WO2021188931A1 - Anti-coronaviral compositions and methods of using the same - Google Patents

Abstract

Methods of inhibiting a coronavirus (CoV) in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a conserved RNA secondary structure of CoV with an effective amount of an agent that specifically binds to the secondary structure to inhibit the CoV. Also provided are methods of treating or preventing CoV infection in a subject. Methods of treating or preventing a neoplastic condition in a subject are also provided. The neoplastic condition can include lung cancer. Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a secondary structure of a CoV that find use in the subject methods.

Description

ANTI-CORONA VIRAL COMPOSITIONS AND METHODS OF

USING THE SAME

CROSS-REFERENCE

Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 62/992,659 filed March 20, 2020, the disclosure of which application is incorporated herein by reference in its entirety. This application also claims priority to the filing date of PCT Application Serial No. PCT/US2021/018025 filed on February 12, 2021, the disclosure of which is herein incorporated by reference.

GOVERNMENT RIGHTS

This invention was made with Government support under contract AI132191 awarded by the National Institutes of Health. The Government has certain rights in the invention.

INTRODUCTION

Coronaviruses (CoV) are enveloped RNA viruses which typically cause self-limited respiratory tract infection in humans. However, in the past 2 decades, there have been life- threatening conditions caused by new strains of CoV such as those found in the 2003 severe respiratory syndrome (SARS) outbreak, Middle East Respiratory Syndrome (MERS) in 2012, and most recently the 2019 CoV outbreak originated from Wuhan, China (SARS-CoV-2). New therapies are critically needed to contain the current outbreak and also future outbreaks.

SUMMARY

Aspects of the present disclosure provide agents and compositions designed to disrupt an RNA secondary structure of a coronavirus (CoV). Disruption of an RNA secondary structure of a CoV can inhibit the CoV virus. Aspects of the disclosure also include agents and compositions for inhibiting a microRNA (miRNA) interaction with a CoV. Agents and compositions are also provided for the treatment of a neoplastic condition. The neoplastic condition can include lung cancer.

Methods of inhibiting a CoV in a sample are provided. Aspects of the methods include contacting a sample comprising viral RNA (vRNA) having a conserved RNA secondary structure of CoV with an effective amount of an agent that specifically binds to the secondary structure to inhibit the CoV. In some cases, the vRNA is isolated from a virion or a cell. In some cases, the vRNA is in a virion. In some cases, the vRNA is in an infected cell. Also provided are methods of treating or preventing CoV infection in a subject. Also provided are methods for screening a candidate agent for the ability to inhibit CoV in a cell, the method comprising: contacting a sample with a candidate agent; and determining whether the candidate agent specifically binds to secondary structure of vRNA.

Methods of treating or preventing a neoplastic condition in a subject are also provided. Cancers in the lungs include small cell lung cancer, non-small cell lung cancer, lymphoma, carcinoid, mesothelioma, and metastatic cancers to the lung. Cancers that can metastasize to the lungs can initially arise from any anatomic site in the body including but not limited to breast, colon, endometrial, cervix, testicle, liver ovarian, prostate, pancreatic, brain, thyroid, stomach, kidney, melanoma, urothelial tract, muscle.

Also provided are compounds and pharmaceutical compositions comprising an oligonucleotide sequence complementary to a secondary structure of CoV that find use in the subject methods.

BRIEF DESCRIPTION OF THE FIGURES

The skilled artisan will understand 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.

FIG. 1 illustrates predicted RNA secondary structures conserved across corona B viruses.

FIG. 2 illustrates caspase activity of exemplary agents.

FIG. 3 Antiviral efficacy of miRNA-directed LNAs designed to disrupt respiratory viral infection. Huh7 cells were pre-treated with 25 nM of miRNA-directed LNAs 12-24 hours prior to infection with 0.3 MOI of a fully-replicating, BSL3 SARS-CoV-2-nLuc reporter virus. 48 hr post-infection, luciferase signal was read. Results shown as log 10 luciferase signal. Samples performed in duplicate, N=2; controls in quadruplicate, N=4. Statistical analysis performed by GraphPad Prism software and calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test between samples and the Scrambled LNA (Scr. LNA) control mean. A positive control nucleoside analog, EIDD-2801 (EIDD), was included as positive control. Anti- miR-29 is LNA-191.12 and Anti-miR-57 is LNA-6810.2.

Fig. 4 Effect of LNA combination treatment on respiratory virus infection. Huh7 cells were pretreated with 25 nM of a combination of LNAs (12.5 nM each LNA = 25 nM total) 12-24 hours prior to infection with 0.3 MOI of a fully-replicating, BSL3 SARS-CoV-2-nLuc reporter virus. 48 hr post-infection, luciferase signal was read. Results shown as loglO luciferase signal. Samples performed in duplicate, N=2; controls in quadruplicate, N=4. Statistical analysis performed by GraphPad Prism software and calculated using an ordinary one-way ANOVA with Dunnet’s multiple comparisons test between samples and the DMSO control mean. A positive control nucleoside analog, EIDD-2801 (EIDD), was included as positive control. LNA-12.8 is Cov 12.8; LNA-8.5 is Cov 8.5; LNA-18.1 is Cov 18.1 and Anti-miR-29 is LNA-191.12.

FIG. 5 In vivo efficacy of combination of LNAs against a respiratory virus. Mice transgenic for human ACE2 were treated with vehicle, small molecule A, or a combination of LNAs administered intranasally once 5 days prior to infection with a lethal inoculum of SARS- Cov-2. Following infection, animals were monitored by clinical score daily where 1= asymptomatic and higher score indicates worsening clinical status. Vehicle: Glyceryltrioctanoate:octanoic acidNMP (80:10:10)

FIG. 6 Antiviral efficacy of CoV-2 LNAs designed to disrupt respiratory viral infection. ACE2-A549 cells were pre-treated with either 50 nM, 25 nM, or 5 nM of CoV-2 positive-strand targeted, CoV-2 negative-strand targeted, or miRNA-directed LNAs 12-24 hours prior to infection with 0.3 MOI of a fully-replicating, BSL3 SARS-CoV-2-nLuc reporter virus. 48 hr post-infection, luciferase signal was read. Results shown as log 10 luciferase signal. Samples performed in replicates of 6, N=6. Statistical analysis performed by GraphPad Prism software and calculated using an ordinary one-way ANOVA with Dunnett’s multiple comparisons test between samples and the Scrambled LNA (Scr. LNA) control mean. A positive control nucleoside analog, EIDD-2801 (EIDD), was included as positive control. 6.7 is Cov 6.7; 12.8 is Cov 12.8; 10.1 is Cov 10.1; 13.9 is Cov 13.9; 11.3 is Cov 11.3; 14.3 is Cov 14.3 and 18.1 is Cov 18.1.

DEFINITIONS

Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the 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.Y. (1991) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.

It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “a primer” refers to one or more primers, i.e., a single primer and multiple primers. It is further noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

As used herein, the term “effective amount” refers to that amount of a substance (e.g., an agent of interest) that produces some desired local or systemic effect. Effective amounts of active agents of interest 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 manner of administration and the like, and can readily be determined, e.g., determined empirically using data such as that data provided in the experimental section below.

The term “sample” as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid, i.e., aqueous, form, containing one or more components of interest. Samples may be derived from a variety of sources such as from a biological sample or solid, such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components). Components in a sample are termed “analytes” herein. In many embodiments, the sample is a complex sample containing at least about 10², 5x10², 10³, 5x10³, 10⁴, 5x10⁴, 10^s, 5x10^s, 10⁶, 5x10⁶, 10⁷, 5x10⁷, 10^s, 10⁹, 10¹⁰, 10¹¹, 10¹² or more species of analyte.

"Antibody fragments" comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et ah, Protein Eng. 8(10): 1057-1062 (1995)); single-chain antibody molecules; nanobodies, and multispecific and multifunctional antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. 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 a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term “fusion protein” or grammatical equivalents thereof is meant a protein composed of a plurality of polypeptide components, that while typically unjoined in their native state, typically are joined by their respective amino and carboxyl termini through a linkage, e.g., a peptide linkage, to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins. The term polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N- terminal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, b- galactosidase, luciferase, etc.; and the like. In addition, the term “protein” can further encompass the post-translational modification including but not limited to glycosylation, phosphorylation, methylation, and acetylation.

In general, polypeptides may 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 500 or 1000 or more amino acids. “Peptides” are 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 between 5 and 30 amino acids in length.

The term “specific binding” refers to the ability of an agent to preferentially bind to a particular target (e.g., an RNA secondary structure in the CoV vRNA or its negative sense strand) that is present in a homogeneous mixture of different analytes. In some cases, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, typically more than about 10 to 100-fold or more (e.g., more than about 1000-fold). Specific binding can include hybridization, polypeptide-nucleic acid interactions or small molecule -nucleic acid interactions.

“Oligonucleotide” refers to ribose and/or deoxyribose nucleoside subunit polymers having between about 2 and about 200 contiguous subunits. The nucleoside subunits can be joined by a variety of intersubunit linkages, including, but not limited to, phosphodiester, phosphotriester, an alkylphosphonate, e.g., methylphosphonate, P3'→N5' phosphoramidate, N3'→P5' phosphoramidate, N3'→P5' thiophosphoramidate, phosphorodiamidate, and phosphorothioate linkages. In certain cases, intersubunit linkage has a chiral atom. Representative chiral intersubunit linkages include, but are not limited to, alkylphosphonates, phosphorodiamidates and phosphorothioates. Further, “oligonucleotides” includes chemical and biochemical modifications, such as those known to one skilled in the art, e.g., to the sugar (e.g., 2' substitutions), the base (see the definition of “nucleoside” below), and/or the 3' and 5' termini. In embodiments where the oligonucleotide moiety includes a plurality of intersubunit linkages, each linkage may be formed using the same chemistry or a mixture of linkage chemistries may be used. In embodiments where the oligonucleotide moiety includes a plurality of intersubunit linkages, one or more of the linkages may be chiral. Linkages having a chiral atom 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 with gene, cDNA, DNA and RNA encoded by a gene.

A “bicyclic nucleic acid” or a “bridged nucleic acid” (BNA) refers to a modified RNA nucleotide where the ribose moiety is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon, thereby forming a bicyclic ring system. BNA monomers can contain a five-membered, six-membered or a seven-membered bridge structure with a fixed 3’-endo conformation. Bridged nucleic acids include without limitation, locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA) and constrained ethyl (cEt). A “bridge” refers to a chain of atoms or a valence bond connecting two bridgeheads, where a “bridgehead” is any skeletal atom of a ring system (e.g., the ribose ring system) which is bonded to three or more skeletal atoms (excluding hydrogen). In some embodiments, the bridge in a BNA has 7-12 ring members and 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Unless otherwise specified, a BNA is optionally substituted with one or more substituents, e.g., 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 where the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, thereby forming a bicyclic ring system. The bridge "locks" the ribose in the 3'-endo conformation, which is often found in the A-form duplexes. Locked nucleic acids are also encompassed by the term “bicyclic nucleic acids” or “bridged nucleic acids” (BNA). LNA nucleotides can be mixed with any convenient nucleotides or nucleotide analogs, such as DNA or RNA residues in an oligonucleotide whenever desired. LNA’s hybridize with DNA or RNA according to Watson-Crick base-pairing rules. Such oligomers can be synthesized chemically. In general, the locked ribose conformation enhances base stacking and backbone pre-organization to increase the hybridization properties (melting temperature) of the oligonucleotide. A locked nucleic acid, in some cases can be Alpha-l-locked nucleic acid (a-1-LNA), a stereoisomeric analogue of locked nucleic acid (LNA) with the inverted stereochemistry at C2', C3' and C4' positions.

An “ethylene-bridged nucleic acid” (ENA) refers to an LNA modified RNA nucleotide where the ribose moiety is modified with an extra bridge containing two carbon atoms between the 2' oxygen and the 4' carbon (see, e.g., Morita et al, Bioorganic Medicinal Chemistry, 2003,

11(10), 2211-2226). Ethylene-bridged nucleic acids are also encompassed by the term “bicyclic nucleic acids” or “bridged nucleic acids” (BNA).

A “constrained ethyl (cEt)” refers to an LNA modified RNA nucleotide where the ribose moiety is modified with an extra bridge connecting the 2' oxygen and 4' carbon, wherein the carbon atom of the bridge includes a methyl group. In some cases, the cEt is (S)-constrained ethyl. In other cases, the cEt is (R)-constrained ethyl (see, e.g., Pallan et al, Chem. Commun. (Camb)., 2012, 48(66), 8195-8197). Constrained ethyl nucleic acids are also encompassed by the term “bicyclic nucleic acids” or “bridged nucleic acids” (BNA).

As used herein, the term “2'-modified” or “2'-substituted” means a sugar comprising a substituent at the 2'-position other than H or OH. 2'-modified nucleotides, include moieties with 2' substituents selected from alkyl, allyl, amino, azido, fluoro, thio, O-alkyl, e.g., O-methyl, O- allyl, OCF₃, O-(CH₂)₂-O-CH₃ (e.g., 2'-0-methoxyethyl (MOE)), O-(CH₂)₂SCH₃, )-(CH₂)₂-ONR₂, and O-CH₂C(0)-NR₂, where each R is independently selected from H, alkyl, and substituted alkyl.

The disclosure encompasses isolated or substantially purified nucleic acid nucleic acid molecules and compositions containing those molecules. In the context of the present disclosure, an “isolated” or “purified” DNA molecule or RNA molecule is a DNA molecule or RNA molecule that exists apart from its native environment and is therefore not a product of nature.

An isolated DNA molecule or RNA molecule may exist in a purified form or may 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 substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (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. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. Fragments and variants of the disclosed nucleotide sequences are also encompassed by the present disclosure. By “fragment” or “portion” is meant a full length or less than full length of the nucleotide sequence. The siRNAs of the present disclosure can be generated by any method known to the art, for example, by in vitro transcription, recombinantly, or by synthetic means. In one example, the siRNAs can be generated in vitro by using a recombinant enzyme, such as T7 RNA polymerase, and DNA oligonucleotide templates.

A "small interfering” or “short interfering RNA" or siRNA is a RNA duplex of nucleotides that is targeted to a gene interest. A "RNA duplex" refers to the structure formed by the complementary pairing between two regions of a RNA molecule. siRNA is "targeted" to a gene in that the nucleotide sequence of the duplex portion of the siRNA is complementary to a nucleotide sequence of the targeted gene. In some embodiments, the length of the duplex of siRNAs is less than 30 nucleotides. In some embodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 nucleotides in length. In some embodiments, the length of the duplex is 19 - 25 nucleotides in length. 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 positioned between the two sequences that form the duplex. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12 or 13 nucleotides in length. The hairpin structure can also contain 3' or 5' overhang portions. In some embodiments, the overhang is a 3' or a 5' overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

The term “genome(s)” refers to the hereditary information of an individual typically encoded in nucleic acids, either single-stranded RNA, or double-stranded DNA, and including both genes and non-coding sequences.

The term “lipid” is used broadly herein to encompass substances that are soluble in organic solvents, but sparingly soluble, if at all, in water. The term lipid includes, but is not limited to, hydrocarbons, oils, fats (such as fatty acids, glycerides), sterols, steroids and derivative forms of these compounds. Preferred lipids are fatty acids and their deri vatives, hydrocarbons and their derivatives, and sterols, such as cholesterol. As used herein, the term lipid also includes amphipathic compounds which contain both lipid and hydrophilic moieties. Fatty acids usually contain even numbers of carbon atoms in a straight chain (commonly 12-24 carbons) and may be saturated or unsaturated, and can contain, or be modified to contain, a variety of substituent groups. For simplicity, the term “fatty acid” also encompasses fatty acid derivatives, such as fatty amides produced by the conjugation reactions, e.g., with a modified terminal of an oligonucleotide.

Other definitions of terms may appear throughout the specification. DETAILED DESCRIPTION

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

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 will be appreciated by those of skill 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 also be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

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

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

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

METHODS FOR INHIBITING A CORONA VIRUS (COV)

As summarized above, aspects of the present disclosure include agents and compositions designed to disrupt an RNA secondary structure of a coronavirus (CoV). Disruption of an RNA secondary structure of a CoV can inhibit the CoV virus. The agents and compositions are effective across a variety of different types of coronaviruses (CoV) where RNA secondary structures are 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 the anti-viral activity of a single moiety that is active against two or more different viruses, such as three or more, four or more, five or more, six or more, eight or more, 10 or more different viruses. The two or more different viruses may be selected from different virus sub-groups (e.g., human coronavirus 229E (HCoV-229E), human coronavirus OC43 (HCoV-OC43), SARS-CoV (the causative 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 (the coronavirus disease 2019).

Disruption of an RNA secondary structure of a CoV can dramatically inhibit the CoV. Figure 1 shows an example of the predicted RNA secondary structures conserved across corona B viruses that can be targeted in the subject methods. In some cases, the subject compositions have broad spectrum activity against Co Vs, such as activity against 1 or more Co Vs selected from HCoV-299E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, and SARS-CoV-2 or COVID-19. 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 a target CoV conserved RNA secondary structure, without induction of caspase or interferon.

Aspects of the present disclosure include methods for inhibiting a coronavirus (CoV) in a sample. In some embodiments, the method includes contacting a sample comprising viral RNA (vRNA) having a conserved RNA secondary structure of CoV with an effective amount of an agent that specifically binds the secondary structure to inhibit the CoV. In some embodiments, the method includes contacting a sample comprising viral RNA (vRNA), having a conserved RNA secondary structure of CoV, with an effective amount of an agent that inhibits a host microRNA (miRNA) interaction with the CoV. In some cases, the miRNA is microRNA 191 (miR191). In some cases, the miRNA can be one of the followings: miR663a, miR-381, miR-744, miR-4508, miR-4730, miR-6777, miR-10396, miR-4749, miR-6787, miR-4706, miR-6810, miR-3675, miR- 6812, miR-6796. In some cases, the 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 microRNA listed above with a predicted binding sites in the viral RNA. In some cases, the subject agent or composition is designed to sequester miR191 in cells transfected with a CoV. In some cases, the cells are transfected with a CoV 5’ terminal RNA segment linked to a luciferase report. In some cases, the CoV is SARS-CoV-2 or COVID-19. In some cases, the miRNA can directly bind to coronavirus viral RNA including SARS-Cov; SARS- Cov2; MERS in virions or in the infected cells. In some cases, the miRNA can regulate a host cell process that modulate the level of coronavirus viral RNA including SARS-Cov; SARS-Cov2; MERS viral RNA in virions or in the infected cells in other ways. In some cases, the conserved RNA secondary structure includes the presence of miR191 binding sites. In some cases, the subject agent or composition is designed to sequester miR191 in cells transfected with a CoV.

In some cases, the sample is in vitro. In certain cases, the sample is in vivo. The vRNA in the sample can be comprised in a virion. In some cases, the vRNA is comprised in a cell, such as a cell infected with the virus.

Aspects of the present disclosure include methods for inhibiting a coronavirus (CoV) in a cell. Aspects of the present disclosure include methods for inhibiting a coronavirus (CoV) in a human. In some embodiments, the method includes contacting a cell comprising viral RNA (vRNA) having a conserved RNA secondary structure of CoV with an effective amount of an agent that specifically binds the secondary structure to inhibit the CoV. In some cases, the cell is in vitro. In certain cases, the cell is in vivo. In some embodiments, the agent (e.g., as described herein) can bind to particular sites of the conserved RNA secondary structure motif to disrupt the overall structure of the vRNA thereby inhibiting the virus. In some cases, the agent inhibits the packaging ability of the vRNA, thereby inhibiting the vims.

In some embodiments, contacting the sample (e.g., cell) with an agent results in 1 logio or more titer deficits of the vims, such as 1.5 or more, 2 logio or more titer deficits of the virus, such as 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, 9 or more, 10 logio or even more titer deficits of the vims. In some embodiments, the agent is an oligonucleotide compound (e.g., as described herein) comprising a sequence complementary to a conserved RNA secondary stmcture motif of the CoV vRNA, or a salt thereof.

In some instances of the methods, binding of the oligonucleotide compound (e.g., one of the sequences included below) to the conserved RNA secondary stmcture region of vRNA dismpts the overall secondary RNA stmcture of the vRNA. In some cases, binding of the oligonucleotide compound inhibits the packaging ability of the vRNA, thereby inhibiting the CoV. In some cases, binding of the oligonucleotide compound inhibits translation of the vRNA into protein, thereby inhibiting the CoV. In some cases, binding of the oligonucleotide compound inhibits replication of CoV. In some cases, binding of the oligonucleotide compound inhibits the production of CoV virus particles, thereby inhibiting the CoV. In some cases, the subject compound targets at least part of the region in the (-)-sense notation of the 5’ terminal coding region of the conserved RNA secondary structure. In some cases, the subject compound targets a miRNA binding site at the 5’ terminal coding region of the conserved RNA secondary structure.

