAGACAGCGGA3′ (SEQ ID NO: 108),
5′GACAGCCAGACAGCG3′ (SEQ ID NO: 109),
5'CATCAATTAGTGTCG3' (SEQ ID NO: 110),
5′CCATCAATTAGTGTCG3′ (SEQ ID NO: 111),
5′GCCATCAATTAGTGTG3′ (SEQ ID NO: 112),
5′AAGAATTCGGATGGC3′ (SEQ ID NO: 113),
5'CAGACAGCGACCAA3' (SEQ ID NO: 114),
12. The compound of any one of clauses 1-11, comprising a sequence selected from 5'TGACAGCCAGACAGC3' (SEQ ID NO: 115).
Item 13. the oligonucleotide
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′AGCCAGACAGCGA3′ (SEQ ID NO: 65),
5'CGACCAAAAGAATT3' (SEQ ID NO: 98),
5'GACCAAAAGAATTCGG3' (SEQ ID NO: 99),
5′AGCATACTTACTGACA3′ (SEQ ID NO: 100),
5'CATACTTACTGACA3' (SEQ ID NO: 101),
5'ATACTTACTGACAG3' (SEQ ID NO: 102),
5'CATACTTACTGACAGC3' (SEQ ID NO: 103),
5′AGACAGCGACCAAAAG3′ (SEQ ID NO: 104),
5′ACAGCGACCAAAAG (SEQ ID NO: 105),
5′CAGCCAGACAGCGAC3′ (SEQ ID NO: 106),
5′CAGCCAGACAGCGA3′ (SEQ ID NO: 107),
5′ACAGCCAGACAGCGGA3′ (SEQ ID NO: 108),
5′GACAGCCAGACAGCG3′ (SEQ ID NO: 109),
5'CATCAATTAGTGTCG3' (SEQ ID NO: 110),
5′CCATCAATTAGTGTCG3′ (SEQ ID NO: 111),
5′GCCATCAATTAGTGTG3′ (SEQ ID NO: 112),
5′AAGAATTCGGATGGC3′ (SEQ ID NO: 113),
13. The compound of paragraph 12, comprising a sequence selected from 5'CAGACAGCGACCAA3' (SEQ ID NO: 114), and 5'TGACAGCCAGACAGC3' (SEQ ID NO: 115).
Item 14. 13. The compound of paragraph 12, comprising an oligonucleotide having at least 70% sequence identity with a sequence selected from (SEQ ID NOs:45-66), and (SEQ ID NOs:98-115).
Item 15. 15. The compound of paragraph 14, comprising an oligonucleotide having at least 80% sequence identity with a sequence selected from (SEQ ID NOs:45-66), and (SEQ ID NOs:98-115).
Item 16. 16. The compound of paragraph 15, comprising an oligonucleotide having at least 90% sequence identity with a sequence selected from (SEQ ID NOs:45-66), and (SEQ ID NOs:98-115).
Item 17. 13. The compound of Paragraph 12, wherein one or more of the nucleotides of the oligonucleotide are modified nucleotides (eg, as described herein).
Item 18. 13. The compound of Paragraph 12, wherein one or more of the nucleotides of the oligonucleotide are bridging nucleic acid (BNA) nucleotides.
Item 19. 13. The compound of Paragraph 12, wherein all nucleotides of the oligonucleotide are Locked Nucleic Acid (LNA) nucleotides.
Item 20. 13. The compound of Paragraph 12, wherein one or more of the nucleotides of the oligonucleotide are ethylene-bridged nucleic acid (ENA) nucleotides.
Item 21. 13. The compound of Paragraph 12, wherein one or more of the nucleotides of the oligonucleotide are constrained ethyl nucleic acid (cEt) nucleotides.
Item 22. 13. The compound of Paragraph 12, wherein one or more of the nucleotides of the oligonucleotide comprise 2' modified nucleotides.