In some instances, the method further includes recruiting an RNase to degrade the vRNA.

Aspects of the present disclosure include a method of treating or preventing a coronavirus (CoV) 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 a conserved RNA secondary structure of a viral RNA (vRNA) (e.g., as described herein). As such, in some cases, the subject is one who has been infected with the vims. In certain cases, the subject is one who is at risk of being infected, or is suspected of being infected with the virus. In some cases, the CoV is selected from HCoV-299E, HCoV-OC43, SARS-CoV, HCoV-NL63, HKU1, MERS-CoV, and SARS-CoV-2 or COVID-19.

Any convenient protocol for administering the agent to a subject may be employed. The particular protocol that is employed may vary, e.g., depending on the site of administration and whether the agents are e.g., oligonucleotides, antibodies, proteins, peptides or small molecules.

For in vivo protocols, any convenient administration protocol may be employed. Depending upon the identity and binding affinity of the agent, the response desired, the manner of administration, e.g. locally or systemic, intraocular, periocular, retrobalbar, intramuscular, intravenous, intraperitoneal, subcutaneous, subconjunctival, intranasal, topical, eye drops, i.v. s.c., i.p., oral, and the like, the half-life, the number of cells or size of the graft bed or transplanted tissue, various protocols may be employed. In some cases, the agent is administered nasally. In some cases, the agent is administered as an aerosol. In certain cases, the agent is administered by a nebulizer. In certain cases, the agent is administered via the assistance of breathing-assisting devices including but not limited to non-invasive positive pressure ventilation or mechanical ventilation. In certain cases, the agent is administered intravenously. In certain other cases, the agent is administered subcutaneously. In certain cases, the agent is administered by intramuscular injection. In certain cases, the agent is administered in a deproteinized pollen formulation (see, e.g., Glenn et al, PCT Application Serial No. PCT/US2021/021855, “Pulmonary Agent Delivery Methods and Compositions for Practicing the Same”, the disclosure of which is incorporated herein by reference).

In some cases, the oligonucleotides can inhibit the growth cancer cells. In some cases, the oligonucleotides can inhibit the growth of cancers that are present in the lungs. In some cases, the oligonucleotides modulate the level and or activity of a factor that has a differential expression in cancer cells compared to normal cells. Such factor can be an mRNA, a microRNA, a protein, a glycan. In some cases, the microRNA can be selected from the list above. In some cases, the oligonucleotides inhibit the growth of cancers in the lung through activation of the caspase pathway.

Also provided are pharmaceutical compositions including the subject agents. Any convenient excipients, carriers, etc. can be utilized in the compositions. Pharmaceutically acceptable carriers that find use in the compositions may include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, corn oil, sunflower 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 may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like. The agent composition may also be lyophilized, for subsequent reconstitution and use. The composition can also include a carrier as described here. Examples of carriers which may be used include, but are not limited to, alum, microparticles, liposomes, and nanoparticles. Any convenient additives can be included in the subject compositions to enhance the delivery of the subject active agent. Additives of interest include, cellular uptake enhancers, carrier proteins, lipids, dendrimer carriers, carbohydrates, and the like.

In some cases, the pharmaceutical composition further includes one or more additional active agents. Active agents of interest include an additional oligonucleotide compound of the present disclosure and any convenient antiviral compounds or drugs of interest including but not limited to Amantadine, Rimantadine, Zanamivir, Oseltamivir, Peramivir and the like.

Agents

Any convenient agents may be utilized as an agent of a target of interest (e.g., a conserved RNA secondary structure of a CoV or a miRNA) in the subject methods and compositions. Agents of interest include, but are not limited to, a ligand of CoV, a CoV-binding antibody, a scaffolded protein binder of CoV, an oligonucleotide, a small molecule, and a peptide; or a fragment, variant, or derivative thereof; or combinations of any of the foregoing.

Antibodies that may be used as agents in connection with the present disclosure can encompass, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, Fab antibody fragments, F(ab)₂ antibody fragments, Fv antibody fragments (e.g., V_H or V_L), single chain Fv antibody fragments and dsFv antibody fragments. Furthermore, the antibody molecules may be fully human antibodies, humanized antibodies, or chimeric antibodies. The antibodies that may be used in connection with the present disclosure can include any antibody variable region, mature or unprocessed, linked to any immunoglobulin constant region. Minor variations in the amino acid sequences of antibodies or immunoglobulin molecules are encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain 75% or more, e.g., 80% or more, 90% or more, 95% or more, or 99% or more of the sequence. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in 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 (e.g., as described herein).

In some embodiments, the agent is a scaffolded polypeptide binder. A scaffold refers to an underlying peptidic framework (e.g., a consensus sequence or structural motif) from which a polypeptide agent arose. The underlying scaffold sequence includes those residues that are fixed and variant residues that may confer on the resulting polypeptide agents different functions, such as specific binding to a target receptor. Such structural motifs may be characterized and compared structurally as a combination of particular secondary and tertiary structural elements, or alternatively, as a comparable primary sequence of amino acid residues. Any convenient scaffolds and scaffolded polypeptides may be utilized as agents in the subject methods. In some embodiments, such agents may be identified utilizing a recombinant screening method such as phage display screening. Scaffolded polypeptide binders 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 a conserved RNA secondary structure of a CoV. Small molecules of interest include, but are not limited to, small organic or inorganic compounds having a molecular weight (MW) of more than 50 and less than about 2,500 daltons (Da), such as more than 50 and less than about 1000 Da, or more than 50 and less than about 500 Da. “Small molecules” encompasses numerous biological and chemical classes, including synthetic, semi-synthetic, or naturally-occurring inorganic or organic molecules, including synthetic, recombinant or naturally-occurring polypeptides and nucleic acids. Small molecules of interest can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, and can contain at least two of the functional chemical groups. The 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 a salt thereof (e.g., a pharmaceutically acceptable salt). In some instances, the oligonucleotide is complementary to a particular segment of a conserved RNA secondary structure motif of a CoV (e.g., as described herein). Complementary oligonucleotides that find use in the subject methods will in some cases be 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 even more. In some cases, the complementary oligonucleotide is 75 nucleotides or less in length, such as 50 nucleotides or less in length, 45 nucleotides or less in length, 40 nucleotides or less in length, or 35 nucleotides or less in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross reactivity, and the like. In some cases, the complementary oligonucleotide is 30 nucleotides or less in length, such as 25 nucleotides or less in length, 20 nucleotides or less in length, or 15 nucleotides or less in length, where the length is governed by efficiency of inhibition, specificity, including absence of cross-reactivity, and the like. The present disclosure provides for short oligonucleotides, e.g., of from 7, 8 to 15, or 15 to 16 nucleotides in length, can be strong and selective inhibitors of CoV function. In some embodiments, the active agent is a compound comprising an oligonucleotide sequence comprising at least 8 nucleoside subunits complementary to an RNA secondary structure of a CoV. In some embodiments, the active agent is a compound comprising an oligonucleotide sequence comprising at least 8 and 20 or less (e.g., 15 or less) nucleoside subunits complementary to an RNA secondary structure of a CoV.

A specific region or regions of the endogenous strand RNA secondary structure of a CoV sequence is chosen to be complemented by the oligonucleotide agent. Selection of a specific sequence for the oligonucleotide may use an empirical method, where based on the structural analysis (e.g., as described herein) several candidate sequences are assayed for inhibition of the target CoV in an in vitro or animal model. A combination of oligonucleotides and sequences may also be used, where several regions of the target RNA secondary structure of a CoV are selected for antisense complementation.

In some embodiments, the agent is an oligonucleotide compound comprising at least 5 nucleoside subunits (e.g., at least 6, at least 7, at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, or at least 20) complementary to a RNA secondary structure of a CoV, or a salt thereof. In certain cases, the linkages of the oligonucleotides are modified phosphate groups, e.g., where one or more oxygens of phosphate has been replaced with a different substituent. Without being bound to any particular theory, such modification can increase resistance of the oligonucleotide to nucleolytic breakdown. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. In some cases, the linkages of the oligonucleotides are modified phosphate groups where one or more of the non-bridging phosphonate oxygen atoms in the linkage has been replaced by a group selected from, S, Se, BR^a ₃, alkyl, substituted alkyl, aryl, substituted 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 of the linkages of the oligonucleotide the phosphorous atom is chiral, e.g., a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (referred to herein as Rp), or the “S” configuration (referred to herein as Sp). In certain embodiments, the linkages of the oligonucleotide include one or more stereogenic phosphorus atoms with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more enantiomeric excess of the Sp configuration. In certain other , the linkages of the oligonucleotide include one or more stereogenic phosphorus atoms with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,

80%, 90%, 95%, or more enantiomeric excess of the Rp configuration.

In certain embodiments, one or more of the linkages of the oligonucleotide are selected from methylphosphonate, P3'→N5' phosphoramidate, N3'→P5' phosphoramidate, N3'®P5' thiophosphoramidate, phosphorodithioate and phosphorothioate linkages. In certain cases, one or more linkages of the oligonucleotide is a phosphorothioate linkage. In certain cases, the phosphorus atom in one or more of the phosphorothioate linkages is chiral. In some cases, the chiral phosphorus atom in the one or more phosphorothioate linkages has Rp configuration. In some cases, the chiral phosphorus atom in the one or more phosphorothioate linkages has Sp configuration. In certain embodiments, the linkages of the oligonucleotide include one or more phosphorothioate linkages with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more enantiomeric excess of the Sp configuration. In certain other embodiments, the linkages of the oligonucleotide includes one or more phosphorothioates with at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more enantiomeric excess of the Rp configuration.

In certain instances, the oligonucleotide sequence is a bridged nucleic acid (e.g., as described herein). In certain instances, the oligonucleotide sequence includes one or more bridged nucleic acid nucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more.

In certain instances, the oligonucleotide sequence is a locked nucleic acid. In certain instances, the oligonucleotide sequence includes one or more locked nucleic acid nucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more.

In certain instances, the oligonucleotide sequence is an ethylene-bridged nucleic acid (ENA). In certain instances, the oligonucleotide sequence includes one or more ENA nucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more.

In certain instances, the oligonucleotide sequence is constrained ethyl (cEt) nucleic acid. In certain instances, the oligonucleotide sequence includes one or more cEt nucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more. In certain instances, the oligonucleotide sequence includes one or more (S)- constrained ethyl nucleic acids, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more. In certain instances, the oligonucleotide sequence includes one or more (R) -constrained ethyl nucleic acids, such as 2 or more, 3 or more,

4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more. In certain instances, the oligonucleotide sequence includes one or more ribose modifications. In some cases, the oligonucleotide sequence includes one or more 2'-modified ribose sugars (also referred to herein as 2'-modified nucleotides). In certain instances, the oligonucleotide sequence includes one or more 2'-modified nucleotides, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more. 2'-modified nucleotides, include, but are not limited to moieties with 2' substituents selected from alkyl, allyl, amino, azido, fluoro, thio, O-alkyl, e.g., O-methyl, O-allyl, OCF₃, O-(CH₂)₂-O-CH₃ (e.g., 2'-O- methoxyethyl (MOE)), O-(CH₂)₂SCH₃, )-(CH₂)₂-ONR₂, and 0-CH₂C(O)-NR₂, where each R is independently selected from H, alkyl, and substituted alkyl. In certain instances, the oligonucleotide sequence includes one or more 2'-O-methoxyethyl (MOE) modifications, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, or even more.

In some embodiments, the agent is an oligonucleotide that comprises at least 5 deoxyribonucleotide units (e.g., units complementary to a target motif) (e.g., 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 an RNase. In some case, the oligonucleotide recruits an RNase to catalyze the degradation of the target vRNA into smaller components. Any convenient methods and moieties for recruiting an RNase can be incorporated into the subject agents (e.g., oligonucleotides), in some instances, the oligonucleotide agent further includes a sequence that recruits an RNase of interest. It is understood that unless indicated otherwise, an oligonucleotide sequence as depicted herein is meant to include DNA sequences, RNA sequences (e.g., where U can optionally replace T ), mixed RNA/DNA sequences, and analogs thereof, including analogs where one or more nucleotides of the sequence are modified nucleotides, such as BNA analogs, LNA analogs, ENA analogs, cEt analogs, 2'-modified analogs, and/or analogs where one or more internucleoside linkages are replaced, e.g., with a non-naturally occurring linkage such as a phosphorothioate, phosphorodithioate, phosphoramidate or thiophosphorarnidate linkage. It will be understood that in embodiments where one or more of the linkages of the oligonucleotide include a chiral phosphorous atom, e.g., a stereogenic center, the stereogenic phosphorus atom can possess either the “R” configuration (referred to herein as Rp), or the “S” configuration (referred to herein as Sp).

In some embodiments, the oligonucleotide comprises a sequence selected from:

5' GACGTGATATATGTGG 3' (SEQ ID NO:1);

5' CGTGATATATGTGGTA 3' (SEQ ID NO:2);

5' GATATGTGGTACCAT 3' (SEQ ID NO:3);

5' TGGTACCATGTCAC 3' (SEQ ID NO:4);

5' CCTCAGCAGCAGATTT 3' (SEQ ID NO:5);

5' TCAGCAGCAGATTTC 3' (SEQ ID NO:6);

5' CAGCAGCAGATTTC 3' (SEQ ID NO:7);

5' CAGATTTCTTAGTGAC 3' (SEQ ID NO:8);

5' TGCAACACGGACGAAA 3' (SEQ ID NO:9);

5' GCAACACGGACGAAAC 3' (SEQ ID NO:10);

5' ACGAAACCCGTAAGCA 3' (SEQ ID NO:11);

5' AACATGTCTGGACCTA 3' (SEQ ID NO:12);

5' ACAT GTCTG G ACCTAT 3' (SEQ ID NO:13);

5' TGAATATGACATAGT 3' (SEQ ID NO:14);

5' AATATGACATAGTC 3' (SEQ ID NO:15);

5' CACAGATTTTAAAGTT 3' (SEQ ID NO:16);

5' GATTTTAAAGTTCGT 3' (SEQ ID NO:17);

5' TAAAGTTCGTTTAGA 3' (SEQ ID NO:18);

5' AAAGTTCGTTTAGA 3' (SEQ ID NO:19);

5' TCGTTTAGAGAACAGAT 3' (SEQ ID NO:20);

5' AGAGAACAGATCTACA 3' (SEQ ID NO:21);

5' AGATCTACAAGAGA 3' (SEQ ID NO:22);

5' CAGGCAAACTGAGTTG 3' (SEQ ID NO:23);

5' CAGGCAAACTGAGT 3' (SEQ ID NO:24);

5' AAACTGAGTTGGAC 3' (SEQ ID NO: 25);

5' GAGTTGGACGTGTGT 3' (SEQ ID NO: 26);

5' GGACGTGTGTTTTCTC 3' (SEQ ID NO: 27);

5' TTTCGGTCACACCCGG (SEQ ID NO: 28);

5' CGGTCACACCCGGACG 3' (SEQ ID NO:29); 5' TCACACCCGGACGAAA 3' (SEQ ID N0:30);

5' CACCCG GACGAAAC 3' (SEQ ID NO:31);

5' CACCCG GACGAAACC 3' (SEQ ID NO:32);

5' CACCCG GACGAAACCT 3' (SEQ ID NO:33);

5' CCGGACGAAACCTA 3' (SEQ ID NO:34);

5' TCGATCGTACTCCGC 3' (SEQ ID NO:35);

5' ACTCGATCGTACTC 3' (SEQ ID NO:36);

5' CGTGGCCTCGGTGAA 3' (SEQ ID NO:37);

5' GTGGCCTCGGTGAA 3' (SEQ ID NO:38);

5' TAAAGATTGCTATGTG 3' (SEQ ID NO:39);

5' AAGATTGCTATGTGAG 3' (SEQ ID N0:40);

5' TATGTGAGTTAAAGTT 3' (SEQ ID N0:41);

5' TGTGAGTTAAAGTTAA 3' (SEQ ID NO:42);

5' TCGTAGAAGCCTTTTG 3' (SEQ ID NO:43);

5' AAGCCTTTTGGCAATG 3' (SEQ ID NO: 44)

5' TTGGCAATGTTGTTCC 3' (SEQ ID NO:45)

5' CTTGAGGAAGTTGT 3' (SEQ ID NO:46);

5' AGGAAGTTGTAGCACG 3' (SEQ ID NO:47);

5' CTACTAAAATTAATT 3' (SEQ ID. NO:48);

5' AATTAATTTT ACACAT 3' (SEQ ID NO:49);

5' ATTTT ACACATT AG G G 3' (SEQ ID N0:50);

5' TACACATTAGGGCTC 3' (SEQ ID NO:51);

5' ACATTAGGGCTCTTC 3' (SEQ ID NO:52);

5' GCTCTTCCATATAGG 3' (SEQ ID NO:53);

5' AAG G CTCTCC AT CTT A 3' (SEQ ID NO:54);

5' AAGGCTCTCCATCT 3' (SEQ ID NO:55);

5' GCTCTCCATCTTACCT 3' (SEQ ID NO:56);

5' TCCATCTTACCTTTCG 3' (SEQ ID NO:57);

5' CCTCCCTAATGTTACA 3' (SEQ ID NO:58);

5' CTCCCTAATGTTACAG 3' (SEQ ID NO:59);

5' TAAAACCAACACTACC 3' (SEQ ID NO:60);

5' AAACCAACACTACCAC 3' (SEQ ID NO:61);

5' ACCAACACTACCACAT 3' (SEQ ID NO:62); and 5' CAACACTACCACATGA 3' (SEQ ID NO:63).

In some embodiments, the oligonucleotide comprises a sequence selected from (COV2):

5' GGTGACATGGTACCACATATATCACGTC 3' (SEQ ID NO:218)

5' GACGTGATATATGTGGTACCATGTCACC 3' (SEQ ID NO:219)

5' GACGTGATATATGTGG 3' (SEQ ID NO:220)

5' CGTGATATATGTGGTA 3' (SEQ ID NO:221)

5' GATATGTGGTACCAT 3' (SEQ ID NO:222)

5' TGGTACCATGTCAC 3' (SEQ ID NO:223)

5' GTCACTAAGAAATCTGCTGCTGAGG 3' (SEQ ID NO:224)

5' CCTCAGCAGCAGATTTCTTAGTGAC 3' (SEQ ID NO:225)

5' CCTCAGCAGCAGATTT 3' (SEQ ID NO:226)

5' TCAGCAGCAGATTTC 3' (SEQ ID NO:227)

5' CAGCAGCAGATTTC 3' (SEQ ID NO:228)

5' CAGATTTCTTAGTGAC 3' (SEQ ID NO:229)

5' TGCTTACGGTTTCGTCCGTGTTGCA 3' (SEQ ID NO:230)

5' TGCAACACGGACGAAACCGTAAGCA 3' (SEQ ID NO:231)

5' TGCAACACGGACGAAA 3' (SEQ ID NO:232)

5' GCAACACGGACGAAAC 3' (SEQ ID NO:233) 5' ACACGGACGAAACCG 3' (SEQ ID NO:234)

5' GGACGAAACCCGTAA 3' (SEQ ID NO:235)

5' ACGAAACCGTAAGCA 3' (SEQ ID NO:236)

5' ACGAAACCGTAAGCA 3' (SEQ ID NO:237)

5' GCAACACGGACGAAAC 3' (SEQ ID NO:238)

5' GCAACACGGACGAAA 3' (SEQ ID NO:239)

5' GCAACACGGACGAAAC 3' (SEQ ID NO:240)

5' GCAACACGGACGAAAC 3' (SEQ ID NO:241)

5' GCAACACGGACGAAAC 3' (SEQ ID NO:242)

5' GCAACACGGACGAA 3' (SEQ ID NO:243)

5' ACACGGACGAAACCG 3' (SEQ ID NO:244)

5' ACACGGACGAAACCG 3' (SEQ ID NO:245)

5' AACACGGACGAAACCG 3' (SEQ ID NO:246)

5' AACACGGACGAAACCG 3' (SEQ ID NO:247)

5' ACGAAACCGTAAGCA 3' (SEQ ID NO:248)

5' ACGAAACCGTAAGCA 3' (SEQ ID NO:249)

5' ATAGGTCCAGACATGTTCC 3' (SEQ ID NO:250)

5' GGAACATGTCTGGACCTAT 3' (SEQ ID NO:251)

5' AACATGTCTGGACCTA 3' (SEQ ID NO:252)

5' ACAT GTCTG G ACCTAT 3' (SEQ ID NO:253)

5' TATGACTATGTCATATTCA 3' (SEQ ID NO:254)

5' AGTG AAT AT G ACAT AGT CAT ATT 3' (SEQ ID NO:255)

5' TGAATATGACATAGT 3' (SEQ ID NO:256)

5' AATATGACATAGTC 3' (SEQ ID NO:257)

5' GATCTCTTGTAGATCTGTTCTCTAAACGAACTTTAAAATCTGTG 3' (SEQ ID NO:258) 5' CACAGATTTTAAAGTTCGTTTAGAGAACAGATCTACAAGAGATC 3' (SEQ ID NO:259) 5' CACAGATTTTAAAGTT 3' (SEQ ID NO:260)

5' CACAGATTTTAAAGTT 3' (SEQ ID NO:261)

5' CAGATTTTAAAGTTCG 3' (SEQ ID NO:262)

5' GATTTTAAAGTTCGT 3' (SEQ ID NO:263)

5' TAAAGTTCGTTTAGA 3' (SEQ ID NO:264)

5' AAAGTTCGTTTAGA 3' (SEQ ID NO:265)

5' TCGTTTAGAGAACAGAT 3' (SEQ ID NO:266)

5' AGAGAACAGATCTACA 3' (SEQ ID NO:267)

5' AGATCTACAAGAGA 3' (SEQ ID NO:268)

5' ATCTACAAGAGATC 3' (SEQ ID NO:269)

5' AGATTTTAAAGTTCGT 3' (SEQ ID NO:270)

5' GATTTTAAAGTTCGT 3' (SEQ ID NO:271)

5' GATTTTAAAGTTCGT 3' (SEQ ID NO:272)

5' AGATTTTAAAGTTCG 3' (SEQ ID NO:273)

5' TCGTTTAGAGAACAGAT 3' (SEQ ID NO:274)

5' TTCGTTTAGAGAACAG 3' (SEQ ID NO:275)

5' TTCGTTTAGAGAACAG 3' (SEQ ID NO:276)

5' G AG AAAACACACGT CCAACT CAGTTTGCCTG 3' (SEQ ID NO:277)

5' CAGGCAAACTGAGTTGGACGTGTGTTTTCTC 3' (SEQ ID NO:278)

5' CAGGCAAACTGAGTTG 3' (SEQ ID NO:279)

5' CAGGCAAACTGAGTTG 3' (SEQ ID NO:280)

5' CAGGCAAACTGAGT 3' (SEQ ID NO:281)

5' CAAACTGAGTTGGACG 3' (SEQ ID NO:282)

5' AAACTGAGTTGGAC 3' (SEQ ID NO:283)

5' AAACTGAGTTGGACGT 3' (SEQ ID NO:284)

5' GAGTTGGACGTGTGT 3' (SEQ ID NO:285)

5' TTGGACGTGTGTTTTC 3' (SEQ ID NO:286) 5' GGACGTGTGTTTTCTC 3' (SEQ ID NO:287)

5' GGTTTCGTCCGGGTGTGACCG 3' (SEQ ID NO:288)

5' TTTCGGTCACACCCGGACGAAACCTAGAT 3' (SEQ ID NO:289)

5' TTTCGGTCACACCCGG 3' (SEQ ID NO:290)

5' CGGTCACACCCGGACG 3' (SEQ ID NO:291)

5' TCACACCCGGACGAAA 3' (SEQ ID NO:292)

5' CACCCG GACGAAAC 3' (SEQ ID NO:293)

5' CACCCG GACGAAACC 3' (SEQ ID NO:294)

5' CACCCG GACGAAACCT 3' (SEQ ID NO:295)

5' CCGGACGAAACCTA 3' (SEQ ID NO:296)

5' CGGTCACACCCGGACGA 3' (SEQ ID NO:297)

5' CGGTCACACCCGGACG 3' (SEQ ID NO:298)

5' CGGTCACACCCGGACG 3' (SEQ ID NO:299)

5' CGGTCACACCCGGACG 3' (SEQ ID NO:300)

5' GTCACACCCGGACG 3' (SEQ ID NO:301)

5' GTCACACCCGGACG 3' (SEQ ID NO:302)

5' CACCCG GACGAAACC 3' (SEQ ID NO:303)

5' CACCCG GACGAAACC 3' (SEQ ID NO:304)

5' CACCCG GACGAAAC 3' (SEQ ID NO:305)

5' CACACCCGGACGAAAC 3' (SEQ ID NO:306)

5' CACACCCGGACGAAA 3' (SEQ ID NO:307)

5' CACACCCGGACGAA 3' (SEQ ID NO:308)

5' CGAGGCCACGCGGAGTACGATCGA 3' (SEQ ID NO:309)

5' ACTCGATCGTACTCCGCGTGGCCTCGGTGAA 3' (SEQ ID NO:310)

5' TCGATCGTACTCCGC 3' (SEQ ID NO:311)

5' TCGATCGTACTCCG 3' (SEQ ID NO:312)

5' ATCGTACTCCGCGTG 3' (SEQ ID NO:313)

5' ACTCGATCGTACTC 3' (SEQ ID NO:314)

5' CGTGGCCTCGGTGAA 3' (SEQ ID NO:315)

5' GTGGCCTCGGTGAA 3' (SEQ ID NO:316)

5' T AGTTAACTTT AAT CT CAC AT AG CAAT CTTT AAT C 3' (SEQ ID NO:317)

5' GATTAAAGATTGCTATGTGAGATTAAAGTTAACTA 3' (SEQ ID NO:318)

5' GATTAAAGATTGCTAT 3' (SEQ ID NO:319)

5' TAAAGATTGCTATGTG 3' (SEQ ID NO:320)

5' AAGATTGCTATGTGAG 3' (SEQ ID NO:321)

5' GCTATGTGAGTTAAAG 3' (SEQ ID NO:322)

5' TATGTGAGTTAAAGTT 3' (SEQ ID NO:323)

5' TGTGAGTTAAAGTTAA 3' (SEQ ID NO:324)

5' CGTGCTACAACTTCCTCAAGGAACAACATTGCCAAAAGGCTTCTACGCAG 3' (SEQ ID NO:325) 5' CTGCGTAGAAGCCTTTTGGCAATGTTGTTCCTTGAGGAAGTTGTAGCACG 3' (SEQ ID NO:326) 5' TCGTAGAAGCCTTTTG 3' (SEQ ID NO:327)

5' CGTAGAAGCCTTTTGGC 3' (SEQ ID NO:328)

5' TAGAAGCCTTTTGGCA 3' (SEQ ID NO:329)

5' AAGCCTTTTGGCAATG 3' (SEQ ID NO:330)

5' TTGGCAATGTTGTTCC 3' (SEQ ID NO:331)

5' GCAATGTTGTTCCT 3' (SEQ ID NO:332)

5' CTTGAGGAAGTTGT 3' (SEQ ID NO:333)

5' AGGAAGTTGTAGCACG 3' (SEQ ID NO:334)

5' GCCTATATGGAAGAGCCCTAATGTGTAAAATTAATTTTAGTAG 3' (SEQ ID NO:335)

5' CTACTA AAATT AATTTT ACACATT AGGGCTCTTCCATATAGGC 3' (SEQ ID NO:336)

5' CTACTAAAATTAATT 3' (SEQ ID NO:337)

5' TACTAAAATTAATTT 3' (SEQ ID NO:338)

5' ACT AAAATT A ATTT 3' (SEQ ID NO:339) 5' AATTAATTTT ACACAT 3' (SEQ ID NO:340)

5' ATTTT ACACATT AG G G 3' (SEQ ID NO:341)

5' TACACATTAGGGCTC 3' (SEQ ID NO:342)

5' ACATTAGGGCTCTTC 3' (SEQ ID NO:343)

5' TAGGGCTCTTCCATA 3' (SEQ ID NO:344)

5' G CT CTT CCAT ATAG G 3' (SEQ ID NO:345)

5' AGGTAAGATGGAGAGCCTT 3' (SEQ ID NO:346)

5' AAG G CTCTCC AT CTT ACCTTT CG G 3' (SEQ ID NO:347)

5' AAG G CTCTCC AT CTT A 3' (SEQ ID NO:348)

5' AAGGCTCTCCATCT 3' (SEQ ID NO:349)

5' GCTCTCCATCTTACCT 3' (SEQ ID NO:350)

5' TCCATCTTACCTTTCG 3' (SEQ ID NO:351)

5' TGTGTAACATTAGGGAGG 3' (SEQ ID NO:352)

5' CCTCCCTAATGTTACACA 3' (SEQ ID NO:353)

5' CCTCCCTAATGTTACA 3' (SEQ ID NO:354)

5' CTCCCTAATGTTACAG 3' (SEQ ID NO:355)

5' CTCCCTAATGTTACAG 3' (SEQ ID NO:356)

5' TCATGTGGTAGTGTTGGTTTTA 3' (SEQ ID NO:357)

5' TAAAACCAACACTACCACATGA 3' (SEQ ID NO:358)

5' TAAAACCAACACTACC 3' (SEQ ID NO:359)

5' AAACCAACACTACCAC 3' (SEQ ID NO:360)

5' AAACCAACACTACCAC 3' (SEQ ID NO:361)

5' ACCAACACTACCACAT 3' (SEQ ID NO:362)

5' CAACACTACCACATGA 3' (SEQ ID NO:363)

In some embodiments, the oligonucleotide comprises a sequence selected from: (miRNA targeting sequences)

5' GGTTTCGTCCGTGTT 3' (SEQ ID NO:364) 5' GTTTCGTCCGTGTT 3' (SEQ ID NO:365)

5' GCTGTCGCCCGTGTC 3' (SEQ ID NO:366) 5' GCTGTCGCCCGTGTC 3^; (SEQ ID NO:367) 5' GCTGTCGCCCGTGTC 3' (SEQ ID NO:368) 5' GCTGTCGCCCGTGT 3' (SEQ ID NO:369) 5' TTCGTCCGTG 3' (SEQ ID NO:370)

5' TTCGTCCGTG 3' (SEQ ID NO:371)

5' TTCGTCCGTG 3' (SEQ ID NO:372)

5' CCCACCCAC 3' (SEQ ID NO:373)

5' CCCACCCAC 3' (SEQ ID NO:374)

5' CCCACCCAC 3' (SEQ ID NO:375)

5' TTTCGTCCGT 3' (SEQ ID NO:376)

5' TTTCGTCCGT 3' (SEQ ID NO:377)

5' TTTCGTCCGT 3' (SEQ ID NO:378)

5' CCCCACCCAC 3' (SEQ ID NO:379)

5' CCCCACCCAC 3' (SEQ ID NO:380)

5' CCCCACCCAC 3' (SEQ ID NO:381)

5' TTTCGTCCGTGT 3' (SEQ ID NO:382)

5' TTTCGTCCGTGT 3' (SEQ ID NO:383)

5' TTTCGTCCGTGT 3' (SEQ ID NO:384)

5' TTCCATCCATGT 3' (SEQ ID NO:385)

5' TTCCATCCATGT 3' (SEQ ID NO:386) 5' TTCCATCCATGT 3' (SEQ ID NO:387)

5' TTCCATCCATGT 3' (SEQ ID NO:388)

5' GTTTCGTCCGTGTT 3' (SEQ ID NO:389)

5' GTTTCGTCCGTGTT 3' (SEQ ID NO:390)

5' GTTTCGTCCGTGTT 3' (SEQ ID NO:391)

5' GTTTCGGCCATGTG 3' (SEQ ID NO:392)

5' CGTCCGTGp 3' (SEQ ID NO:393)

5' CGTCCGTGTT 3' (SEQ ID NO:394)

5' CGTCCGTGTT 3' (SEQ ID NO:395)

5' CGTCCGTGTT 3' (SEQ ID NO:396)

5' CCTCCGTGTC 3' (SEQ ID NO:397)

5' CCTCCGTGTC 3' (SEQ ID NO:398)

5' CCTCCGTGTC 3' (SEQ ID NO:399)

5' TCCGTGTTGC 3' (SEQ ID NO:400)

5' TCCGTGTTGC 3' (SEQ ID NO:401)

5' TCCGTGTTGC 3' (SEQ ID NO:402)

5' TCCGTCTTGC 3' (SEQ ID NO:403)

5' TCCGTCTTGC 3 (SEQ ID NO:404)

5' TCCGTTTGC 3' (SEQ ID NO:405)

5' TTTCGTCCGTGTT 3' (SEQ ID NO:406)

5' TTTCGTCCGTGTT 3' (SEQ ID N0:407)

5' TTTCCTCTATGTT 3' (SEQ ID NO:408)

5' TTTCCTCTATGTT 3' (SEQ ID NO:409)

5' GTTTCGTCCGTGT 3' (SEQ ID NO:410)

5' GGTTTCGTCCGTGTT 3' (SEQ ID NO:411)

5' GGTTTCGTCCGTGTT 3' (SEQ ID NO:412)

5' AAGTACGTCCTACTT 3' (SEQ ID NO:413)

5' GGTTTCGTCC 3' (SEQ ID NO:414)

5' GGTTTCGTCC 3' (SEQ ID NO:415)

5' TGGGTTTCGTCCGTT 3' (SEQ ID NO:416)

5' TTTTGGGATTCCGT 3' (SEQ ID NO:417)

5' GCTTTTGGGATTCCGT 3' (SEQ ID NO:418)

5' GCTTTTGGGATTCCGT 3' (SEQ ID NO:419)

5' AGCTGCTTTTGGGATT 3' (SEQ ID NO:420)

5' GAGCTGCTTTTGGGAT 3' (SEQ ID NO:421)

5' CTGCTTTTGGGATTCC 3' (SEQ ID NO:422)

5' TGCTTTTGGGATTC 3' (SEQ ID NO:423)

5' AGCTGCTTTTGGGATT 3' (SEQ ID NO:424)

5' AGCTGCTTTTGGGA 3' (SEQ ID NO:425)

5' CAGCTGCTTTTGGGAT 3' (SEQ ID NO:426)

5' AGCTGCTTTTGGGATT 3' (SEQ ID NO:427)

5' AGCTGCTTTTGGGA 3' (SEQ ID NO:428)

5' AGCTGCTTTTGGGA 3' (SEQ ID NO:429)

5' AGCTGCTTTTGGGATT 3' (SEQ ID NO:430)

5' TGCTTTTGGGATTC 3' (SEQ ID NO:431)

5' GCGGCGCCCCGCCT 3' (SEQ ID NO:432)

5' GCGGCGCCCCGCCT 3' (SEQ ID NO:433)

5' CGGTCCCGCGGCGCCCCGCCT 3' (SEQ ID NO:434) 5' TTTGGGCTTCCTCGCT 3' (SEQ ID NO:435)

5' TTTGGGCTTCCTCG 3' (SEQ ID NO:436)

5' TTTGGGCTTCCTCG 3' (SEQ ID NO:437)

5' TGGGCTTCCTCGCT 3' (SEQ ID NO:438)

5' ATATACTTTGGGCTTC 3' (SEQ ID NO:439) 5' ATATACTTTGGGCTTC 3' (SEQ ID NO:440)

5' ATATACTTTGGGCT 3' (SEQ ID NO:441)

5' ATATACTTTGGGCTT 3' (SEQ ID NO:442)

5' TAGCCCTAGCCCCGCA 3' (SEQ ID NO:443)

5' TAGCCCTAGCCCCGCA 3' (SEQ ID NO:444)

5' TAGCCCTAGCCCCG 3' (SEQ ID NO:445)

5' TGCTGTTAGCCCTA 3' (SEQ ID NO:446)

5' TGCTGTTAGCCCTA 3' (SEQ ID NO:447)

5' GTGTTAGCCCTAGCC 3' (SEQ ID NO:448)

5' GCGCCCAGCCCCGC 3' (SEQ ID NO:449)

5' GCGCCCAGCCCCGC 3' (SEQ ID NO:450)

5' CGCGCGCCCAGCCC 3' (SEQ ID NO:451)

5' CGCGCGCCCAGCCCCGC 3' (SEQ ID NO:452)

5' TGGCATGGAATGGG 3' (SEQ ID NO:453)

5' ATGGAATGGGCTCCGC 3' (SEQ ID NO:454)

5' ATGGAATGGGCTCC 3' (SEQ ID NO:455)

5' AATGGGCTCCGCCAG 3' (SEQ ID NO:456)

5' AATGGGCTCCGCCAG 3' (SEQ ID NO:457)

5' AAT G G G CTCCG CCA 3' (SEQ ID NO:458)

5' AAT G G G CTCCG CCA 3' (SEQ ID NO:459)

5' CTTACCCGAGGCGGTC 3' (SEQ ID NO:460)

5' TTACCCGAGGCGGT 3' (SEQ ID NO:461)

5' ACCGTACCTTACCCGA 3' (SEQ ID NO:462)

5' CGTACCTTACCCGA 3' (SEQ ID NO:463)

5' CTCCG AGCCCCGCC 3' (SEQ ID NO:464)

5' CCCGGCTCCGAGCCCCGCC 3' (SEQ ID NO:465)

5' TGGCCTGTCCCCGCA 3' (SEQ ID NO:466)

5' TGGCCTGTCCCCGCA 3' (SEQ ID NO:467)

5' GATGCCCTGGCCTG 3' (SEQ ID NO:468)

5' GATGCCCTGGCCTG 3' (SEQ ID NO:469)

5' GCAGCCAGCTCTAC 3' (SEQ ID NO:570)

5' GCAGCCAGCTCTAC 3' (SEQ ID NO:471)

5' AGCCAGCTCTACCC 3' (SEQ ID NO:472)

5' AGCTCTACCCCCGCCA 3' (SEQ ID NO:473)

5' AGCTCTACCCCCGCCA 3' (SEQ ID NO:474)

5' AAAGCAGCGCCCACTT 3' (SEQ ID NO:475)

5' ACTTCCTCCCCGCT 3' (SEQ ID NO:476)

5' CTACAGAAGCCCCATA 3' (SEQ ID N 0:477)

5' CTACAGAAGCCCCATA 3' (SEQ ID N 0:478)

5' GAAATCTCTACAGAAG 3' (SEQ ID NO:479)

5' GAAATCTCTACAGAAG 3' (SEQ ID NO:480)

5' TGATCCCTGTCCCCAT 3' (SEQ ID NO:481)

5' ATGCTGATCCCTGTC 3' (SEQ ID NO:482)

5' GCCATGCTGATCCCTGTCCCCAT 3' (SEQ ID NO:483) 5' CCATCTCACCCCAT 3' (SEQ ID NO:484)

5' GCTGCTCCTCCCCAT 3' (SEQ ID NO:485)

5' TCTCCAACCCCACAA 3' (SEQ ID NO:486)

5' CTCCAACCCCACAA 3' (SEQ ID NO:487)

5' CAGCCAGCTCTCCAA 3' (SEQ ID NO:488)

5' AGCCAGCTCTCCAA 3' (SEQ ID NO:489) In some embodiments, the oligonucleotide comprises a sequence selected from:

(Negative-strand targeted LNA)

5’ ATCTGTTCTCTAAACGA 3’ (SEQ ID NO: 490) 5’ ATCTGTTCTCTAAACG 3’ (SEQ ID NO:491)

5’ AGATCTGTTCTCTAAA 3’ (SEQ ID NO:492)

5’ TCTCTAAACGAACTTT 3’ (SEQ ID NO:493)

5’ TCTCTAAACGAACTTT 3’ (SEQ ID NO:494)

5’ CTCTAAACGAACTTTA 3’ (SEQ ID NO:495) 5’ ACTTTAAAATCTGT 3’ (SEQ ID NO:496)

5’ GGTTTCGTCCGTGTT 3’ (SEQ ID NO:497)

5’ GGTTTCGTCCGTGTT 3’ (SEQ ID NO:498)

5' GGTTTCGTCCGTGTT 3' (SEQ ID NO:499)

5' TGCTTACGGTTTCGTCC 3’ (SEQ ID NO: 500) 5’ GTTTCGTCCGTGTTGC 3’ (SEQ ID NO:501)

5’ TAGGTTTCGTCCGG 3’ (SEQ ID NO: 502)

5’ TCGTCCGGGTGTGA 3’ (SEQ ID NO: 503)

5’ TTCGTCCGGGTGTGA 3’ (SEQ ID NO:504)

5’ TTTCGTCCGGGTGTGA 3’ (SEQ ID NO: (SEQ ID N0:505) 5’ AGGTTTCGTCCGGGT 3’ (SEQ ID NO:506)

5’ AGGTTTCGTCCGGG 3’ (SEQ ID NO: 507)

5’ AGGTTTCGTCCGGGT 3’ (SEQ ID NO:508)

5’ TTTCGTCCGGGTGTG 3’ (SEQ ID NO:509)

5’ AGGCCACGCGGAGTA 3’ (SEQ ID N0:510) 5’ CACGCGGAGTACGATC 3’ (SEQ ID NO:511)