Item 23. the oligonucleotide
LNA1: 5'AaaAGaaT3' (SEQ ID NO: 67),
LNA2: 5'TggCcATcaaT3' (SEQ ID NO: 68),
LNA3: 5'TagCAtActtA3' (SEQ ID NO: 69),
LNA4: 5'CCAAAGA3' (SEQ ID NO: 70),
LNA5: 5'CATACTTA3' (SEQ ID NO: 71),
LNA6: 5'CagaCaCGaCCaaAA3' (SEQ ID NO: 72),
LNA7: 5'TAcTtaCTgaCagCC3' (SEQ ID NO: 73),
LNA8: 5'AGACacgaccaAAAG3' (SEQ ID NO: 74),
LNA9: 5'TACTtactgacaGCC3' (SEQ ID NO: 75),
LNA9.2: 5'TACttactgacAGCC3' (SEQ ID NO: 76),
LNA10: 5'ACCaaaagAAT3' (SEQ ID NO: 77),
LNA11: 5'TGGccatcAAT3' (SEQ ID NO: 78),
LNA12: 5'TAGcatacTTA3' (SEQ ID NO: 79),
LNA13: 5'CgacCAaaAGaattC3' (SEQ ID NO: 80),
LNA14: 5'CGACcaaaagaATTC3' (SEQ ID NO: 81),
LNA15: 5′GaTGgCcATcaAttA3′ (SEQ ID NO: 82),
LNA16: 5'GATGgccatcaATTA3' (SEQ ID NO: 83),
LNA17: 5'TcTAgCaTActTacT3' (SEQ ID NO: 84),
LNA18: 5'TCTAgcatactTACT3' (SEQ ID NO: 85),
LNA19: 5′GAAttcggatgGCCA3′ (SEQ ID NO: 86),
LNA20: 5'GGCCatcaattaGTG3' (SEQ ID NO: 87),
LNA21: 5'TTCGgatggccaTCA3' (SEQ ID NO: 88),
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO: 89),
LNA23: 5'GACAgccagacaGCA3' (SEQ ID NO: 90),
LNA9. G74C: 5'TACTtactgacaGTC3' (SEQ ID NO: 91), and LNA9. T80C: comprising a sequence selected from 5'TACTtaccgacaGCC3' (SEQ ID NO: 92);
13. The compound of Paragraph 12, wherein capital letters indicate LNA nucleotides and lower case letters indicate DNA nucleotides.
Item 24. the oligonucleotide
LNA19: 5′GAAttcggatgGCCA3′ (SEQ ID NO: 86),
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO: 89),
LNA22.2: 5'CAGCcagacagCGAC3' (SEQ ID NO: 116),
LNA22.3: 5'CAGccagacagCGAC3' (SEQ ID NO: 117),
LNA22.5: 5'CAGccagacaGCGA3' (SEQ ID NO: 118),
LNA22.6: 5'CAGccagacagCGA3' (SEQ ID NO: 119),
LNA22.7: 5'CAGCcagacagCGA3' (SEQ ID NO: 120),
LNA22.8: 5'ACAgccagacagCGA3' (SEQ ID NO: 121),
LNA22.9: 5'ACAGccagacaGCGA3' (SEQ ID NO: 122),
LNA22.10: 5'ACAgccagacaGCGA3' (SEQ ID NO: 123),
LNA22.11: 5'GACAgccagacaGCG3' (SEQ ID NO: 124),
13. The compound of paragraph 12, comprising a sequence selected from LNA22.13: 5'GACagccagacaGCG3' (SEQ ID NO: 125), and LNA22.14: 5'GACAgccagacAGCG (SEQ ID NO: 126).
Item 25. the oligonucleotide
LNA24: 5'CATcaattagtgTCG3' (SEQ ID NO: 127),
LNA25: 5'CCAtcaattagTCG3' (SEQ ID NO: 128),
LNA26: 5′GCCatcaattagtGTG3′ (SEQ ID NO: 129),
LNA27: 5'AAGAattcggaTGGC3' (SEQ ID NO: 130),
13. The compound of paragraph 12, comprising a sequence selected from LNA28: 5'CAGacagcgacCAA3' (SEQ ID NO: 131), and LNA29: 5'TGAcagccagacAGC3' (SEQ ID NO: 132).