In certain embodiments, the oligonucleotide comprises the sequence: 5’ GACGTGATATATGTGG 3’ (SEQ ID NO:l). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CGTGATATATGTGGTA 3’ (SEQ ID NO:2). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GATATGTGGTACCAT 3’ (SEQ ID NOG). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TGGTACCATGTCAC 3’ (SEQ ID NO:4). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CCTCAGCAGCAGATTT 3’ (SEQ ID NOG). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCAGCAGCAGATTTC 3’ (SEQ ID NOG). In certain embodiments, the oligonucleotide comprises the sequence: 5’

CAGCAGCAGATTTC 3’ (SEQ ID NO:7). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGATTTCTTAGTGAC 3’ (SEQ ID NO:8) In certain embodiments, the oligonucleotide comprises the sequence: 5’ TGCAACACGGACGAAA 3’ (SEQ ID NO:9). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCAACACGGACGAAAC 3’ (SEQ ID NO:10). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACGAAACCCGTAAGCA 3’ (SEQ ID NO:ll). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AACATGTCTGGACCTA 3’ (SEQ ID NO: 12). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACATGTCTGGACCTAT 3’ (SEQ ID NO: 13). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TGAATATGACATAGT 3’ (SEQ ID NO: 14). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AATATGACATAGTC 3’ (SEQ ID NO:15). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CACAGATTTTAAAGTT 3’ (SEQ ID NO: 16). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GATTTTAAAGTTCGT 3’ (SEQ ID NO: 17). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TAAAGTTCGTTTAGA 3’ (SEQ ID NO: 18). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAAGTTCGTTTAGA 3’ (SEQ ID NO: 19). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCGTTTAGAGAACAGAT 3’ (SEQ ID NO:20). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AGAGAACAGATCTACA 3’ (SEQ ID NO:21). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AGATCTACAAGAGA 3’ (SEQ ID NO:22). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGGCAAACTGAGTTG 3’ (SEQ ID NO:23). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGGCAAACTGAGT 3’ (SEQ ID NO:24). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAACTGAGTTGGAC 3’ (SEQ ID NO: 25). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GAGTTGGACGTGTGT 3’ (SEQ ID NO: 26). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GGACGTGTGTTTTCTC 3’ (SEQ ID NO: 27). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TTTCGGTCACACCCGG (SEQ ID NO: 28). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CGGTCACACCCGGACG 3’ (SEQ ID NO:29). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCACACCCGGACGAAA 3’ (SEQ ID NO:30). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CACCCGGACGAAAC 3’ (SEQ ID NO:31). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CACCCGGACGAAACC 3’ (SEQ ID NO:32). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CACCCGGACGAAACCT 3’

(SEQ ID NO:33). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CCGGACGAAACCTA 3’ (SEQ ID NO:34). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCGATCGTACTCCGC 3’ (SEQ ID NO:35). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACTCGATCGTACTC 3’ (SEQ ID NO:36). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CGTGGCCTCGGTGAA 3’ (SEQ ID NO:37). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GTGGCCTCGGTGAA 3’ (SEQ ID NO:38). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TAAAGATTGCTATGTG 3’ (SEQ ID NO:39). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAGATTGCTATGTGAG 3’ (SEQ ID NO:40). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TATGTGAGTTAAAGTT 3’ (SEQ ID NO:41). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TGTGAGTTAAAGTTAA 3’

(SEQ ID NO:42). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCGTAGAAGCCTTTTG 3’ (SEQ ID NO:43). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAGCCTTTTGGCAATG 3’ (SEQ ID NO: 44). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TTGGCAATGTTGTTCC 3’ (SEQ ID NO:45). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CTTGAGGAAGTTGT 3’ (SEQ ID NO:46). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AGGAAGTTGTAGCACG 3’ (SEQ ID NO:47). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CTACTAAAATTAATT 3’ (SEQ ID. NO:48). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AATTAATTTTACACAT 3’ (SEQ ID NO:49). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ATTTTACACATTAGGG 3’ (SEQ ID NO:50). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TACACATTAGGGCTC 3’ (SEQ ID NO:51). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACATTAGGGCTCTTC 3’ (SEQ ID NO:52). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCTCTTCCATATAGG 3’ (SEQ ID NO:53). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAGGCTCTCCATCTTA 3’ (SEQ ID NO:54). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAGGCTCTCCATCT 3’ (SEQ ID NO:55). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCTCTCCATCTTACCT 3’ (SEQ ID NO:56). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TCCATCTTACCTTTCG 3’ (SEQ ID NO:57). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CCTCCCTAATGTTACA 3’ (SEQ ID NO:58). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CTCCCTAATGTTACAG 3’ (SEQ ID NO:59). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TAAAACCAACACTACC 3’

(SEQ ID NO:60). In certain embodiments, the oligonucleotide comprises the sequence: 5’

A A ACC A AC ACT ACC AC 3’ (SEQ ID NO:61). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACC A AC ACT ACC AC AT 3’ (SEQ ID NO:62). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAACACTACCACATGA 3’

(SEQ ID NO:63).

In some instances, binding (e.g., via hybridization) of the oligonucleotide compound (e.g., one of the sequences described above) to the region of vRNA having a conserved RNA secondary structure of CoV disrupts the overall secondary RNA structure of the vRNA. In certain cases, binding of the oligonucleotide compound to the conserved RNA secondary structure of CoV inhibits the packaging ability of the vRNA, thereby inhibiting the CoV virus.

The oligonucleotide sequences can include any convenient number of DNA, RNA BNA, LNA, ENA, cEt, 2’-modified nucleotides, or other chemically modified nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed RNA/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed BNA/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed BNA/RNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence includes only BNA nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed LNA/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed LNA/RNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence includes only LNA nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed ENA/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed ENA/RNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence includes only ENA nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed cEt/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed cEt/RNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence includes only cEt nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed 2’-modifed nucleotide/DNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence is a mixed 2’-modified nucleotide/RNA sequence. In some instances of the oligonucleotide sequences described herein, the sequence includes only 2’-modified nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence includes only DNA nucleotides. In some instances of the oligonucleotide sequences described herein, the sequence includes only RNA nucleotides.

In certain instances, the subject oligonucleotide has one of the following arrangements of types of nucleotide in the sequence (e.g., one of SEQ ID NOs:l-63): A;

A-B-A;

A-B-A-B-A;

A-B-A-B-A-B-A;

A-B-A-B-A-B-A-B-A;

B;

B-A-B;

B-A-B-A-B;

B-A-B-A-B-A-B; or

B-A-B-A-B-A-B-A-A; where each A is independently a sequence of modified nucleotides, and each B is a sequence of DNA nucleotides. In certain instances, A is a sequence of modified nucleotides where the ribose moiety is modified to include a modification selected from BNA, LNA, ENA, cEt, and 2’-modified nucleotides. In certain instances, each A is a sequence from 1 to 50 nucleotides, such as 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5. In certain instances, each B is a sequence from 1 to 50 nucleotides, such as 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5.

In certain instances, the subject oligonucleotide has one of the following arrangements of types of nucleotide in the sequence (e.g., one of SEQ ID NOs:l-63):

(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 ; where A is a modified nucleotide, B is a DNA nucleotide, nl is 8 or more, n2 is 3-4, n3 is 6-8, n4 is 1-2, and n5 is 1-3. In certain instances, A is a modified nucleotide where the ribose moiety is modified to include a modification selected from BNA, LNA, ENA, cEt, and 2’- modified nucleotides.

In certain instances, the subject oligonucleotide has one of the following arrangements of types of nucleotide in the sequence (e.g., one of SEQ ID NOs:1-63):

A, where A is a sequence of 8 or more LNA nucleotides;

A-B-A, where B is a sequence of 6-8 DNA nucleotides, and each A is a sequence of 3-4 LNA nucleotides;

A-B-A, where B is a sequence of 7-8 DNA nucleotides, and each A is a sequence of 4 LNA nucleotides; L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, and each D is a sequence of 2 DNA nucleotides;

L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, and each D is a sequence of 1-2 DNA nucleotides;

L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, and each D is a sequence of 1-3 DNA nucleotides;

L-D-L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, and each D is a sequence of 1-3 DNA nucleotides; and

L-D-L-D-L-D-L-D-L, where each L is a sequence of 1-2 LNA nucleotides, and each D is a sequence of 1-2 DNA nucleotides.

The subject oligonucleotide sequences may further include one or more modified internucleoside linkages, such as phosphorothioate, phosphorodithioate, phosphoramidate and/or thiophosphoramidate linkages. A non-naturally occurring internucleoside linkage may be included at any convenient position(s) of the sequence of the subject oligonucleotide. In embodiments where the internucleoside linkage includes a chiral phosphorous atom, e.g., a stereogenic center, the stereogenic phosphorus atom can possess either the “R” configuration (referred to herein as Rp), or the “S” configuration (referred to herein as Sp). The subject oligonucleotide sequences including a chiral phosphorous atom may be prepared as racemic mixtures, or as separate enantiomers.

In certain embodiments, the oligonucleotide has one of the following sequences where a “+” before the letter denote LNA nucleotides and other letters denote DNA nucleotides (i.e., deoxyribonucleotide units):

LNA 1.1 5' +G+A+CGTGATATATG+T+G+G 3' (SEQ ID NO:64);

LNA 1.2 5' +C+G+TGATATATGTG+G+T+A 3' (SEQ ID NO:65);

LNA 1.3 5' +G+A+TATGTGGTAC+C+A+T 3' (SEQ ID NO:66);

LNA 1.4 5' +T+G+GTACCATGT+C+A+C 3' (SEQ ID NO:67);

LNA 2.1 5' +C+C+TCAG CAG CAG+ A+T +T +T 3' (SEQ ID NO:68);

LNA 2.2 5' +T+C+AGCAGCAGAT+T+T+C 3' (SEQ ID NO:69);

LNA 2.3 5' +C+A+G+CAGCAGAT+T+T+C 3' (SEQ ID NO:70);

LNA 2.4 5' +C+A+GATTTCTTAGT+G+A+C 3' (SEQ ID NO:71);

LNA 3.1 5' +T+G+CAACACGGAC+G+A+A+A 3' (SEQ ID NO:72);

LNA 3.2 5' +G+C+AACACGGACG+A+A+A+C 3' (SEQ ID NO:73);

LNA 3.5 5' +A+C+GAAACCCGTA+A+G+C+A 3' (SEQ ID NO:74);

LNA 3.6 5' +A+C+G+AAACCCGTA+A+G+C+A 3' (SEQ ID NO:75); LNA 4.1 5' +A+A+CATGTCTGGAC+C+T+A 3' (SEQ ID NO:76);

LNA 4.2 5' +A+C+ATGTCTGGAC+C+T+A+T 3' (SEQ ID NO:77);

LNA 5.1 5' +T+G +AAT ATG ACAT+ A+G +T 3' (SEQ ID NO:78);

LNA 5.2 5' +A+A+TATGACATA+G+T+C 3' (SEQ ID NO:79);

LNA 6.1 5' +C+A+CAGATTTTAAA+G+T+T 3' (SEQ ID N0:80);

LNA 6.2 5' +C+A+C+AGATTTTAA+A+G+T+T 3' (SEQ ID N0:81);

LNA 6.4 5' +G+ A+TPT AAAGT +T+C+G +T 3' (SEQ ID NO:82);

LNA 6.5 5' +T+A+A+AGTTCGTT+T+A+G+A 3' (SEQ ID NO:83);

LNA 6.6 5' +A+A+A+GTTCGTTT+A+G+A 3' (SEQ ID NO:84);

LNA 6.7 5' +T+C+GTTTAGAGAAC+A+G+A+T 3' (SEQ ID NO:85); LNA 6.8 5' +A+G+AGAACAGATCT+A+C+A 3' (SEQ ID NO:86);

LNA 6.9 5' +A+G+ATCTACAAG+A+G+A 3' (SEQ ID NO:87);

LNA 7.2 5' +C+A+G+GCAAACTGAG+T+T+G 3' (SEQ ID NO:88);

LNA 7.3 5' +C+A+G+GCAAACTG+A+G+T 3^; (SEQ ID NO:89);

LNA 7.5 5' +A+A+ACTGAGTTG+G+A+C 3' (SEQ ID NO: 90);

LNA 7.7 5' +G+A+GTTGGACGTG+T+G+T 3' (SEQ ID NO: 91);

LNA 7.9 5' +G+G+ACGTGTGTTTT+C+T+C 3' (SEQ ID NO: 92);

LNA 8.1 5' +T+T+TCGGTCACACC+C+G+G (SEQ ID NO: 93);

LNA 8.2 5' +C+G+GTCACACCCGG+A+C+G 3' (SEQ ID NO:94);

LNA 8.3 5' +T+C+ACACCCGGACG+A+A+A 3' (SEQ ID NO:95);

LNA 8.4 5' +C+A+CCCGGACGA+A+A+C 3' (SEQ ID NO:96);

LNA 8.5 5' +C+A+CCCGGACGAA+A+C+C 3' (SEQ ID NO:97);

LNA 8.6 5' +C+A+CCCGGACGAAA+C+C+T 3' (SEQ ID NO:98);

LNA 8.7 5' +C+C+GGACGAAAC+C+T+A 3' (SEQ ID NO:99);

LNA 10.1 5' +T+C+GATCGTACTC+C+G+C 3' (SEQ ID NO: 100);

LNA 10.4 5' +A+C+TCGATCGT+A+C+T+C 3' (SEQ ID NO:101);

LNA 10.5 5' +C+G+TGGCCTCGG+T+G+A+A 3' (SEQ ID NO:102); LNA 10.6 5' +G+T+GGCCTCGG+T+G+A+A 3' (SEQ ID NO:103);

LNA 11.1 5' +G+T+GGCCTCGG+T+G+A+A 3' (SEQ ID NO:104);

LNA 11.2 5' +T+A+A+AGATTGCT+A+T+G+T+G 3' (SEQ ID NO:105); LNA 11.3 5' +A+A+G+ATTGCTATG+T+G+A+G 3' (SEQ ID NO:106); LNA 11.5 5' +T+A+T+GTGAGTTAA+A+G+T+T 3' (SEQ ID NO: 107); LNA 11.6 5' +T+G+T+GAGTTAAAGT+T+A+A 3' (SEQ ID NO:108); LNA 12.1 5' +T+C+GTAGAAGCCTT+T+T+G 3' (SEQ ID NO:109);

LNA 12.4 5' +A+A+G+CCTTTTGGC+A+A+T+G 3' (SEQ ID NO:110); LNA 12.5 5' +T+T+G+GCAATGTTG+T+T+C+C 3' (SEQ ID NO:111); LNA 12.7 5' +C+T+TGAGGAAG+T+T+G+T 3' (SEQ ID NO:112);

LNA 12.8 5' +A+G+G+AAGTTGTAG+C+A+C+G 3' (SEQ ID NO:113); LNA 13.1 5' +C+T +A+CT AAAATT A+ A+T +T 3' (SEQ ID. NO:114);

LNA 13.4 5' +A+A+T+TAATTTTAC+A+C+A+T 3' (SEQ ID NO:115); LNA 13.5 5' + A+T +T +TT ACAC ATT +A+G+G +G 3' (SEQ ID NO: 116); LNA 13.6 5' +T+A+CACATTAGGG+C+T+C 3' (SEQ ID NO:117);

LNA 13.7 5' + A+C+ ATT AG G G CTC+T+T+C 3' (SEQ ID NO:118);

LNA 13.9 5' +G+C+T+CTTCCATAT+A+G+G 3' (SEQ ID NO:119);

LNA 14.1 5' +A+A+GGCTCTCCATC+T+T+A 3' (SEQ ID NO:120);

LNA 14.2 5' +A+A+G+GCTCTCC+A+T+C+T 3' (SEQ ID NO: 121);

LNA 14.3 5' + G + C+TCTCC AT CTT A+ C+ C+T 3' (SEQ ID NO:122);

LNA 14.4 5' +T+C+C AT CTT ACCTT +T+C+G 3' (SEQ ID NO:123);

LNA 16.1 5' +C+C+T +CCCT AAT GT +T +A+C+ A 3' (SEQ ID NO: 124); LNA 16.2 5' +C+T +C+CCTAAT GTT +A+C+A+G 3' (SEQ ID NO: 125); LNA 16.3 5' +C+T+CCCTAATGTTA+C+A+G 3' (SEQ ID NO:126);

LNA 18.1 5' +T +A+A+ AACCAACAC+T + A+C+C 3' (SEQ ID NO: 127); LNA 18.2 5' +A+A+A+CCAACACTA+C+C+A+C 3' (SEQ ID NO: 128); LNA 18.3 5' +A+A+ACCAACACTAC+C+A+C 3' (SEQ ID NO:129);

LNA 18.4 5' +A+C+C+AACACTACCA+C+A+T 3' (SEQ ID NO:130); and LNA 18.5 5' +C+A+A+CACTACCAC+A+T+G+A 3' (SEQ ID NO:131).

In certain embodiments, the oligonucleotide has the sequence miR, or a derivative thereof, as shown in Table 1, where a “+” before the letter denotes LNA nucleotides, or other chemically modified nucleotides (e.g., as described herein), and other letters denote DNA nucleotides (i.e., deoxyribonucleotide units):

Table 1: miR-Targeting LNAs

In certain embodiments, the oligonucleotide has the sequence Cov, or a derivative thereof, as shown in Table 8, where a “+” before the letter denotes LNA nucleotides, or other chemically modified nucleotides (e.g., as described herein), and other letters denote DNA nucleotides (i.e., deoxyribonucleotide units):

Table 2: Cov Targeting LNAs

In certain embodiments, the oligonucleotide comprises the sequence: LNA 1.1 5’ +G+A+CGTGATATATG+T+G+G 3’ (SEQ ID NO:64). In certain embodiments, the oligonucleotide comprises the sequence: LNA 1.2 5’ +C+G+TGATATATGTG+G+T+A 3’ (SEQ ID NO:65). In certain embodiments, the oligonucleotide comprises the sequence: LNA 1.3 5’ +G+A+TATGTGGTAC+C+A+T 3’ (SEQ ID NO:66). In certain embodiments, the oligonucleotide comprises the sequence: LNA 1.45’ +T+G+GTACCATGT+C+A+C 3’ (SEQ ID NO:67). In certain embodiments, the oligonucleotide comprises the sequence: LNA 2.1 5’ +C+C+TCAGCAGCAG+A+T+T+T 3’ (SEQ ID NO:68). In certain embodiments, the oligonucleotide comprises the sequence: LNA 2.2 5’ +T+C+AGCAGCAGAT+T+T+C 3’ (SEQ ID NO:69). In certain embodiments, the oligonucleotide comprises the sequence: LNA 2.3 5’ +C+A+G+CAGCAGAT+T+T+C 3’ (SEQ ID NO:70). In certain embodiments, the oligonucleotide comprises the sequence: LNA 2.45’ +C+A+GATTTCTTAGT+G+A+C 3’ (SEQ ID NO:71). In certain embodiments, the oligonucleotide comprises the sequence: LNA 3.1 5’ +T+G+CAACACGGAC+G+A+A+A 3’ (SEQ ID NO:72). In certain embodiments, the oligonucleotide comprises the sequence: LNA 3.2 5’ +G+C+AACACGGACG+A+A+A+C 3’ (SEQ ID NO:73). In certain embodiments, the oligonucleotide comprises the sequence: LNA 3.5 5’ +A+C+GAAACCCGTA+A+G+C+A 3’ (SEQ ID NO:74). In certain embodiments, the oligonucleotide comprises the sequence: LNA 3.6 5’ +A+C+G+AAACCCGTA+A+G+C+A 3’ (SEQ ID NO:75). In certain embodiments, the oligonucleotide comprises the sequence: LNA 4.1 5’ +A+A+CATGTCTGGAC+C+T+A 3’ (SEQ ID NO:76). In certain embodiments, the oligonucleotide comprises the sequence: LNA 4.2 5’ +A+C+ATGTCTGGAC+C+T+A+T 3’ (SEQ ID NO:77). In certain embodiments, the oligonucleotide comprises the sequence: LNA 5.1 5’ +T+G+AATATGACAT+A+G+T 3’ (SEQ ID NO:78). In certain embodiments, the oligonucleotide comprises the sequence: LNA 5.2 5’ +A+A+TATGACATA+G+T+C 3’ (SEQ ID NO:79). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.1 5’ +C+A+CAGATTTTAAA+G+T+T 3’ (SEQ ID NO:80). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.2 5’ +C+A+C+AGATTTTAA+A+G+T+T 3’ (SEQ ID NO:81). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.4 5’ +G+A+TTTTAAAGT+T+C+G+T 3’ (SEQ ID NO:82). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.5 5’ +T+A+A+AGTTCGTT+T+A+G+A 3’