Item 26. the oligonucleotide
LNA14: 5'CGACcaaaagaATTC3' (SEQ ID NO: 81),
LNA14.5: 5'CGACcaaaagaATT3' (SEQ ID NO: 135),
LNA14.8: 5'CGACcaaaagaaTTC3' (SEQ ID NO: 137),
LNA14.28: 5′GACcaaagaatTCGG3′ (SEQ ID NO: 148),
LNA14.30: 5′GACCaaaagaattCGG3′ (SEQ ID NO: 149),
LNA9: 5'TACTtactgacaGCC3' (SEQ ID NO: 75),
LNA9.1: 5'AGCAtacttactGACA3' (SEQ ID NO: 159),
LNA9.2a: 5'CATacttactgACA3' (SEQ ID NO: 160),
LNA9.8: 5'ATActactgACAG (SEQ ID NO: 164),
LNA9.12: 5′CATActtactgacAGC (SEQ ID NO: 167),
LNA8a: 5′AGAcagcgaccaaAAG (SEQ ID NO: 188),
LNA8a. 1: 5'AGACagcgaccaAAAG (SEQ ID NO: 189), and LNA8a. 13. The compound of paragraph 12, comprising a sequence selected from 2:5'ACAGcgaccaAAAG (SEQ ID NO: 190).
Item 27. 27. The compound of any one of paragraphs 23-26, comprising an oligonucleotide having at least 70% sequence identity with a sequence selected from (SEQ ID NOs:67-92), and (SEQ ID NOs:116-191).
Item 28. 28. The compound of Paragraph 27, comprising an oligonucleotide having at least 80% sequence identity with a sequence selected from (SEQ ID NOs:67-92), and (SEQ ID NOs:116-191).
Item 29. 29. The compound of Paragraph 28, comprising an oligonucleotide having at least 90% sequence identity with a sequence selected from (SEQ ID NOs:67-92), and (SEQ ID NOs:116-191).
Item 30. 30. The compound of any one of clauses 1-29, wherein the oligonucleotide comprises at least 5 deoxyribonucleotide units and is capable of recruiting an RNase.
Item 31. Clause 31. The compound of any one of clauses 1-30, wherein binding of the compound to a region of PB2 vRNA disrupts the overall secondary RNA structure of PB2 vRNA.
Item 32. Clause 31. The compound of any one of Clauses 1-30, wherein binding of the compound to a region of PB2 vRNA inhibits the packaging ability of PB2 vRNA.
Item 33. Clause 33. The compound of any one of clauses 1-32, wherein the compound is an oligonucleotide conjugate with enhanced cellular uptake.
Item 34. 34. The compound of Paragraph 33, wherein the compound is an oligonucleotide-lipid conjugate.
Item 35. 34. The compound of any one of clauses 1-33, wherein the compound is an oligonucleotide conjugate with a cell-specific protein.
Item 36. A method of inhibiting influenza A virus in a cell 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 influenza A virus. method, including
Item 37. 37. The method of Paragraph 36, wherein the agent is an oligonucleotide compound or salt thereof comprising at least eight nucleoside subunits complementary to the PSL2 motif of vRNA.
Item 38. 38. The method of paragraphs 36 or 37, wherein the agent is an oligonucleotide compound of any one of paragraphs 1-35.
Item 39. 39. The method of any one of paragraphs 36-38, wherein the vRNA in the sample is PB2 vRNA.
Item 40. 40. The method of any one of paragraphs 36-39, wherein contacting the sample with the agent results in a loss of at least 1 log ₁₀ titer of the virus.
Item 41. 40. The method of any one of paragraphs 36-39, wherein contacting the sample with the agent results in a titer loss of at least 2 log ₁₀ of the virus.
Item 42. 42. The method of any one of paragraphs 36-41, wherein the agent disrupts the overall structure of the PSL2 motif of vRNA.