(SEQ ID NO:83). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.6 5’ +A+A+A+GTTCGTTT+A+G+A 3’ (SEQ ID NO:84). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.7 5’ +T+C+GTTTAGAGAAC+A+G+A+T 3’ (SEQ ID NO: 85). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.8 5’ +A+G+AGAACAGATCT+A+C+A 3’ (SEQ ID NO:86). In certain embodiments, the oligonucleotide comprises the sequence: LNA 6.9 5’ +A+G+ATCTACAAG+A+G+A 3’ (SEQ ID NO:87). In certain embodiments, the oligonucleotide comprises the sequence: LNA 7.2 5’ +C+A+G+GCAAACTGAG+T+T+G 3’ (SEQ ID NO:88). In certain embodiments, the oligonucleotide comprises the sequence: LNA 7.3 5’ +C+A+G+GCAAACTG+A+G+T 3’ (SEQ ID NO:89). In certain embodiments, the oligonucleotide comprises the sequence: LNA 7.5 5’ +A+A+ACTGAGTTG+G+A+C 3’ (SEQ ID NO: 90). In certain embodiments, the oligonucleotide comprises the sequence: LNA 7.7 5’ +G+A+GTTGGACGTG+T+G+T 3’ (SEQ ID NO: 91). In certain embodiments, the oligonucleotide comprises the sequence: LNA 7.9 5’ +G+G+ACGTGTGTTTT+C+T+C 3’ (SEQ ID NO: 92). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.1 5’ +T+T+TCGGTCACACC+C+G+G (SEQ ID NO: 93). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.2 5’ +C+G+GTCACACCCGG+A+C+G 3’ (SEQ ID NO:94). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.3 5’ +T+C+ACACCCGGACG+A+A+A 3’

(SEQ ID NO:95). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.4 5’ +C+A+CCCGGACGA+A+A+C 3’ (SEQ ID NO:96). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.5 5’ +C+A+CCCGGACGAA+A+C+C 3’ (SEQ ID NO:97). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.6 5’ +C+A+CCCGGACGAAA+C+C+T 3’ (SEQ ID NO:98). In certain embodiments, the oligonucleotide comprises the sequence: LNA 8.7 5’ +C+C+GGACGAAAC+C+T+A 3’ (SEQ ID NO:99). In certain embodiments, the oligonucleotide comprises the sequence: LNA 10.1 5’ +T+C+GATCGTACTC+C+G+C 3’ (SEQ ID NO: 100). In certain embodiments, the oligonucleotide comprises the sequence: LNA 10.45’ +A+C+TCGATCGT+A+C+T+C 3’ (SEQ ID NO:101). In certain embodiments, the oligonucleotide comprises the sequence: LNA 10.5 5’ +C+G+TGGCCTCGG+T+G+A+A 3’ (SEQ ID NO: 102). In certain embodiments, the oligonucleotide comprises the sequence: LNA 10.6 5’ +G+T+GGCCTCGG+T+G+A+A 3’ (SEQ ID NO:103). In certain embodiments, the oligonucleotide comprises the sequence: LNA 11.1 5’ +G+T+GGCCTCGG+T+G+A+A 3’ (SEQ ID NO:104). In certain embodiments, the oligonucleotide comprises the sequence: LNA 11.2 5’ +T+A+A+AGATTGCT+A+T+G+T+G 3’ (SEQ ID NO: 105). In certain embodiments, the oligonucleotide comprises the sequence: LNA

11.3 5’ +A+A+G+ATTGCTATG+T+G+A+G 3’ (SEQ ID NO:106). In certain embodiments, the oligonucleotide comprises the sequence: LNA 11.5 5’ +T+A+T+GTGAGTTAA+A+G+T+T 3’ (SEQ ID NO: 107). In certain embodiments, the oligonucleotide comprises the sequence: LNA

11.6 5’ +T+G+T+GAGTTAAAGT+T+A+A 3’ (SEQ ID NO: 108). In certain embodiments, the oligonucleotide comprises the sequence: LNA 12.1 5’ +T+C+GTAGAAGCCTT+T+T+G 3’

(SEQ ID NO: 109). In certain embodiments, the oligonucleotide comprises the sequence: LNA

12.4 5’ +A+A+G+CCTTTTGGC+A+A+T+G 3’ (SEQ ID NO: 110). In certain embodiments, the oligonucleotide comprises the sequence: LNA 12.5 5’ +T+T+G+GCAATGTTG+T+T+C+C 3’ (SEQ ID NO: 111). In certain embodiments, the oligonucleotide comprises the sequence: LNA

12.7 5’ +C+T+TGAGGAAG+T+T+G+T 3’ (SEQ ID NO: 112). In certain embodiments, the oligonucleotide comprises the sequence: LNA 12.8 5’ +A+G+G+AAGTTGTAG+C+A+C+G 3’ (SEQ ID NO: 113). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.1 5’ +C+T+A+CTAAAATTA+A+T+T 3’ (SEQ ID. NO: 114). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.45’ +A+A+T+TAATTTTAC+A+C+A+T 3’ (SEQ ID NO: 115). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.5 5’ +A+T+T+TTACACATT+A+G+G+G 3’ (SEQ ID NO:116). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.6 5’ +T+A+CACATTAGGG+C+T+C 3' (SEQ ID NO:117). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.7 5’ +A+C+ATTAGGGCTC+T+T+C 3’ (SEQ ID NO: 118). In certain embodiments, the oligonucleotide comprises the sequence: LNA 13.9 5’ +G+C+T+CTTCCATAT+A+G+G 3’

(SEQ ID NO: 119). In certain embodiments, the oligonucleotide comprises the sequence: LNA

14.1 5’ +A+A+GGCTCTCCATC+T+T+A 3’ (SEQ ID NO: 120). In certain embodiments, the oligonucleotide comprises the sequence: LNA 14.2 5’ +A+A+G+GCTCTCC+A+T+C+T 3’

(SEQ ID NO: 121). In certain embodiments, the oligonucleotide comprises the sequence: LNA

14.3 5’ +G+C+TCTCCATCTTA+C+C+T 3’ (SEQ ID NO: 122). In certain embodiments, the oligonucleotide comprises the sequence: LNA 14.45’ +T+C+CATCTTACCTT+T+C+G 3’ (SEQ ID NO:123). In certain embodiments, the oligonucleotide comprises the sequence: LNA 16.1 5’ +C+C+T+CCCTAATGT+T+A+C+A 3’ (SEQ ID NO:124). In certain embodiments, the oligonucleotide comprises the sequence: LNA 16.2 5’ +C+T+C+CCTAATGTT+A+C+A+G 3’ (SEQ ID NO: 125). In certain embodiments, the oligonucleotide comprises the sequence: LNA

16.3 5’ +C+T+CCCTAATGTTA+C+A+G 3’ (SEQ ID NO: 126). In certain embodiments, the oligonucleotide comprises the sequence: LNA 18.1 5’ +T+A+A+AACCAACAC+T+A+C+C 3’ (SEQ ID NO: 127). In certain embodiments, the oligonucleotide comprises the sequence: LNA

18.2 5’ +A+A+A+CCAACACTA+C+C+A+C 3’ (SEQ ID NO: 128). In certain embodiments, the oligonucleotide comprises the sequence: LNA 18.3 5’ +A+A+ACCAACACTAC+C+A+C 3’ (SEQ ID NO: 129). In certain embodiments, the oligonucleotide comprises the sequence: LNA

18.4 5’ +A+C+C+AACACTACCA+C+A+T 3’ (SEQ ID NO: 130). In certain embodiments, the oligonucleotide comprises the sequence: LNA 18.5 5’ +C+A+A+CACTACCAC+A+T+G+A 3’ (SEQ ID NO:131).

It will be understood that for any of the sequences LNA1.1-LNA18.5 (SEQ ID NO: 64)- (SEQ ID NO: 131), one or more of the LNA nucleotides can be replaced with a modified nucleotide 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 can be replaced with a BNA nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with an ENA nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a cEt nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a 2'-modified nucleotide (e.g., substituted with MOE and other substituents as described herein).

In some embodiments, the oligonucleotide comprises a sequence selected from:

5' GCGACCAAAAGAATT 3' (SEQ ID NO:132);

5' CGACCAAAAGAATTC 3' (SEQ ID NO: 133);

5' GCGACCAAAAGAATTC 3' (SEQ ID NO: 134);

5' GACCAAAAGAATTC 3' (SEQ ID NO:135);

5' CGACCAAAAGAATTCGG 3' (SEQ ID NO:136);

5' GACCAAAAGAATTCGG 3' (SEQ ID NO:137);

5' ACCAAAAGAATTCGGA 3' (SEQ ID NO:138);

5' CAAAAGAATTCGGA 3' (SEQ ID NO:139);

5' CAAAAGAATTCGGAT 3' (SEQ ID NO:140);

5' AGCATACTTACTGACA 3' (SEQ ID NO:141);

5' CATACTTACTGACAG 3' (SEQ ID NO:142);

5' ATACTTACTGACAG 3' (SEQ ID NO:143);

5' ATACTTACTGACAGC 3' (SEQ ID NO:144);

5' TACTTACTGACAGC 3' (SEQ ID NO:145);

5' ATACTTACTGACAGCC 3' (SEQ ID NO: 146);

5' TACTTACTGACAGCC 3' (SEQ ID NO:147);

5' TACTTACTGACAGCCA 3' (SEQ ID NO:148);

5' ACTTACTGACAGCCA 3' (SEQ ID NO:149);

5' CTTACTGACAGCCAG 3' (SEQ ID N0:150);

5' TTACTGACAGCCAGA 3' (SEQ ID NO:151);

5' AGCCAGACAGCGA 3' (SEQ ID NO:152);

5' CAGCCAGACAGCGAC 3' (SEQ ID NO:153);

5' CAGCCAGACAGCGA 3' (SEQ ID NO:154);

5' ACAGCCAGACAGCGA 3' (SEQ ID NO:155);

5' GACAGCCAGACAGCG 3' (SEQ ID NO:156);

5' CCATCAATTAGTGTCG 3' (SEQ ID NO:157);

5' GCCATCAATTAGTGTG 3' (SEQ ID NO:158);

5' AAGAATTCGGATGGC 3' (SEQ ID NO: 159);

5' CAGACAGCGACCAA 3' (SEQ ID NO:160); and 5' TGACAGCCAGACAGC 3' (SEQ ID NO:161);

In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCGACCAAAAGAATT 3’ (SEQ ID NO:132). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CGACCAAAAGAATTC 3’ (SEQ ID NO: 133). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCGACCAAAAGAATTC 3’ (SEQ ID NO: 134). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GACCAAAAGAATTC 3’ (SEQ ID NO: 135). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CGACCAAAAGAATTCGG 3’ (SEQ ID NO: 136). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GACCAAAAGAATTCGG 3’

(SEQ ID NO: 137). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACCAAAAGAATTCGGA 3’ (SEQ ID NO:138). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAAAAGAATTCGGA 3’ (SEQ ID NO: 139) In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAAAAGAATTCGGAT 3’ (SEQ ID NO:140). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AGCATACTTACTGACA 3’ (SEQ ID NO: 141). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CATACTTACTGACAG 3’ (SEQ ID NO: 142). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ATACTTACTGACAG 3’ (SEQ ID NO: 143). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ATACTTACTGACAGC 3’ (SEQ ID NO: 144). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TACTTACTGACAGC 3’ (SEQ ID NO: 145). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ATACTTACTGACAGCC 3’ (SEQ ID NO: 146). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TACTTACTGACAGCC 3’ (SEQ ID NO: 147). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TACTTACTGACAGCCA 3’ (SEQ ID NO: 148). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACTTACTGACAGCCA 3’ (SEQ ID NO:149). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CTTACTGACAGCCAG 3’ (SEQ ID NO: 150). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TTACTGACAGCCAGA 3’ (SEQ ID NO: 151). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AGCCAGACAGCGA 3’ (SEQ ID NO: 152). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGCCAGACAGCGAC 3’ (SEQ ID NO: 153). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGCCAGACAGCGA 3’ (SEQ ID NO:154). In certain embodiments, the oligonucleotide comprises the sequence: 5’ ACAGCCAGACAGCGA 3’ (SEQ ID NO: 155). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GACAGCCAGACAGCG 3’ (SEQ ID NO: 156). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CCATCAATTAGTGTCG 3’ (SEQ ID NO: 157). In certain embodiments, the oligonucleotide comprises the sequence: 5’ GCCATCAATTAGTGTG 3’ (SEQ ID NO: 158). In certain embodiments, the oligonucleotide comprises the sequence: 5’ AAGAATTCGGATGGC 3’ (SEQ ID NO:159). In certain embodiments, the oligonucleotide comprises the sequence: 5’ CAGACAGCGACCAA 3’ (SEQ ID NO:160). In certain embodiments, the oligonucleotide comprises the sequence: 5’ TGACAGCCAGACAGC 3’ (SEQ ID NO:161).

In certain embodiments, the oligonucleotide has one of the following sequences where a “+” before the letter denote LNA nucleotides and other letters denote DNA nucleotides (i.e., deoxyribonucleotide units):

IV14 1 5' +G+C+G+ACCAAAAGA+A+T+T 3' (SEQ ID NO:162);

IV14 2 5' +G+C+G+ACCAAAAG+A+A+T+T 3' (SEQ ID NO:163);

IV14_7 5' +C+G+ACCAAAAGA+A+T+T+C 3' (SEQ ID NO:164);

IV14_9 5' +C+G+ACCAAAAGAA+T+T+C 3' (SEQ ID NO:165);

IV14_145' +G+C+G+ACCAAAAGA+A+T+T+C 3' (SEQ ID NO: 166);

IV14 15 5' +G+C+GACCAAAAGA+A+T+T+C 3' (SEQ ID NO:167);

IV14_16 5' +G+C+G ACCAAAAG AA+T +T +C 3' (SEQ ID NO:168);

IV14 22 5' +G+A+CCAAAAGAA+T+T+C 3' (SEQ ID NO:169);

IV14_27 5' +C+G+ACCAAAAG AAT +T +C+G+G 3' (SEQ ID NO: 170);

IV14 31 5' +G+A+CCAAAAGAATT+C+G+G 3' (SEQ ID NO:171);

IV14 32 5' +A+C+C+AAAAG AATT +C+G+G+A 3' (SEQ ID NO: 172);

IV14_37 5' +C+A+A+AA6AATT+G+G+A 3' (SEQ ID NO: 173);

IV14_45 5' +C+A+A+AAGAATTC+G+G+A+T 3' (SEQ ID NO:174);

IV14_47 5' +C+A+AAAGAATTC+G+G+A+T 3' (SEQ ID NO:175);

IV9 1 5' +A+G+C+ATACTTACT+G+A+C+A 3' (SEQ ID NO:176);

IV9 3 5' +C+A+T+ACTTACTG+A+C+A+G 3' (SEQ ID NO:177);

IV9_5 5' +A+T+ACTTACTGA+C+A+G 3' (SEQ ID NO:178);

IV9_6 5' +A+T+A+CTTACTG+A+C+A+G 3' (SEQ ID NO:179);

IV9 13 5' +A+T+ACTTACTGAC+A+G+C 3' (SEQ ID NO:180);

IV9 145' +A+T+A+CTTACTGA+C+A+G+C 3' (SEQ ID NO:181);

IV9_15 5' + A+T+ A+ CTT ACTGAC+A+G+C 3' (SEQ ID NO: 182);

IV9 16 5' +A+T +ACTT ACT G A+C+A+G +C 3' (SEQ ID NO: 183);

IV9 18 5' +T+A+CTTACTGAC+A+G+C 3' (SEQ ID NO:184);

IV9 19 5' +T+A+CTTACTGA+C+A+G+C 3' (SEQ ID NO:185);

IV9 205' +T+A+C+TTACTGA+C+A+G+C 3' (SEQ ID NO: 186);

IV9_21 5' +A+T+A+CTTACTGAC+A+G+C+C 3' (SEQ ID NO: 187);

IV9 22 5' +A+T+ACTTACTGACA+G+C+C 3' (SEQ ID NO: 188); IV9 23 5' +A+T+ACTTACTGAC+A+G+C+C (SEQ ID NO: 189);

IV9 25 5' +T+A+C+TTACTGAC+A+G+C+C 3' (SEQ ID N0:190);

IV9 26 5' +T+A+CTTACTGACA+G+C+C 3' (SEQ ID NO:191);

IV9 28 5' +T+A+C+TTACTGACA+G+C+C+A 3' (SEQ ID NO:192);

IV9 29 5' +T+A+CTTACTGACA+G+C+C+A 3' (SEQ ID NO:193);

IV9 30 5' +T+A+C+TTACTGACAG+C+C+A 3' (SEQ ID NO:194);

IV9 31 5' +T+A+CTTACTGACAG+C+C+A 3' (SEQ ID NO:195);

IV9 32 5' +A+C+TTACTGACA+G+C+C+A 3' (SEQ ID NO: 196);

IV9_33 5' +A+C+TTACTGACAG+C+C+A 3' (SEQ ID NO:197);

IV9 35 5' +A+C+T+TACTGACA+G+C+C+A 3' (SEQ ID NO:198);

IV9_41 5' +C+T +T +ACTG ACAG +C+C+ A+G 3' (SEQ ID NO:199);

IV9_45 5' +T+T+ACTGACAGCC+A+G+A 3' (SEQ ID NO:200);

IV22 5' +A+G+C+CAGACAG+C+G+A 3' (SEQ ID NO:201);

IV22_2 5' +C+A+G+CCAGACAG+C+G+A+C 3' (SEQ ID NO:202);

IV22 3 5' +C+A+GCCAGACAG+C+G+A+C 3' (SEQ ID N0:203);

IV22 5 5' +C+A+GCCAGACA+G+C+G+A 3' (SEQ ID NO:204);

IV22 6 5' +C+A+GCCAGACAG+C+G+A 3' (SEQ ID NO: 205)

IV22 7 5' +C+A+G+CCAGACAG+C+G+A 3' (SEQ ID NO:206)

IV22 8 5' +A+C+AG CCAGACAG+C+G+A 3' (SEQ ID NO:207);

IV22 9 5' +A+C+A+GCCAGACA+G+C+G+A 3' (SEQ ID N0:208);

IV22 10 5' +A+C+AGCCAGACA+G+C+G+A 3' (SEQ ID N0:209);

IV22 11 5' +G+A+C+AGCCAGACA+G+C+G 3' (SEQ ID NO:210);

IV22_13 5' +G+A+CAGCCAGACA+G+C+G 3' (SEQ ID NO:211);

IV22 14 5' +G+A+C+AGCCAGAC+A+G+C+G 3' (SEQ ID NO:212);

IV25 5' +C+C+ATCAATTAGTG+T+C+G 3' (SEQ ID NO:213);

IV26 5' +G+C+CATCAATTAGT+G+T+G 3' (SEQ ID NO:214);

IV27 5' +A+A+G+AATTCGGA+T+G+G+C 3' (SEQ ID NO:215);

IV28 5' +C+A+GACAGCGAC+C+A+A 3' (SEQ ID NO:216); and IV29 5' +T+G+ACAGCCAGAC+A+G+C 3' (SEQ ID NO:217).