Item 43. 42. The method of any one of paragraphs 36-41, wherein the agent inhibits the packaging ability of the PSL2 motif of vRNA.
Item 44. 42. The method of any one of paragraphs 36-41, wherein the vRNA is isolated from virions or cells.
Item 45. 42. The method of any one of paragraphs 36-41, wherein the vRNA is contained in virions or infected cells.
Item 46. The method of any one of claims 36-45, wherein the sample is in vitro.
Item 47. 46. The method of any one of claims 36-41 or 45, wherein the sample is in vivo.
Item 48. A method of treating or preventing influenza A virus infection in a subject, comprising an effective amount of activity that specifically binds to the PSL2 motif of viral RNA (vRNA) in a subject in need of treatment or prevention of influenza A virus infection. A method comprising administering a pharmaceutical composition comprising the agent.
Item 49. 49. The method of Paragraph 48, wherein the vRNA is PB2 vRNA.
Item 50. 50. The method of Paragraph 48 or 49, wherein the active agent is a compound comprising an oligonucleotide sequence comprising at least eight nucleoside subunits complementary to a region of PB2 vRNA.
Item 51. 51. The method of any one of paragraphs 48-50, wherein the agent is an oligonucleotide compound of any one of paragraphs 1-35.
Item 52. The subject is at risk for influenza A virus infection and administration of the oligonucleotide compound has infected the subject for 1 week or more (e.g., 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, etc.) 52. The method of any one of paragraphs 48-51, which protects against disease.
Clause 53. The method of Clause 52, wherein administering comprises administering a weekly, biweekly, or monthly effective dose of the oligonucleotide compound.
Item 54. 54. The method of any one of paragraphs 48-53, wherein administration results in a loss of at least 1 log ₁₀ titer of virus in the subject's sample.
Item 55. 54. The method of any one of paragraphs 48-53, wherein administration results in at least a 2 log ₁₀ titer loss of virus in the subject's sample.
Item 56. 54. The method of any one of paragraphs 48-53, wherein the active agent is an oligonucleotide conjugate with enhanced cellular uptake.
Item 57. 56. The method of any one of paragraphs 48-55, wherein the active agent is an oligonucleotide conjugate with a cell-specific protein.
Item 58. 56. The method of any one of paragraphs 48-55, wherein the pharmaceutical composition comprises an enhancer of cellular uptake.
Item 59. 56. The method of any one of paragraphs 48-55, wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral agent.
Item 60. 56. The method of any one of Paragraphs 48-55, wherein the active agent is siRNA, shRNA, antisense RNA, or antisense DNA.
Item 61. 56. The method of any one of paragraphs 48-55, wherein the subject is at risk for influenza A virus infection and the method prevents infection.
Item 62. 56. The method of any one of paragraphs 48-55, wherein the subject is diagnosed or suspected of having an influenza A virus infection and the method treats the infection.
Item 63. A method for screening a candidate agent for the ability to inhibit influenza A virus in a cell, the method comprising:
contacting a sample containing viral RNA (vRNA) containing a PSL2 motif with a candidate agent;
determining whether the candidate agent specifically binds to the PSL2 motif;
A method, wherein an agent that specifically binds to the PSL2 motif inhibits influenza A virus in the cell.
Item 64. 64. The method of Paragraph 63, wherein the candidate agent is selected from small molecules, nucleic acids, and polypeptides.
Item 65. Section 64, wherein the determining step comprises detecting a cellular parameter, wherein a change in the parameter in the cell compared to cells not contacted with the candidate agent indicates that the candidate agent specifically binds to the PSL2 motif, paragraph 64 The method described in .
Item 66. 66. The method of any one of paragraphs 63-65, wherein the agent that specifically binds to the PSL2 motif treats a subject with influenza A virus infection.
Item 67. A method of treating or preventing a respiratory viral infection in a subject, comprising:
A subject in need of treatment or prevention of a respiratory viral infection is provided with a pharmaceutical composition comprising an effective amount of an active agent that specifically binds to the target motif of the subject's viral RNA (vRNA) or miRNA associated with the target motif. A method comprising administering.