In certain embodiments, the oligonucleotide comprises the sequence: IV14_1 5’ +G+C+G+ACCAAAAGA+A+T+T 3’ (SEQ ID NO: 162). In certain embodiments, the oligonucleotide comprises the sequence: IV14_25’ +G+C+G+ACCAAAAG+A+A+T+T 3’

(SEQ ID NO:163). In certain embodiments, the oligonucleotide comprises the sequence: IV14_7 5’ +C+G+ACCAAAAGA+A+T+T+C 3’ (SEQ ID NO: 164). In certain embodiments, the oligonucleotide comprises the sequence: IV14_95’ +C+G+ACCAAAAGAA+T+T+C 3’ (SEQ ID NO:165). In certain embodiments, the oligonucleotide comprises the sequence: IV14_145’ +G+C+G+ACCAAAAGA+A+T+T+C 3’ (SEQ ID NO: 166). In certain embodiments, the oligonucleotide comprises the sequence: IV14_15 5’ +G+C+GACCAAAAGA+A+T+T+C 3’ (SEQ ID NO: 167). In certain embodiments, the oligonucleotide comprises the sequence:

IV14_165’ +G+C+GACCAAAAGAA+T+T+C 3’ (SEQ ID NO: 168). In certain embodiments, the oligonucleotide comprises the sequence: IV14_22 5’ +G+A+CCAAAAGAA+T+T+C 3’ (SEQ ID NO: 169). In certain embodiments, the oligonucleotide comprises the sequence:

IV14_275’ +C+G+ACCAAAAGAAT+T+C+G+G 3’ (SEQ ID NO:170). In certain embodiments, the oligonucleotide comprises the sequence: IV 14_31 5’ +G+A+CCAAAAGAATT+C+G+G 3’ (SEQ ID NO:171). In certain embodiments, the oligonucleotide comprises the sequence: IV14_32 5’ +A+C+C+AAAAGAATT+C+G+G+A 3’ (SEQ ID NO: 172). In certain embodiments, the oligonucleotide comprises the sequence:

IV14_375’ +C+A+A+AAGAATTC+G+G+A 3’ (SEQ ID NO: 173). In certain embodiments, the oligonucleotide comprises the sequence: IV14_45 5’ +C+A+A+AAGAATTC+G+G+A+T 3’ (SEQ ID NO: 174). In certain embodiments, the oligonucleotide comprises the sequence:

IV14_475’ +C+A+AAAGAATTC+G+G+A+T 3^' (SEQ ID NO: 175). In certain embodiments, the oligonucleotide comprises the sequence: IV9_1 5’ +A+G+C+ATACTTACT+G+A+C+A 3’ (SEQ ID NO: 176). In certain embodiments, the oligonucleotide comprises the sequence: IV9_3 5’ +C+A+T+ACTTACTG+A+C+A+G 3’ (SEQ ID NO: 177). In certain embodiments, the oligonucleotide comprises the sequence: IV9_5 5’ +A+T+ACTTACTGA+C+A+G 3’ (SEQ ID NO: 178). In certain embodiments, the oligonucleotide comprises the sequence: IV9_6 5’ +A+T+A+CTTACTG+A+C+A+G 3’ (SEQ ID NO: 179). In certain embodiments, the oligonucleotide comprises the sequence: IV9_13 5’ +A+T+ACTTACTGAC+A+G+C 3’ (SEQ ID NO: 180). In certain embodiments, the oligonucleotide comprises the sequence: IV9_145’ +A+T+A+CTTACTGA+C+A+G+C 3’ (SEQ ID NO:181). In certain embodiments, the oligonucleotide comprises the sequence: IV9_15 5’ +A+T+A+CTTACTGAC+A+G+C 3’ (SEQ ID NO:182). In certain embodiments, the oligonucleotide comprises the sequence: IV9_16 5’ +A+T+ACTTACTGA+C+A+G+C 3’ (SEQ ID NO: 183). In certain embodiments, the oligonucleotide comprises the sequence: IV9_18 5' +T+A+CTTACTGAC+A+G+C 3’ (SEQ ID NO: 184). In certain embodiments, the oligonucleotide comprises the sequence: IV9_19 5’ +T+A+CTTACTGA+C+A+G+C 3’ (SEQ ID NO: 185). In certain embodiments, the oligonucleotide comprises the sequence: IV9_205’ +T+A+C+TTACTGA+C+A+G+C 3’ (SEQ ID NO: 186). In certain embodiments, the oligonucleotide comprises the sequence: IV9_21 5’ +A+T+A+CTTACTGAC+A+G+C+C 3’ (SEQ ID NO: 187). In certain embodiments, the oligonucleotide comprises the sequence: IV9_225’ +A+T+ACTTACTGACA+G+C+C 3’ (SEQ ID NO: 188). In certain embodiments, the oligonucleotide comprises the sequence: IV9_23 5’ +A+T+ACTTACTGAC+A+G+C+C (SEQ ID NO: 189). In certain embodiments, the oligonucleotide comprises the sequence: IV9_25 5' +T+A+C+TTACTGAC+A+G+C+C 3’ (SEQ ID NO:190). In certain embodiments, the oligonucleotide comprises the sequence: IV9_26 5’ +T+A+CTTACTGACA+G+C+C 3’ (SEQ ID NO:191). In certain embodiments, the oligonucleotide comprises the sequence: IV9_28 5’ +T+A+C+TTACTGACA+G+C+C+A 3’ (SEQ ID NO: 192). In certain embodiments, the oligonucleotide comprises the sequence: IV9_29 5’ +T+A+CTTACTGACA+G+C+C+A 3’ (SEQ ID NO:193). In certain embodiments, the oligonucleotide comprises the sequence: IV9_305’ +T+A+C+TTACTGACAG+C+C+A 3’ (SEQ ID NO: 194). In certain embodiments, the oligonucleotide comprises the sequence: IV9_31 5’ +T+A+CTTACTGACAG+C+C+A 3’ (SEQ ID NO: 195). In certain embodiments, the oligonucleotide comprises the sequence: IV9_325’ +A+C+TTACTGACA+G+C+C+A 3’ (SEQ ID NO:196). In certain embodiments, the oligonucleotide comprises the sequence: IV9_33 5’ +A+C+TTACTGACAG+C+C+A 3’ (SEQ ID NO:197). In certain embodiments, the oligonucleotide comprises the sequence: IV9_35 5’ +A+C+T+TACTGACA+G+C+C+A 3’ (SEQ ID NO: 198). In certain embodiments, the oligonucleotide comprises the sequence: IV9_41 5’ +C+T+T+ACTGACAG+C+C+A+G 3’ (SEQ ID NO: 199). In certain embodiments, the oligonucleotide comprises the sequence: IV9_45 5’ +T+T+ACTGACAGCC+A+G+A 3’ (SEQ ID NO:200). In certain embodiments, the oligonucleotide comprises the sequence: IV225’ +A+G+C+CAGACAG+C+G+A 3’ (SEQ ID NO:201). In certain embodiments, the oligonucleotide comprises the sequence: IV22_25’ +C+A+G+CCAGACAG+C+G+A+C 3’

(SEQ ID NO:202). In certain embodiments, the oligonucleotide comprises the sequence: IV22_3 5’ +C+A+GCCAGACAG+C+G+A+C 3’ (SEQ ID NO:203). In certain embodiments, the oligonucleotide comprises the sequence: IV22_5 5’ +C+A+GCCAGACA+G+C+G+A 3’ (SEQ ID NO:204). In certain embodiments, the oligonucleotide comprises the sequence: IV22_6 5’ +C+A+GCCAGACAG+C+G+A 3’ (SEQ ID NO: 205). In certain embodiments, the oligonucleotide comprises the sequence: IV22_75’ +C+A+G+CCAGACAG+C+G+A 3’ (SEQ ID NO:206). In certain embodiments, the oligonucleotide comprises the sequence: IV22_8 5’ +A+C+AGCCAGACAG+C+G+A 3' (SEQ ID NO:207). In certain embodiments, the oligonucleotide comprises the sequence: IV22_95’ +A+C+A+GCCAGACA+G+C+G+A 3’

(SEQ ID NO:208). In certain embodiments, the oligonucleotide comprises the sequence:

IV22J05’ +A+C+AGCCAGACA+G+C+G+A 3’ (SEQ ID. NO:209). In certain embodiments, the oligonucleotide comprises the sequence: IV22_11 5’ +G+A+C+AGCCAGACA+G+C+G 3’ (SEQ ID NO:210). In certain embodiments, the oligonucleotide comprises the sequence:

IV22J3 5’ +G+A+CAGCCAGACA+G+C+G 3’ (SEQ ID NO:211). In certain embodiments, the oligonucleotide comprises the sequence: IV22_14 5’ +G+A+C+AGCCAGAC+A+G+C+G 3’ (SEQ ID NO:212). In certain embodiments, the oligonucleotide comprises the sequence: IV25 5’ +C+C+ATCAATTAGTG+T+C+G 3’ (SEQ ID NO:213). In certain embodiments, the oligonucleotide comprises the sequence: IV26 5’ +G+C+CATCAATTAGT+G+T+G 3’ (SEQ ID NO:214). In certain embodiments, the oligonucleotide comprises the sequence: IV27 5’ +A+A+G+AATTCGGA+T+G+G+C 3’ (SEQ ID NO:215). In certain embodiments, the oligonucleotide comprises the sequence: IV28 5’ +C+A+GACAGCGAC+C+A+A 3’ (SEQ ID NO:216). In certain embodiments, the oligonucleotide comprises the sequence: IV29 5’

+T +G+ AC AGCC AGAC+ A+G+C 3’ (SEQ ID NO:217).

It will be understood that for any of the sequences (SEQ ID NO: 162)-(SEQ ID NO:

217), one or more of the LNA nucleotides can be replaced with a modified nucleotide 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 can be replaced with a BNA nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with an ENA nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a cEt nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a 2'- modified nucleotide (e.g., substituted with MOE and other substituents as described herein).

It will also be understood that for any of the oligonucleotide sequences described above, one or more of the DNA nucleotides can be modified to provide a corresponding BNA, LNA, ENA, cEt, or 2’-modified nucleotide. In certain cases, 1, 2, 3, 4, or more of the DNA nucleotides can be replaced with a BNA nucleotide. In certain cases, 1, 2, 3, 4, or more of the DNA nucleotides can be modified to provide the corresponding LNA nucleotides. In certain cases, 1,

2, 3, 4, or more of the DNA nucleotides can be modified to provide the corresponding ENA nucleotides. In certain cases, 1, 2, 3, 4, or more of the DNA nucleotides can be modified to provide the corresponding cEt nucleotides. In certain cases, 1, 2, 3, 4, or more of the DNA nucleotides can be modified to provide a corresponding 2'-modified nucleotides (e.g., as described herein).

Sequence mutants of the oligonucleotide sequences described above are also encompassed by the present disclosure. It is understood that in any of the sequences described herein that 1, 2, 3, 4 or more of the nucleotide may be mutated to provide for a desirable property, such as enhanced inhibition activity, conjugation to a modifying agent, etc.

In some cases, any one of the sequences described herein (e.g., one of SEQ ID NO: 1- 217) is comprised in a longer sequence, e.g., includes additional 5’ and/or 3’ nucleotides. In certain instances, the subject oligonucleotide is 75 nucleotides or less in length, such as 50 or less, 40 or less, or 35 nucleotides or less in length. In certain instances, the subject oligonucleotide is 30 nucleotides or less in length, such as 25 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 nucleotides or less in length.

In some cases, the subject oligonucleotide compound comprises a sequence having a deletion relative to one of the sequences described herein (e.g., one of SEQ ID NO: 1-217). For example, a sequence where 1, 2 or 3 nucleotides are deleted from the 5’ and/or 3’ terminal of the sequence, e.g., one of SEQ ID NOs: 1-217. In some cases, the deletion sequence has one nucleotide missing from the 5’ terminal of one of SEQ ID NOs: 1-217. In some cases, the deletion sequence has one nucleotide missing from the 3’ terminal of one of SEQ ID NOs: 1-217. In some cases, the deletion sequence has two nucleotides missing from the 5’ terminal of one of SEQ ID NOs: 1-131. In some cases, the deletion sequence has two nucleotides missing from the 3’ terminal of one of SEQ ID NOs: 1-217.

In certain instances, the oligonucleotide comprises a sequence with at least 70% homology to any one of sequences (SEQ ID NO: 1)-(SEQ ID NO: 217) (e.g., as defined herein). In certain instances, the oligonucleotide comprises a sequence with 70% or more homology to any one of sequences (SEQ ID NO: 1)-(SEQ ID NO: 217), such as 75% or more, 80% or more, 85% or more, 90% or more or even more. In certain cases, the oligonucleotide comprises a sequence with from 70 to 80% homology to any one of the sequences (SEQ ID NO: 1)-(SEQ ID NO: 217). In certain cases, the oligonucleotide comprises a sequence with from 80% to 90% homology to any one of sequences (SEQ ID NO: 1)-(SEQ ID NO: 217). In certain cases, the oligonucleotide comprises a sequence with from 90% to 99% homology to any one of sequences (SEQ ID NO: 1)-(SEQ ID NO: 217).

In certain cases, the oligonucleotide sequence can include a mutation designed to cover single-nucleotide polymorphisms (SNPs) in a target conserved RNA secondary structure sequence. In some cases, the oligonucleotide is a modified version of any one of LNA1.1- LNA18.5 (SEQ ID NO:64-131) with single mutation sites that protect against conserved RNA secondary structure target sequences containing a nucleotide change with the LNA1.1-LNA18.5 target sequence. In some cases, the oligonucleotide is a modified version of any one of sequences (SEQ ID NO: 162-217) with single mutation sites that protect against conserved RNA secondary structure target sequences containing a nucleotide change with the SEQ ID NO: 162-217 target sequence. It is understood that SNP mutations of interest can be applied to any of the sequences described herein.

It will be understood that for any of the sequences (SEQ ID NO:64)-(SEQ ID NO:131) or (SEQ ID NO:162)-(SEQ ID NO: 217), one or more of the LNA nucleotides can be replaced with a modified nucleotide selected from ENA nucleotides, cEt nucleotides, and 2'-modified nucleotides. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with an ENA nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a cEt nucleotide. In certain cases, 1, 2, 3, 4, or more of the LNA nucleotides can be replaced with a 2'-modified nucleotide (e.g., substituted with MOE and other substituents as described herein).

In certain instances, the oligonucleotide has a maximum length that corresponds to the particular region of the conserved RNA secondary structure of CoV (e.g., a sub-region). In certain instances, the oligonucleotide length is 20 nucleotides or less, such as 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 even less.

Oligonucleotides may be chemically synthesized by methods known in the art (see Wagner et al. (1993), supra, and Milligan et al, supra.) Oligonucleotides may be chemically modified from the native phosphodiester structure, in order to increase their intracellular stability and binding affinity. A number of such modifications have been described in the literature, which alter the chemistry of the backbone, sugars or heterocyclic bases.

Among useful changes in the backbone chemistry are phosphorothioates; phosphorodithioates, where both of the non-bridging oxygens are substituted with sulfur; phosphoroamidites; alkyl phosphotriesters and boranophosphates. Achiral phosphate derivatives include 3'-0'-5'-S-phosphorothioate, 3'-S-5'-0-phosphorothioate, 3'-CH₂-5'-0-phosphonate, 3'- NH-5'-0-phosphoroamidate, and thiophosphoramidates. Peptide nucleic acids replace the entire ribose phosphodiester backbone with a peptide linkage. Sugar modifications are also used to enhance stability and affinity. The oc-anomer of deoxyribose may be used, where the base is inverted with respect to the natural b-anomer. In certain cases, the 2'-OH of the ribose sugar may be altered, e.g., as described herein. The 2'-OH of the ribose sugar may be altered to form 2'-0- methyl or 2'-0-allyl sugars, which provides resistance to degradation without comprising affinity. In certain cases, modification of the 2'-OH of the ribose sugar can improve toxicity. Modification of the heterocyclic bases must maintain proper base pairing. Some useful substitutions include deoxyuridine for deoxy thymidine; 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.

The oligonucleotide agents may be derivatized with any convenient modifying agent, e.g., by conjugation of the modifying agent to the 5’- and/or 3’terminal of the oligonucleotide sequence. In some cases, the modifying agent is a moiety that enhances cellular uptake (e.g., a lipid). Any convenient lipids may be conjugated to the subject oligonucleotides. In some instances, the modifying agent is a fatty acid, connected to the 5’ or 3’ terminal via an optional linker. The lipid group can be an aliphatic hydrocarbon or fatty acid, including but not limited to, derivatives of hydrocarbons and fatty acids, with examples being saturated straight chain compounds having 14-20 carbons, such as myristic (tetradecanoic) acid, palmitic (hexadecanoic) acid, and stearic (octadeacanoic) acid, and their corresponding aliphatic hydrocarbon forms, tetradecane, hexadecane and octadecane. Examples of other suitable lipid groups that may be employed are sterols, such as cholesterol, and substituted fatty acids and hydrocarbons, particularly polyfluorinated forms of these groups. The scope of the lipid group includes derivatives such as amine, amide, ester and carbamate derivatives.

In some cases, the modifying agent is a further nucleic acid sequence having a desirable activity (e.g., recruitment of an RNase, as described herein). In certain instances, the modifying agent has a specific binding activity that provides for 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 a cell-specific target of interest. In certain instances, the antibody modifying agent is specifically binds a hemagglutinin (HA) target.

The oligonucleotide active agent can be utilized in any convenient form. In some instances, the oligonucleotide active agent is single stranded. In some instances, the oligonucleotide active agent is double stranded. In some instances, the oligonucleotide active agent is an siRNA. In some instances, the oligonucleotide active agent is an shRNA. In some instances, the oligonucleotide active agent is a ssRNA. In some instances, one or more nucleotides of the ssRNA can be replaced with LNA nucleotides. In some instances, the oligonucleotide active agent is a ssDNA. In some instances, one or more nucleotides of the ssDNA can be replaced with LNA nucleotides.

METHODS OF TREATMENT

Methods of treatment of a Coronavirus

Aspects of the present disclosure include methods of treating or preventing a coronavirus (CoV) infection in a subject. The subject oligonucleotide compounds find use as a new class of antiviral therapeutics that can efficiently disrupt the RNA secondary structures of CoV, and thus treat or prevent the CoV infection in a subject. In some cases, the subject oligonucleotide compounds can inhibit a host miRNA interaction with CoV. In some instances the subject oligonucleotide compounds find use as a new antiviral therapeutic that can efficiently disrupt the RNA secondary structures of CoV, and disrupt packaging and completely prevent otherwise lethal disease in vivo. 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 to a subject in need thereof a therapeutically effective amount of a subject compound to treat the subject for an infection or prevent infection in the subject. By “a therapeutically effective amount” is meant the concentration of a compound that is sufficient to elicit the desired biological effect (e.g., treatment or prevent of the condition or disease, coronavirus infection). By “treatment” is meant that at least an amelioration of the symptoms associated with the condition afflicting the host is achieved, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. symptom, associated with the condition being treated. As such, treatment also includes situations where the pathological condition, or at least symptoms associated therewith, are partially or completely inhibited, e.g., prevented from happening, or stopped, e.g. terminated, such that the host no longer suffers from the condition, or at least the symptoms that characterize the condition. Thus treatment includes: (i) prevention, that is, reducing the risk of development of clinical symptoms, including causing the clinical symptoms not to develop, e.g., preventing disease progression to a harmful state; (ii) inhibition, that is, arresting the development or further development of clinical symptoms, e.g., mitigating or completely inhibiting an active disease (e.g., infection); (iii) relief, that is, causing the regression of clinical symptoms; (iv) prevents hospitalization or the need to be in a critically ill conditions that require intensive care unit (ICU); (v) shortens the length of hospitalization or ICU stay; (vi) prevents or shortens the need to have assisted ventilation including but not limited to supplemental oxygen, non-invasive positive pressure ventilation (CPAP and or BPAP), mechanical ventilation; (vi) prevents or shortens the need to have assisted cardiopulmonary bypass such as extracorporeal membrane oxygenation (ECMO) devices; (vii) prevent the development of super-imposed bacterial infections; and (viii) prevent death. In the context of coronavirus (CoV) infection, the term “treating” includes any or all of: reducing the number of viruses in the body, reducing the number of virally infected cells in a patient, inhibiting replication of intra and/or extracellular viruses and virus-infected cells, and ameliorating one or more symptoms associated with an infection. The subject to be treated can be one that is in need of therapy, where the host to be treated is one amenable to treatment using the subject compound. In some embodiments, the subject is one that is suspected of having a coronavirus infection. In certain embodiments, the subject is diagnosed as having a coronavirus infection. As such, in some cases, the subject is one who has been infected with the virus.

In certain cases, the subject is one who is at risk of being infected, or is suspected of being infected with the virus. The subject methods can be used to prevent infection of the subject with a coronavirus. By “prevention” is meant that the subject at risk of coronavirus infection is not infected despite exposure to the virus under conditions that would normally lead to infection or the subject’s infection severity is attenuated upon infection. In some cases, the administering of the subject active agent (e.g., oligonucleotide compound) protects the subject against infection instantaneously, for 1 week or more, such as 2 weeks or more, 3 weeks or more, 1 month or more, 2 months or more, 3 months or more, etc. Multiple doses of the subject compound can be administered according to the subject methods to provide for protection of the subject form infection for an extended period of time. The timing and dosage amounts can be readily determined using conventional methods.

In some cases, the subject methods of treatment include a step of determining or diagnosing whether the subject has a coronavirus infection. The determining step can be performed using any convenient methods. In some cases, the determining step includes obtaining a biological sample from the subject and assaying the sample for the presence of intra and/or extracellular viruses and virus-infected cells. The sample can be a cellular sample. The determining step can include identification of intra and/or extracellular viruses and virus-infected cells including a particular mutation.

Accordingly, a 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”, with humans being of interest. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys).

The amount of the subject compound administered can be determined using any convenient methods to be an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present disclosure will depend on the particular compound employed and the effect to be achieved, and the pharmacodynamics associated with each compound in the host. In some embodiments, an effective dosage of the subject compound is an effective volume in a mass concentration that ranges from about 50 ng/ml to about 50 pg/ml (e.g., from about 50 ng/ml to about 40 pg/ml, from about 30 ng/ml to about 20 pg/ml, from about 50 ng/ml to about 10 pg/ml, from about 50 ng/ml to about 1 pg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, from about 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml to about 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml).

In some embodiments, an effective amount of a subject compound is an amount that ranges from about 10 pg to about 100 mg, e.g., 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, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 pg, from about 1 pg to about 10 pg, 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, from about 750 pg to about 1 mg, from about 1 mg to about 50 mg, from about 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 amount 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 compound are administered. Where multiple doses are administered over a period of time, the subject compound can be administered twice daily (qid), daily (qd), every other day (qod), every third day, three times per week (tiw), or twice per week (biw) over a period of time. For example, a compound can be administered qid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, a compound can be administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors.

Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated with a subject method can be assayed for the presence and/or level of intra and/or extracellular viruses and virus-infected cells. Assessment of the effectiveness of the methods of treatment on the subject can include assessment of the subject before, during and/or after treatment, using any convenient methods. Aspects of the subject methods further include a step of assessing the therapeutic response of the subject to the treatment.

In some embodiments, the method includes assessing the condition of the subject, including diagnosing or assessing one or more symptoms of the subject which are associated with the disease or condition of interest being treated (e.g., as described herein). In some embodiments, the method includes obtaining a biological sample from the subject and assaying the sample, e.g., for the presence of intra and/or extracellular viruses and virus-infected cells or components thereof that are associated with the disease or condition of interest (e.g., as described herein). The sample can be a cellular sample. The assessment step(s) of the subject method can be performed at one or more times before, during and/or after administration of the subject compounds, using any convenient methods. In certain cases, the assessment step includes identification and/or quantitation of intra and/or extracellular viruses and virus-infected cells. In certain instances, assessing the subject include diagnosing whether the subject has a viral infection or symptoms thereof.

Methods of treatment of a Neoplastic Condition

Aspects of the present disclosure also include a method of treating or preventing a neoplastic condition 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 treats or prevents the neoplastic condition. In some cases, the neoplastic condition comprises lung cancer. In some cases, the pharmaceutical composition for treating the neoplastic condition may further comprise an additional active agent. Non-limiting examples of additional active agents include, an additional active agent or a chemotherapeutic agent (e.g., as described herein).

Examples of neoplastic conditions of interest include, but are not limited to, abdominal neoplasms; adenocarcinoma; adenoma; astrocytoma; blast crisis; brain neoplasms; breast neoplasms; bronchial neoplasms; Burkitt lymphoma; carcinoma; basal cell carcinoma; bronchogenic carcinoma; ductal carcinoma; hepatocellular carcinoma; non-small-cell lung carcinoma; renal cell carcinoma; squamous cell carcinoma; transitional cell carcinoma; neoplastic cell transformation; central nervous system neoplasms; colorectal neoplasms; cysts; digestive system neoplasms; endocrine gland neoplasms; Epstein-Barr virus infections; gastrointestinal neoplasms; genital neoplasms; gestational trophoblastic neoplasms; glioblastoma; glioma; gliosarcoma; hemangioma; capillary hemangioma; hematologic neoplasms; Hodgkin disease; inflammatory breast neoplasms; actinic keratosis; kidney neoplasms; leukemia; B-cell leukemia; hairy cell leukemia; large granular lymphocytic leukemia; lymphocytic, chronic, B-cell leukemia; lymphoid leukemia; myelogenous, chronic, BCR-ABL positive leukemia; myeloid, accelerated phase leukemia; myeloid, chronic, atypical, BCR-ABL negative leukemia; myeloid, chronic- phase leukemia; myelomonocytic, chronic leukemia; myelomonocytic, juvenile leukemia; neutrophilic, chronic leukemia; T-cell leukemia; lipoma; liver neoplasms; lung neoplasms; lymphoma; B-cell lymphoma; follicular lymphoma; large B-cell, diffuse lymphoma; large-cell, anaplastic lymphoma; large-cell, immunoblastic lymphoma; mantle-cell lymphoma; non-Hodgkin lymphoma; T-cell lymphoma; lymphomatoid granulomatosis; mastocytoma; mastocytosis; cutaneous mastocytosis; melanoma; mesothelioma; multiple myeloma; mycosis fungoides; neoplasm metastasis; adipose tissue neoplasms; connective and soft tissue neoplasms; connective tissue neoplasms; germ cell and embryonal neoplasms; glandular and epithelial neoplasms; nerve tissue neoplasms; neuroepithelial neoplasms; plasma cell neoplasms; second primary neoplasms; neoplastic processes; nerve sheath neoplasms; nervous system neoplasms; neurilemmoma; neuroblastoma; neuroectodermal tumors; neuroendocrine tumors; neuroma; acoustic neuroma; nevus; ovarian neoplasms; peritoneal neoplasms; precancerous conditions; precursor cell lymphoblastic leukemia-lymphoma; preleukemia; prostatic neoplasms; respiratory tract neoplasms; rhabdomyosarcoma; sarcoma; Kaposi sarcoma; Sezary syndrome; skin neoplasms; testicular neoplasms; thoracic neoplasms; thymoma; trophoblastic neoplasms; tumor virus infections; ureteral neoplasms; urinary bladder neoplasms; urogenital neoplasms; urologic neoplasms; urticaria pigmentosa; Waldenstrom macroglobulinemia; and Wilms tumor.

In certain embodiments, the neoplastic condition comprises a lung cancer. In some cases, the lung cancer is a lung adenocarcinoma. In some cases the lung cancer is non-small cell lung cancer (NSCLC).

In some embodiments, the subject method is an in vivo method that includes administering to a subject an effective amount of an agent or composition (e.g., as described herein) that specifically inhibits a lung cancer cell. An “effective amount” is an amount of a compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to inhibit a lung cancer by at least about 20% (20% inhibition), such as at least about 30% (30% inhibition), at least about 40% (40% inhibition), at least about 50% (50% inhibition), at least about 60% (60% inhibition), at least about 70% (70% inhibition), at least about 80% (80% inhibition), or at least about 90% (90% inhibition), compared to the lung cancer cell activity in the individual in the absence of treatment with the compound, or alternatively, compared to the lung cancer cell activity in the individual before or after treatment with the compound.

In some embodiments, a “therapeutically effective amount” is an amount of a compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to decrease tumor burden in the subject by at least about 20%, such as at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to tumor burden in the individual in the absence of treatment with the compound, or alternatively, compared to the tumor burden in the subject before treatment with the compound. As used herein the term “tumor burden” refers to the total mass of tumor tissue carried by a subject with a cancer (e.g., lung cancer).

In some embodiments, a “therapeutically effective amount” is an amount of a subject compound that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to reduce the dose of radiotherapy required to observe tumor shrinkage in the subject by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to the dose of radiotherapy required to observe tumor shrinkage in the individual in the absence of treatment with the compound.

In some embodiments, a “therapeutically effective amount” is an amount of a compound that, when admini tered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to decrease metastases burden in the subject by at least about 20%, such as at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to metastases burden in the individual in the absence of treatment with the compound, or alternatively, compared to the metastases burden in the subject before treatment with the compound. As used herein the term “metastases burden” refers to the total mass or number of metastases tissue carried by a subject with cancer (e.g., lung cancer).

In some embodiments, an effective amount of a compound for treating a neoplastic condition is an effective volume that ranges from about 50 ng/ml to about 50 pg/ml (e.g., from about 50 ng/ml to about 40 pg/ml, from about 30 ng/ml to about 20 pg/ml, from about 50 ng/ml to about 10 pg/ml, from about 50 ng/ml to about 1 pg/ml, from about 50 ng/ml to about 800 ng/ml, from about 50 ng/ml to about 700 ng/ml, from about 50 ng/ml to about 600 ng/ml, from about 50 ng/ml to about 500 ng/ml, from about 50 ng/ml to about 400 ng/ml, from about 60 ng/ml to about 400 ng/ml, from about 70 ng/ml to about 300 ng/ml, from about 60 ng/ml to about 100 ng/ml, from about 65 ng/ml to about 85 ng/ml, from about 70 ng/ml to about 90 ng/ml, from about 200 ng/ml to about 900 ng/ml, from about 200 ng/ml to about 800 ng/ml, from about 200 ng/ml to about 700 ng/ml, from about 200 ng/ml to about 600 ng/ml, from about 200 ng/ml to about 500 ng/ml, from about 200 ng/ml to about 400 ng/ml, or from about 200 ng/ml to about 300 ng/ml).

In some embodiments, an effective amount of a compound for treating a neoplastic condition is an amount that ranges from about 10 pg to about 100 mg, e.g., 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, from about 750 pg to about 1 ng, from about 1 ng to about 10 ng, from about 10 ng to about 50 ng, from about 50 ng to about 150 ng, from about 150 ng to about 250 ng, from about 250 ng to about 500 ng, from about 500 ng to about 750 ng, from about 750 ng to about 1 pg, from about 1 pg to about 10 pg, 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, from about 750 pg to about 1 mg, from about 1 mg to about 50 mg, from about 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 amount can range fromlO pg to 100 mg, or can range from 100 mg to about 500 mg, or can range from 500 mg to about 1000 mg or 3000 mg.

In some embodiments of treating a neoplastic condition, a single dose of a compound is administered. In other embodiments, multiple doses are administered. Where multiple doses are administered over a period of time, the compound can be admini tered twice daily (bid), daily (qd), every other day (qod), every third day, once per week(qw), three times per week (tiw), or twice per week (biw) over a period of time. For example, a compound is administered bid, qd, qod, tiw, or biw over a period of from one day to about 2 years or more. For example, a compound is administered at any of the aforementioned frequencies for one week, two weeks, one month, two months, six months, one year, or two years, or more, depending on various factors. In some embodiments, the compound may be administered orally, intravenously, subcutaneously, intramuscularly, via inhalation, topically, or sublingually, among other routes of administration, including depot administration. In some embodiments, the compound is administered in combination with an inhibitor of its metabolism, such as an inhibitor of cytochrome P450 3A/4 (e.g. ritonavir or cobicistat). In some embodiments, the compound may be administered in courses wherein “drug holidays” are allowed that may last from 1-7 days.

Administration of a therapeutically effective amount of a subject compound to an individual with a cancer (e.g. lung cancer) can result in one or more of: 1) a reduction in tumor burden; 2) a reduction in the dose of radiotherapy required to effect tumor shrinkage; 3) a reduction in the spread of a cancer from one location to another in an individual; 4) a reduction of morbidity or mortality in clinical outcomes; 5) shortening the total length of treatment when combined with other anti-cancer treatment modalities; 6) a decrease in the size or number of metastases; 7) an improvement in an indicator of disease response (e.g., a reduction in one or more symptoms of cancer); 8) prolonging disease-free survival of the subject, increasing the progression-free survival of the subject or increasing overall survival of the subject. Any of a variety of methods can be used to determine whether a treatment method is effective. For example, a biological sample obtained from an individual who has been treated with a subject method can be assayed, or an imaging study may be performed.

Any of the agents described herein can be utilized in the subject methods of treatment. In certain instances, the agent is of any one of oligonucleotides with a sequence identity selected from (SEQ ID Nos: 1-63). In certain instances, the oligonucleotide sequence has at least 70% sequence identity with a sequence selected from (SEQ ID NOs: 1-63). In certain instances, the agent is of any one of oligonucleotides with a sequence identity selected from LNA1.1 - LNA18.5 (SEQ ID Nos: 64-131). In certain instances, the oligonucleotide sequence has at least 70% sequence identity with a sequence selected from LNA1.1 - LNA18.5 (SEQ ID Nos: 64- 131). In certain instances, the agent is of any one of oligonucleotides with a sequence identity selected from (SEQ ID Nos: 132-161). In certain instances, the oligonucleotide sequence has at least 70% sequence identity with a sequence selected from (SEQ ID NOs: 132-161). In certain instances, the agent is of any one of oligonucleotides with a sequence identity selected from (SEQ ID Nos: 162-217). In certain instances, the oligonucleotide sequence has at least 70% sequence identity with a sequence selected from (SEQ ID Nos: 162-217).

In some embodiments, the subject with the neoplastic condition is mammalian. In certain instances, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). The subject may be in need of treatment for a neoplastic condition. In some instances, the subject methods include diagnosing a neoplastic condition, including any one of the neoplastic conditions described herein. In some embodiments, the compound is administered as a pharmaceutical preparation.

In certain embodiments, the agent is a modified compound that includes a label, and the method further includes detecting the label in the subject. The selection of the label depends on the means of detection. Any convenient labeling and detection systems may be used in the subject methods, see e.g., Baker, “The whole picture,” Nature, 463, 2010, p977-980. In certain embodiments, the compound includes a fluorescent label suitable for optical detection. In certain embodiments, the compound includes a radiolabel for detection using positron emission tomography (PET) or single photon emission computed tomography (SPECT). In some cases, the compound includes a paramagnetic label suitable for tomographic detection. The subject compound may be labeled, as described above, although in some methods, the compound is unlabeled and a secondary labeling agent is used for imaging.

Combination therapy

For use in the subject methods, subject agents and compositions described herein may be administered in combination with any other treatment modalities in clinical use including but not limited to the use of other pharmaceutically active agents, including other agents that heat the underlying condition or a symptom of the condition. “In combination with” as used herein refers to uses where, for example, the first compound is administered during the entire course of administration of the second compound; where the first compound is administered for a period of time that is overlapping with the administration of the second compound, e.g., where administration of the first compound begins before the administration of the second compound and the administration of the first compound ends before the administration of the second compound ends; where the administration of the second compound begins before the administration of the first compound and the administration of the second compound ends before the administration of the first compound ends; where the administration of the first compound begins before administration of the second compound begins and the administration of the second compound ends before the administration of the first compound ends; where the administration of the second compound begins before administration of the first compound begins and the administration of the first compound ends before the administration of the second compound ends. As such, “in combination” can also refer to regimen involving administration of two or more compounds. “In combination with” as used herein also refers to administration of two or more compounds that may be administered in the same or different formulations, by the same of different routes, and in the same or different dosage form type.

Examples of other agents used in combination therapy of a coronavirus infection include an additional oligonucleotide compound of the present disclosure and any convenient antiviral compounds or drugs of interest, including but not limited to Amantadine, Rimantadine, Zanamivir, Oseltamivir, Peramivir and the like. Examples of treatment modalities used for neoplastic diseases include surgical resection; radiation including proton beam irradiation, ablation, embolization, systemic therapy with chemotherapeutic agents, and combination of these aforementioned modalities.

Chemotherapeutic agents for use in combination therapy of neoplastic disease include, but are not limited to, an additional oligonucleotide compound of the present disclosure, thalidomide, marimastat, COL-3, BMS-275291, squalamine, 2-ME, SU6668, neovastat, Medi-522, EMD121974, CAI, celecoxib, interleukin- 12, IM862, TNP470, avastin, gleevec, herceptin, and mixtures thereof. Chemotherapy agents of the present invention can include any suitable chemotherapy agents or combinations of chemotherapy drugs (e.g., a cocktail). Exemplary' chemotherapy agents include, without limitation, alkylating agents, platinums, anti-metabolites, anthracy dines, taxanes, camptothecins, nitrosoureas, EGFR inhibitors, antibiotics, HER2/neu inhibitors, angiogenesis inhibitors, kinase inhibitors (e.g. sorafenib), proteasome inhibitors, immunotherapies, hormone therapies, photodynamic therapies, cancer vaccines, histone deacetylase inhibitors, sphingolipid modulators, oligomers, other unclassified chemotherapy agents and combinations thereof. Examples of chemotherapeutic agents for use in combination therapy include, but are not limited to, daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside, bis- chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D, mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen, dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea, nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin, 4-hydroxyperoxycyclophosphor- amide, 5- fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan, topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol (DES).

In the context of a combination therapy, combination therapy compounds may be administered by the same route of administration (e.g., intrapulmonary, oral, enteral, etc.) that the subject agents and compositions are administered. In the alternative, the compounds for use in combination therapy with the subject agent or composition may be administered by a different route of administration.

SCREENING METHODS

Aspects of the present disclosure also include screening assays configured to identify agents that find use in methods of the invention, e.g., as reviewed above. Aspects of the present disclosure include methods for screening a candidate agent for the ability to inhibit a coronavirus in a cell. In some instances, the method comprises: contacting a sample comprising viral RNA (vRNA) comprising a conserved RNA secondary structure of CoV with a candidate agent; and determining whether the candidate agent specifically binds to the conserved RNA secondary structure motif. In some cases, an agent that specifically binds to the conserved RNA secondary structure motif will treat the subject having the coronavirus infection. By assessing or determining is meant at least predicting that a given test compound will have a desirable activity, such that further testing of the compound in additional assays, such as animal model and/or clinical assays, is desired.

The candidate agent is selected from: a small molecule, an oligonucleotide, an antibody and a polypeptide. In some instances, the determining step comprises detecting a cellular parameter, wherein a change in the parameter in the cell as compared to in a cell not contacted with candidate agent indicates that the candidate agent specifically binds the conserved RNA secondary structure motif. 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 instances, the candidate agent is an oligonucleotide.

Drug screening may be performed using an in vitro model, a genetically altered cell or animal, or purified protein. One can identify ligands that compete with, modulate or mimic the action of a lead agent. Drug screening identifies agents that bind to particular sites of a conserved RNA secondary structure motif of CoV. A wide variety of assays may be used for this purpose, including labeled in vitro binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, and the like. Knowledge of the 3-dimensional structure of the conserved RNA secondary structure of CoV, derived from the structural studies described herein, can also lead to the rational design of small drugs that specifically inhibit coronavirus activity.

The term "agent" as used herein describes any molecule, e.g., oligonucleotide, protein or pharmaceutical, with the capability of binding a conserved RNA secondary structure of CoV to inhibit CoV. Generally, a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

Candidate agents encompass numerous chemical classes, such as oligonucleotides, antibodies, polypeptides, and organic molecules, e.g., small organic compounds having a molecular weight of more than 50 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. 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 may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Of interest in certain embodiments are compounds that pass the blood-brain barrier.

Where the screening assay is a binding assay, one or more of the molecules may be joined to a member of a signal producing system, e.g., a label, where the label can directly or indirectly provide a detectable signal. Various labels include, but are not limited to: radioisotopes, fluorescers, chemiluminescers, enzymes, specific binding molecules, particles, e.g., magnetic particles, and the like. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc. For the specific binding members, the complementary member would normally be labeled with a molecule that provides for detection, in accordance with known procedures.

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

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. 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 Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly); and the like.

Introduction

There is a critical need for effective antivirals to counter SARS-CoV-2/COVID-2019 infections. In the present disclosure, locked nucleic acid (LNA) therapeutics are developed against critical RNA features identified in SARS-CoV-2. The subject LNAs (e.g., as described herein) have the potential to represent a single dose preventative, therapeutic, and “just-in-time vaccine” for SARS-CoV-2, thereby changing the treatment paradigm for this devastating infection.

Accordingly, the subject LNA compounds represent a new class of anti-SARS-CoV-2 agents for investigational new drug (IND) -enabling studies. The subject LNAs find use in a treatment paradigm for SARS-CoV-2 infections for which no approved effective therapeutic exists.

Example 1: Computational Modeling

Using a suite of computational technology tools (ARNIE), predicted RNA secondary structures in regions of absolute sequence conservation across coronavirus B genomes including SARS-CoV-2 have been identified. Two tandem predicted microRNA 191 (miR191) binding sites within the 5 ’-most such structure were also identified.

Based on the predicted RNA secondary structures, novel LNAs to effectively counter SARS-CoV-2 are being developed. In particular, the identified RNA secondary structures, unique and conserved across corona B viruses, represent ideal candidate targets for disrupting the virus lifecycle, via structure-specific LNAs. Similarly, the miR191 binding sites within the 5’- most conserved RNA secondary structure possibly reflect a potential mechanism for regulating translation of corona B viruses that is amenable to targeting by specifically designed LNAs.

FIG. 1 illustrates the predicted RNA secondary structures conserved across corona B viruses. Genome map of the full NC_045512.2 nCovl9 reference genome (genomic positions shown) with 5’UTR, ORFs, and 3’UTR. Highly conserved regions from the trans-phylogenetic MSA shown as black bars (top); The top 6 scoring secondary structures from the conserved regions are indicated by yellow bars. Light blue = conserved nucleotides, grey = flanking 20 nucleotide context. Green = miR191 seed binding sites. Made in Geneious Prime 2019.

Example 2: Locked nucleic acid (LNA) design

Oligonucleotides containing locked nucleic acids (LNA) are custom synthesized from Exiqon. A “+” before the letter denotes LNA, and other letters denote typical (non-locked) DNA nucleotides. The LNAs are designed to be complementary to different sequences based on the predicted RNA secondary structures. Sequences of all LNAs are shown below.