Item 68. 68. The method of Paragraph 67, wherein the agent is an oligonucleotide compound by comprising a sequence selected from the group consisting of SEQ ID NOs:45-907.
Item 69. 69. The method of Paragraph 68, wherein the method comprises administered two or more sequences selected from the group consisting of SEQ ID NOs:45-907.
Clause 70. The method of Clause 69, wherein the two or more sequences are directed to different respiratory viruses.
Item 71. The subject is at risk for respiratory viral infection and administration of the oligonucleotide compound has infected the subject for ≥1 week (e.g., ≥2 weeks, ≥3 weeks, ≥1 month, ≥2 months, ≥3 months, etc.) 71. The method of any one of paragraphs 67-70, which protects against disease.
Clause 72. The method of Clause 71, wherein administering comprises administering an effective weekly, biweekly, or monthly effective dose of the oligonucleotide compound.
Item 73. 73. The method of any one of paragraphs 67-72, wherein the administration results in a loss of at least 1 log ₁₀ titer of virus in the subject's sample.
Item 74. 74. The method of any one of paragraphs 67-73, wherein the subject is at risk for respiratory viral infection and the method prevents infection.
Item 75. 74. The method of any one of paragraphs 67-73, wherein the subject is diagnosed or suspected of having a respiratory viral infection and the method treats the infection.

In at least some of the foregoing embodiments, one or more elements used in one embodiment may be used interchangeably in another embodiment unless such replacement is technically feasible. It should be appreciated by those skilled in the art that various other omissions, additions, and modifications can be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter defined by the appended claims.

Generally, the terms used herein, particularly in the appended claims (e.g., the body of the appended claims), are generally intended as "open" terms (e.g., "including ' shall be construed as 'including but not limited to', the term 'having' shall be construed as 'having at least', and the term 'including' shall be construed as 'including should be construed as "but not limited to", etc.) will be understood by those skilled in the art. It is understood by those skilled in the art that where a particular number of introduced claim statements is intended, such intent is expressly recited in the claims and that such intent does not exist in the absence of such statement. will be further understood. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases "at least one" and "one or more" to introduce claim recitations. However, the use of such phrases is such that the introduction of a claim recitation by the indefinite article "a" or "an" indicates that a particular claim containing such introduced claim recitation is not subject to such recitation. should not be construed as being meant to be limited to embodiments that include only one. The same claim includes the introductory phrases "one or more" or "at least one" and indefinite articles such as "a" or "an" (e.g., "a" and/or "an" means "at least the same applies to the use of definite articles used to introduce claim recitations. In addition, even though specific numerical statements in the claims that have been introduced are expressly recited, it should be understood by those skilled in the art that such statements should be construed to mean at least the recited number. will recognize (eg, a bare statement of "two statements" without other modifiers means at least two statements, or more than two statements). Further, where conventions similar to "at least one of A, B, and C, etc." are used, generally such configurations are intended in the sense that those skilled in the art will understand the conventions (e.g., " A system having at least one of A, B, and C" includes A only, B only, C only, both A and B, both A and C, both B and C, and/or A, B, and C, etc.). Where conventions similar to "at least one of A, B, or C, etc." are used, generally such configurations are intended in the sense that those skilled in the art will understand the conventions (e.g., "A, A system having at least one of B or C" includes A only, B only, C only, both A and B, both A and C, both B and C, and/or A, B, and C including, but not limited to, systems having both, etc.). Virtually any disjunctive and/or phrase that presents two or more alternative terms, whether in the specification, claims, or drawings, may refer to one of the terms, either term, or both. It will be further understood by those skilled in the art that the terms should be understood to contemplate the possibility of inclusion. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

Additionally, where features or aspects of the disclosure are described with respect to the Markush group, those skilled in the art will recognize that the disclosure is thereby also described with respect to any individual member or subgroup of members of the Markush group. Will.

For any and all purposes, including to provide a written description, as understood by a person of ordinary skill in the art, all ranges disclosed herein also encompass all and all possible subranges and combinations of subranges. do. Any recited range sufficiently describes and allows for the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. can be easily recognized as making By way of non-limiting example, each range discussed herein can be readily broken down into lower thirds, middle thirds, upper thirds, and so on. Also, as understood by those of ordinary skill in the art, all terms such as "up to", "at least", "greater than", "less than" are inclusive of the stated number and sub-ranged thereafter as discussed above. It refers to the range that can be decomposed. Finally, as understood by those of skill in the art, the range includes each individual member. Thus, for example, a group having 1-3 items refers to groups having 1, 2, or 3 items. Similarly, a group having 1-5 items refers to groups having 1, 2, 3, 4, or 5 items, and so on.

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

Accordingly, the foregoing merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements that embody the principles of the invention and that are within its spirit and scope, although not expressly described or shown herein. deaf. Moreover, all examples and conditional language provided herein are primarily intended to assist the reader in understanding the principles of the invention and the concepts that the inventors contribute to advancing the art. is intended and should be construed as not limited to such specifically described examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention, as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. In addition, such equivalents include both now known equivalents and future developed equivalents, regardless of structure, i.e., any element developed to perform the same function regardless of structure. is intended. Moreover, nothing disclosed herein is intended to be dedicated to the public, regardless of whether such disclosure is explicitly recited in the claims.

Accordingly, the scope of the invention is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In a claim, 35 U.S.C. 112(f) or 35 U.S.C. 112(6) preface such limitations in a claim with the precise words "means for" or "of is expressly defined as being enforced for a limitation in a claim only if the precise phrase "a step for" is stated, and such precise phrase is not used in the claim limitation. 35 U.S.C. 112(f) or 35 U.S.C. 112(6) will not be enforced.

Claims (20)

an oligonucleotide compound or salt thereof comprising an oligonucleotide sequence comprising at least eight nucleoside subunits complementary to a region of the packaging stem loop 2 (PSL2) motif of PB2 viral RNA (vRNA) or variants thereof; An oligonucleotide compound or a salt thereof, wherein said oligonucleotide compound inhibits virus production. 2. The compound of claim 1, wherein said oligonucleotide comprises internucleoside linkages selected from phosphorothioate, phosphorodithioate, phosphoramidate, and thiophosphoramidate linkages. 3. The compound of claim 2, wherein said oligonucleotide contains one or more chiral internucleoside linkages. 2. The compound of claim 1, wherein said oligonucleotide comprises bridging nucleic acid (BNA) nucleotides. 5. The compound of Claim 4, wherein said BNA nucleotides are selected from the group consisting of locked nucleic acid (LNA) nucleotides, ethylene bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides. 2. The compound of Claim 1, wherein said oligonucleotide comprises one or more 2' modified nucleotides. The oligonucleotide is
5'ACCAAAAGAAT3' (SEQ ID NO: 45),
5'TGGCCATCAAT3' (SEQ ID NO: 46),
5'TAGCATACTTA3' (SEQ ID NO: 47),
5'CCAAAGA3' (SEQ ID NO: 48),
5'CATACTTA3' (SEQ ID NO: 49),
5′CAGACACGACCAAAA3′ (SEQ ID NO: 50),
5'TACTTACTGACAGCC3' (SEQ ID NO: 51),
5′AGACACGACCAAAG3′ (SEQ ID NO: 52),
5'ACCAAAAGAAT3' (SEQ ID NO: 53),
5'TGGCCATCAAT3' (SEQ ID NO: 54),
5'TAGCATACTTA3' (SEQ ID NO: 55),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 56),
5′CGACCAAAAGAATTC3′ (SEQ ID NO: 57),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 58),
5′GATGGCCATCAATTA3′ (SEQ ID NO: 59),
5'TCTAGCATACTTACT3' (SEQ ID NO: 60),
5'TCTAGCATACTTACT3' (SEQ ID NO: 61),
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′GGCCATCAATTAGTG3′ (SEQ ID NO: 63),
5′TTCGGATGGCCATCA3′ (SEQ ID NO: 64),
5′AGCCAGACAGCGA3′ (SEQ ID NO: 65),
5′GACAGCCAGACAGCA3′ (SEQ ID NO: 66),
5'CGACCAAAAGAATT3' (SEQ ID NO: 98),
5'GACCAAAAGAATTCGG3' (SEQ ID NO: 99),
5′AGCATACTTACTGACA3′ (SEQ ID NO: 100),
5'CATACTTACTGACA3' (SEQ ID NO: 101),
5'ATACTTACTGACAG3' (SEQ ID NO: 102),
5'CATACTTACTGACAGC3' (SEQ ID NO: 103),
5′AGACAGCGACCAAAAG3′ (SEQ ID NO: 104),
5′ACAGCGACCAAAAG (SEQ ID NO: 105),
5′CAGCCAGACAGCGAC3′ (SEQ ID NO: 106),
5′CAGCCAGACAGCGA3′ (SEQ ID NO: 107),
5′ACAGCCAGACAGCGGA3′ (SEQ ID NO: 108),
5′GACAGCCAGACAGCG3′ (SEQ ID NO: 109),
5'CATCAATTAGTGTCG3' (SEQ ID NO: 110),
5′CCATCAATTAGTGTCG3′ (SEQ ID NO: 111),
5′GCCATCAATTAGTGTG3′ (SEQ ID NO: 112),
5′AAGAATTCGGATGGC3′ (SEQ ID NO: 113),
5′CAGACAGCGACCAA3′ (SEQ ID NO:114), and 5′TGACAGCCAGACAGC3′ (SEQ ID NO:115);
2. The compound of claim 1, wherein said oligonucleotide comprises one or more modified nucleic acids (eg, BNA, LNA, ENA, cEt, or 2' modifications). The oligonucleotide is
5′GAATTCGGATGGCCA3′ (SEQ ID NO: 62),
5′AGCCAGACAGCGA3′ (SEQ ID NO: 65),
5'CGACCAAAAGAATT3' (SEQ ID NO: 98),
5'GACCAAAAGAATTCGG3' (SEQ ID NO: 99),
5′AGCATACTTACTGACA3′ (SEQ ID NO: 100),
5'CATACTTACTGACA3' (SEQ ID NO: 101),
5'ATACTTACTGACAG3' (SEQ ID NO: 102),
5'CATACTTACTGACAGC3' (SEQ ID NO: 103),
5′AGACAGCGACCAAAAG3′ (SEQ ID NO: 104),
5′ACAGCGACCAAAAG (SEQ ID NO: 105),
5′CAGCCAGACAGCGAC3′ (SEQ ID NO: 106),
5′CAGCCAGACAGCGA3′ (SEQ ID NO: 107),
5′ACAGCCAGACAGCGGA3′ (SEQ ID NO: 108),
5′GACAGCCAGACAGCG3′ (SEQ ID NO: 109),
5′CATCAATTAGTGTCG3′ (SEQ ID NO: 110),
5′CCATCAATTAGTGTCG3′ (SEQ ID NO: 111),
5′GCCATCAATTAGTGTG3′ (SEQ ID NO: 112),
5′AAGAATTCGGATGGC3′ (SEQ ID NO: 113),
2. The compound of claim 1, comprising a sequence selected from 5'CAGACAGCGACCAA3' (SEQ ID NO: 114), and 5'TGACAGCCAGACAGC3' (SEQ ID NO: 115). 2. The compound of claim 1, comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from (SEQ ID NOs:45-66) and (SEQ ID NOs:98-115). 9. The compound of claim 8, wherein said oligonucleotide comprises at least 5 deoxyribonucleotide units and is capable of recruiting RNase. The oligonucleotide is
LNA19: 5′GAAttcggatgGCCA3′ (SEQ ID NO: 86),
LNA22: 5'AGCCagacagCGA3' (SEQ ID NO: 89),
LNA22.2: 5'CAGCcagacagCGAC3' (SEQ ID NO: 116),
LNA22.3: 5'CAGccagacagCGAC3' (SEQ ID NO: 117),
LNA22.5: 5'CAGccagacaGCGA3' (SEQ ID NO: 118),
LNA22.6: 5'CAGccagacagCGA3' (SEQ ID NO: 119),
LNA22.7: 5'CAGCcagacagCGA3' (SEQ ID NO: 120),
LNA22.8: 5'ACAgccagacagCGA3' (SEQ ID NO: 121),
LNA22.9: 5'ACAGccagacaGCGA3' (SEQ ID NO: 122),
LNA22.10: 5'ACAgccagacaGCGA3' (SEQ ID NO: 123),
LNA22.11: 5'GACAgccagacaGCG3' (SEQ ID NO: 124),
LNA22.13: 5'GACagccagacaGCG3' (SEQ ID NO: 125),
LNA22.14: 5′GACAgccagacAGCG (SEQ ID NO: 126),
LNA24: 5'CATcaattagtgTCG3' (SEQ ID NO: 127),
LNA25: 5'CCAtcaattagTCG3' (SEQ ID NO: 128),
LNA26: 5′GCCatcaattagtGTG3′ (SEQ ID NO: 129),
LNA27: 5'AAGAattcggaTGGC3' (SEQ ID NO: 130),
LNA28: 5'CAGacagcgacCAA3' (SEQ ID NO: 131),
LNA29: 5'TGAcagccagacAGC3' (SEQ ID NO: 132),
LNA14.5: 5'CGACcaaaagaATT3' (SEQ ID NO: 135),
LNA14.8: 5'CGACcaaaagaaTTC3' (SEQ ID NO: 137),
LNA14.28: 5′GACcaaagaatTCGG3′ (SEQ ID NO: 148),
LNA14.30: 5′GACCaaaagaattCGG3′ (SEQ ID NO: 149),
LNA9.1: 5'AGCAtacttactGACA3' (SEQ ID NO: 159),
LNA9.2a: 5'CATacttactgACA3' (SEQ ID NO: 160),
LNA9.8: 5'ATActactgACAG (SEQ ID NO: 164),
LNA9.12: 5′CATActtactgacAGC (SEQ ID NO: 167),
LNA8a: 5′AGAcagcgaccaaAAG (SEQ ID NO: 188),
LNA8a. 1: 5'AGACagcgaccaAAAG (SEQ ID NO: 189), and LNA8a. 2: comprising a sequence selected from 5'ACAGcgaccaAAAG (SEQ ID NO: 190);
8. The compound of claim 7, wherein upper case letters indicate LNA nucleotides and lower case letters indicate DNA nucleotides. 2. The compound of claim 1, comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from LNA1-LNA29 (SEQ ID NOs:67-92 and SEQ ID NOs:116-191). A method of inhibiting influenza A virus in a cell comprising:
11. A method comprising contacting a sample containing viral RNA (vRNA) having a PSL2 motif with an effective amount of the oligonucleotide compound of claim 1. 14. The method of claim 13, wherein contacting the sample with an agent results in a loss of at least 1 log ₁₀ titer of the virus, or wherein the agent disrupts the overall structure of the PSL2 motif of the vRNA. . 14. The method of claim 13, wherein said vRNA is isolated from virions or cells. A method of treating or preventing influenza A virus infection in a subject, comprising:
11. A method comprising administering to a subject in need of treatment or prevention of influenza A virus infection an effective amount of a pharmaceutical composition comprising the oligonucleotide compound of claim 1. 17. The method of claim 16, wherein said subject is at risk for influenza A virus infection and said administration of said oligonucleotide compound protects said subject from infection for more than one week. 18. The method of claim 17, wherein said administration comprises administration of an effective weekly, biweekly, or monthly effective dose of said oligonucleotide compound. 17. The method of Claim 16, wherein said pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral agent. 17. The method of claim 16, wherein the subject has been diagnosed or suspected of having influenza A virus infection.

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