LNA 1.1 5' +G+A+CGTGATATATG+T+G+G 3' (SEQ ID NO:64);

LNA 1.2 5' +C+G+TGATATATGTG+G+T+A 3' (SEQ ID NO:65);

LNA 1.3 5' +G+A+TATGTGGTAC+C+A+T 3' (SEQ ID NO:66);

LNA 1.45' +T+G+GTACCATGT+C+A+C 3' (SEQ ID NO:67);

LNA 2.1 5' +C+C+T CAG CAG CAG+ A+T +T +T 3' (SEQ ID NO:68);

LNA 2.2 5' +T+C+AGCAGCAGAT+T+T+C 3' (SEQ ID NO:69);

LNA 2.3 5' +C+A+G+CAGCAGAT+T+T+C 3' (SEQ ID NO:70);

LNA 2.45' +C+A+GATTTCTTAGT+G+A+C 3' (SEQ ID NO:71);

LNA 3.1 5' +T+G+CAACACGGAC+G+A+A+A 3' (SEQ ID NO:72);

LNA 3.2 5' +G+C+AACACGGACG+A+A+A+C 3' (SEQ ID NO:73);

LNA 3.5 5' +A+C+GAAACCCGTA+A+G+C+A 3' (SEQ ID NO:74);

LNA 3.6 5' +A+C+G+AAACCCGTA+A+G+C+A 3' (SEQ ID NO:75);

LNA 4.1 5' +A+A+CATGTCTGGAC+C+T+A 3' (SEQ ID NO:76);

LNA 4.2 5' + A+C+AT GTCTG G AC+C+T+A+T 3' (SEQ ID NO:77);

LNA 5.1 5' +T+G+AATATGACAT+A+G+T 3' (SEQ ID NO:78);

LNA 5.2 5' +A+A+TATGACATA+G+T+C 3' (SEQ ID NO:79);

LNA 6.1 5' +C+A+CAGATTTTAAA+G+T+T 3' (SEQ ID NO:80);

LNA 6.2 5' +C+A+C+AGATTTTAA+A+G+T+T 3' (SEQ ID NO:81);

LNA 6.45' +G+A+TTTTAAAGT+T+C+G+T 3' (SEQ ID NO:82);

LNA 6.5 5' +T+A+A+AGTTCGTT+T+A+G+A 3' (SEQ ID NO:83); LNA 6.6 5' +A+A+A+GTTCGTTT+A+G+A 3' (SEQ ID NO:84);

LNA 6.7 5' +T+C+GTTTAGAGAAC+A+G+A+T 3' (SEQ ID NO:85); LNA 6.8 5' +A+G+AGAACAGATCT+A+C+A 3' (SEQ ID NO:86);

LNA 6.9 5' +A+G+ATCTACAAG+A+G+A 3' (SEQ ID NO:87);

LNA 7.2 5' +C+A+G+GCAAACTGAG+T+T+G 3' (SEQ ID NO:88);

LNA 7.3 5' +C+A+G+GCAAACTG+A+G+T 3' (SEQ ID NO:89);

LNA 7.5 5' +A+A+ACTGAGTTG+G+A+C 3' (SEQ ID NO: 90);

LNA 7.7 5' +G+A+GTTGGACGTG+T+G+T 3' (SEQ ID NO: 91);

LNA 7.9 5' +G+G+ACGTGTGTTTT+C+T+C 3' (SEQ ID NO: 92);

LNA 8.1 5' +T+T+TCGGTCACACC+C+G+G (SEQ ID NO: 93);

LNA 8.2 5' +C+G+GTCACACCCGG+A+C+G 3' (SEQ ID NO:94);

LNA 8.3 5' +T+C+ACACCCGGACG+A+A+A 3' (SEQ ID NO:95);

LNA 8.45' +C+A+CCCGGACGA+A+A+C 3' (SEQ ID NO:96);

LNA 8.5 5' +C+A+CCCGGACGAA+A+C+C 3' (SEQ ID NO:97);

LNA 8.6 5' +C+A+CCCGGACGAAA+C+C+T 3' (SEQ ID NO:98);

LNA 8.7 5' +C+C+GGACGAAAC+C+T+A 3' (SEQ ID NO:99);

LNA 10.1 5' +T+C+GATCGTACTC+C+G+C 3' (SEQ ID NO:100);

LNA 10.4 5' +A+C+TCGATCGT+A+C+T+C 3' (SEQ ID NO:101);

LNA 10.5 5' +C+G+TGGCCTCGG+T+G+A+A 3' (SEQ ID NO:102); LNA 10.6 5' +G+T+GGCCTCGG+T+G+A+A 3' (SEQ ID NO:103);

LNA 11.1 5' +G+T+GGCCTCGG+T+G+A+A 3' (SEQ ID NO:104);

LNA 11.2 5' +T+A+A+AGATTGCT+A+T+G+T+G 3' (SEQ ID NO:105); LNA 11.3 5' +A+A+G+ATTGCTATG+T+G+A+G 3' (SEQ ID NO: 106); LNA 11.5 5' +T+A+T+GTGAGTTAA+A+G+T+T 3' (SEQ ID NO: 107); LNA 11.6 5' +T+G+T+GAGTTAAAGT+T+A+A 3' (SEQ ID NO:108); LNA 12.1 5' +T+C+GTAGAAGCCTT+T+T+G 3' (SEQ ID NO:109);

LNA 12.4 5' +A+A+G+CCTTTTGGC+A+A+T+G 3' (SEQ ID NO:110); LNA 12.5 5' +T+T+G+GCAATGTTG+T+T+C+C 3' (SEQ ID NO: 111); LNA 12.7 5' +C+T+TGAGGAAG+T+T+G+T 3' (SEQ ID NO:112);

LNA 12.8 5' +A+G+G+AAGTTGTAG+C+A+C+G 3' (SEQ ID NO:113); LNA 13.1 5' +C+T +A+CT AAAATT A+ A+T +T 3' (SEQ ID. NO:114);

LNA 13.4 5' +A+A+T+TAATTTTAC+A+C+A+T 3' (SEQ ID N0:115); LNA 13.5 5' + A+T +T +TT ACAC ATT +A+G +G +G 3' (SEQ ID NO: 116); LNA 13.6 5' +T+A+CACATTAGGG+C+T+C 3' (SEQ ID NO: 117);

LNA 13.7 5' +A+C+ATTAGGGCTC+T+T+C 3' (SEQ ID NO:118); LNA 13.9 5' +G+C+T+CTTCCATAT+A+G+G 3' (SEQ ID NO:119); LNA 14.1 5' +A+A+GGCTCTCCATC+T+T+A 3' (SEQ ID NO:120); LNA 14.2 5' +A+A+G+GCTCTCC+A+T+C+T 3' (SEQ ID NO:121); LNA 14.3 5' +G+C+TCTCCATCTTA+C+C+T 3' (SEQ ID NO:122); LNA 14.4 5' +T+C+CATCTTACCp+T+C+G 3' (SEQ ID NO:123); LNA 16.1 5' +C+C+T+CCCT AAT GT+T+A+C+ A 3' (SEQ ID NO: 124); LNA 16.2 5' +C+T +C+CCTAAT GTT +A+C+A+G 3' (SEQ ID NO:125); LNA 16.3 5' +C+T+CCCTAATGTTA+C+A+G 3' (SEQ ID NO:126); LNA 18.1 5' +T+A+A+AACCAACAC+T+A+C+C 3' (SEQ ID NO:127); LNA 18.2 5' +A+A+A+CCAACACTA+C+C+A+C 3' (SEQ ID NO: 128); LNA 18.3 5' +A+A+ACCAACACTAC+C+A+C 3' (SEQ ID NO:129); LNA 18.4 5' +A+C+C+AACACTACCA+C+A+T 3' (SEQ ID NO:130); LNA 18.5 5' +C+A+A+CACTACCAC+A+T+G+A 3' (SEQ ID NO:131); IV14_1 5' +G+C+G+ACCAAAAGA+A+T+T 3' (SEQ ID NO:162); IV14_2 5' +G+C+G+ACCAAAAG+A+A+T+T 3' (SEQ ID NO: 163); IV14_7 5' +C+G+ ACCAA AAG A+ A+T +T+C 3' (SEQ ID NO:164); IV14 9 5' +C+G+ACCAAAAGAA+T+T+C 3' (SEQ ID NO:165); IV14_145' +G+C+G+ACCAAAAGA+A+T+T+C 3' (SEQ ID NO: 166); IV14_15 5' +G+C+GACCAAAAGA+A+T+T+C 3' (SEQ ID NO:167); IV14 16 5' +G+C+GACCAAAAGAA+T+T+C 3' (SEQ ID NO:168); IV14 22 5' +G+A+CCAAAAGAA+T+T+C 3' (SEQ ID NO:169); IV14_27 5' +C+G+ACCAAAAG AAT +T +C+G+G 3' (SEQ ID NO: 170); IV14_31 5' +G+A+CCAAAAGAATT+C+G+G 3' (SEQ ID NO:171); IV14 32 5' +A+C+C+AAAAGAATT+C+G+G+A 3' (SEQ ID NO: 172); IV14_37 5' +C+A+A+AAGAATTC+G+G+A 3' (SEQ ID NO: 173); IV14_45 5' +C+A+A+AAGAATTC+G+G+A+T 3' (SEQ ID NO:174); IV14_47 5' +C+A+AAAGAATTC+G+G+A+T 3' (SEQ ID NO:175); IV9_1 5' +A+G+C+ATACTTACT+G+A+C+A 3' (SEQ ID NO:176);

IV9 3 5' +C+A+T+ACTTACTG+A+C+A+G 3' (SEQ ID NO:177); IV9_5 5' +A+T+ACTTACTGA+C+A+G 3' (SEQ ID NO:178);

IV9 6 5' +A+T+A+CTTACTG+A+C+A+G 3' (SEQ ID NO:179); IV9 13 5' +A+T+ACTTACTGAC+A+G+C 3' (SEQ ID NO:180); IV9_145' +A+T+A+CTTACTGA+C+A+G+C 3' (SEQ ID NO:181); IV9_15 5' +A+T+A+CTTACTGAC+A+G+C 3' (SEQ ID NO: 182); IV9 16 5' +A+T + ACTT ACTG A+C+A+G +C 3' (SEQ ID NO: 183); IV9 18 5' +T+A+CTTACTGAC+A+G+C 3' (SEQ ID NO:184); IV9 19 5' +T+A+CTTACTGA+C+A+G+C 3' (SEQ ID NO:185);

IV9 205' +T+A+C+TTACTGA+C+A+G+C 3' (SEQ ID NO: 186);

IV9 21 5' +A+T+A+CTTACTGAC+A+G+C+C 3' (SEQ ID NO: 187); IV9 22 5' +A+T+ACTTACTGACA+G+C+C 3' (SEQ ID NO: 188); IV9 23 5' +A+T+ACTTACTGAC+A+G+C+C (SEQ ID NO: 189);

IV9 25 5' +T+A+C+TTACTGAC+A+G+C+C 3' (SEQ ID NO:190); IV9 26 5' +T+A+CTTACTGACA+G+C+C 3' (SEQ ID NO:191); IV9_28 5' +T+A+C+TTACTGACA+G+C+C+A 3' (SEQ ID NO:192); IV9 29 5' +T+A+CTTACTGACA+G+C+C+A 3' (SEQ ID NO:193); IV9 305' +T+A+C+TTACTGACAG+C+C+A 3' (SEQ ID NO:194); IV9 31 5' +T+A+CTTACTGACAG+C+C+A 3' (SEQ ID NO:195);

IV9 32 5' +A+C+TTACTGACA+G+C+C+A 3' (SEQ ID NO: 196); IV9_33 5' +A+C+TTACTGACAG+C+C+A 3' (SEQ ID NO:197);

IV9 35 5' +A+C+T+TACTGACA+G+C+C+A 3' (SEQ ID NO:198); IV9_41 5' +C+T+T+ACTGACAG+C+C+A+G 3' (SEQ ID NO:199); IV9 45 5' +T+T+ACTGACAGCC+A+G+A 3' (SEQ ID NO:200);

IV22 5' +A+G+C+CAGACAG+C+G+A 3' (SEQ ID NO:201);

IV22 2 5' +C+A+G+CCAGACAG+C+G+A+C 3' (SEQ ID NO:202); IV22 3 5' +C+A+GCCAGACAG+C+G+A+C 3' (SEQ ID NO:203); IV22 5 5' +C+A+GCCAGACA+G+C+G+A 3' (SEQ ID NO:204); IV22 6 5' +C+A+GCCAGACAG+C+G+A 3' (SEQ ID NO: 205) IV22_7 5' +C+A+G+CCAGACAG+C+G+A 3' (SEQ ID NO:206)

IV22 8 5' +A+C+AG CCAG ACAG+C+G+A 3' (SEQ ID NO:207); IV22 9 5' +A+C+ A+G CCAG AC A+G +C+G +A 3' (SEQ ID NO:208); IV22 105' +A+C+AG CCAG ACA+G+C+G+A 3' (SEQ ID. NO:209); IV22 11 5' +G+A+C+AGCCAGACA+G+C+G 3' (SEQ ID NO:210); IV22_13 5' +G+A+CAGCCAGACA+G+C+G 3' (SEQ ID NO:211); IV22 145' +G+A+C+AGCCAGAC+A+G+C+G 3' (SEQ ID NO:212); IV25 5' +C+C+ATCAATTAGTG+T+C+G 3' (SEQ ID NO:213);

IV26 5' +G+C+CATCAATTAGT+G+T+G 3' (SEQ ID NO:214);

IV27 5' +A+A+G+AATTCGGA+T+G+G+C 3' (SEQ ID NO:215); IV28 5' +C+A+GACAGCGAC+C+A+A 3' (SEQ ID NO:216); and IV29 5' +T+G+ACAGCCAGAC+A+G+C 3' (SEQ ID NO:217). Example 3: Identifying LNAs most disruptive of identified conserved RNA secondary structure targets

To test the hypothesis that the identified RNA secondary structures, unique and conserved across corona B viruses, represent ideal candidate targets for disrupting the virus lifecycle, via structure-specific LNAs, exemplary LNA gapmers (from a screening panel synthesized against the identified conserved RNA secondary structure targets), that are disruptive to the target secondary structures integrity, as assessed by SHAPE, REVI, and Mutate-and-Map, is determined.

Methods: 14-16 nucleotide long LNAs designed to target the conserved RNA secondary structures of FIG. 1 are synthesized by a commercial supplier (Exiqon). LNA mediated disruption of targeted RNA secondary structures: SARS-CoV-2 RNA templates encoding the identified RNA secondary structures of FIG. 1 are transcribed in vitro, essentially as previously described (Glenn et ah, U.S. Patent Publication No. 2019/0136242, the disclosure of which is incorporated herein by reference). These are incubated with individual designed LNAs and the extent of RNA secondary structure disruption is determined by SHAPE-MaP and Mutate-and- Map-seq analysis as described (Cheng et al., (2017), RNA structure inference through chemical mapping after accidental or intentional mutations. Proc Natl Acad Sci U S A 114:9876-9881; Siegfried et al, (2014), RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat Methods 11:959-65).

Accordingly, LNAs with maximal disruption of targeted SARS-CoV-2 RNA secondary structures are determined and optimized.

Example 4: Optimizing the sequence of top performing LNAs

To test the hypothesis that the most disrupting LNAs can be further optimized for efficacy and safety, the sequence (total LNA length, fine nucleotide target position, and length of single stranded DNA gapmer) of the top performing LNAs is refined and a panel of LNA analogs are tested to identify the most potent disrupters of targeted SARS-CoV-2 conserved RNA secondary structure, with the least activation of caspase or interferon.

Methods: The length, fine nucleotide target position, and length of single stranded DNA gapmer are modified slightly for each top performing LNA identified in Example 3.

Accordingly, optimal LNAs against SARS-CoV-2 are rapidly identified in an accelerated fashion for subsequent testing of antiviral efficacy. Example 5: Determining the effect of sequestering miR191 on SARS-CoV-25’ RNA directed translation

To test the hypothesis that the miR191 binding sites within the 5 ’-most conserved RNA secondary structure of SARS-CoV-2 reflect an important translational regulatory mechanism of corona B viruses amenable to targeting with designed LNAs, the effect of LNAs designed to sequester miR191 in cells transfected with a SARS-CoV-25’ terminal RNA segment linked to a luciferase reporter is determined.

Methods: Exemplary subject LNAs are designed to sequester miR191.

SARS-CoV-2 translation reporter vector assays: A plasmid encoding the 5’ end of SARS- CoV-2 RNA fused to a luciferase reporter gene is transcribed in vitro, and the resulting RNA is transfected into Calu-3 human lung cells with our without a subject miR191 -sequestering LNA (e.g., an LNA as described herein), followed by luciferase assays of cell lysates harvested at various time post transfection.

Accordingly, the effect of depleting miR191 on SARS-CoV-25’ terminal RNA mediated translation is determined.

Without being bound to any particular theory, because it appears that depletion of miR191 is well tolerated, no significant side effects are expected by such transient depletion during an anticipated relatively short course of future treatment in infected patients.

Example 6: Determining efficacies of lead LNAs against SARS-CoV-2 and of the optimal in vivo formulation

To determine if exemplary subject LNAs (e.g., as described herein) can effectively inhibit SARS-CoV-2, and to determine the ideal mode of delivery of the subject LNAS, the effect of the identified LNAs (targeting conserved SARS-CoV-2 RNA secondary structure, and sequestering miR191) in vitro on cells infected with SARS-CoV-2, and in vivo when delivered intranasally by current lung-targeting transfection reagent (i.e. jetPEi) vs. pollen shells to SARS- CoV-2 -infected mice is investigated.

Methods: In vitro efficacy studies are performed using Calu-3 human lung cells, MDCK, or primary human lung cells, infected with virus followed by transfection of optimized exemplary LNAs. Initially the virus tested is SARS-CoV, but this can be replaced with another CoV, such as SARS-CoV-2, or another CoV virus (e.g., as described herein). In vivo efficacy studies are performed as exemplified in previous work (see, e.g., Glenn et al, US Patent Publication No. 2019/0136242). The LNAs are formulated with either Jet PEI or deproteinized pollen (see, e.g., Glenn et al, U.S. Provisional No. 62/988,847, “Pulmonary Agent Delivery Methods and Compositions for Practicing the Same”, the disclosure of which is incorporated herein by reference ). The pharmacokinetics and tissue distribution of LNAs are determined following IN administration in uninfected mice, and this is used to guide the subsequent studies where LNAs are administered to mice at various time points before and after inoculation with SARS-CoV-2.

Example 7: Determining efficacies of lead LNAs against lung cancer cells

To determine if exemplary subject LNAs (e.g., as described herein) can effectively treat a lung cancer, the effect of exemplary LNAs in vitro on lung cancer cells, and in vivo when delivered intranasally by current lung-targeting transfection reagent (i.e. jetPEi) vs. pollen shells to lung cancer infected mice is investigated.

Methods: In vitro efficacy studies are performed using lung cancer cells followed by transfection of optimized exemplary LNAs. The cytotoxic effects against lung cancer cells in culture is determined. In vivo efficacy studies are performed as exemplified in previous work (see, e.g., Glenn et al, US Patent Publication No. 2019/0136242). The LNAs are formulated with either Jet PEI or deproteinized pollen (see, e.g., Glenn et al, U.S. Provisional No. 62/988,847, “Pulmonary Agent Delivery Methods and Compositions for Practicing the Same”, the disclosure of which is incorporated herein by reference). The pharmacokinetics and tissue distribution of LNAs are determined following IN administration in healthy mice, and this is used to guide the subsequent studies where LNAs are administered to mice infected with a lung cancer. FIG 2. illustrates the caspase activity of exemplary LNAs.

Example 8. Cellular SARS-CoV-2 replication assays

For cellular replication assays, LNA ASOs were reconstituted in RNase-free water at 100 mM stock solutions, aliquoted and stored at -20°C prior to single-use. One day prior to transfection, Huh-7, Vero E6, or ACE-A549 cells were plated in 96-well clear bottom plates to be 60-70% confluency at the time of treatment with LNA ASOs or Scrambled LNA. Lipofectamine 3000® (Life Technologies) was used to transfect LNA ASO’s into cells at 25 nM or 100 nM final concentration per manufacturer's protocol. Cells were then infected with SARS-CoV-2 reporter virus expressing nanoluciferase (SARS-CoV-2 nLUC) at an MOI of 0.3 for 1 h after which the virus was removed and fresh medium was added. Recombinant SARS-CoV-2 nLUC is an authentic fully replicating virus where ORF7 has been deleted and replaced with nLUC. Thus, the measurement of nLUC expression is a surrogate marker of virus replication enabling the screening of antiviral compounds. Example 9-Anti- viral activity of miR-targeting LNA: Determining the in vitro antiviral efficacy of LNAs designed against microRNAs hypothesized to mediate respiratory virus replication.

Individual LNAs designed to target microRNAs were transfected into Huh7 cells, followed by infection with a fully replicating SARS-CoV-2-nLuc virus, which harbors a nanoluciferase reporter in ORF7. 48 hours post-infection, the effect on replication was measured by luciferase activity, as compared to control cells treated with negative control Scrambled LNA (Scr. LNA), or positive control nucleoside analog EIDD (Figure 3). While LNAs targeting the frame shift element (FSE) region of the SARS-CoV-2 RNA genome had minimal effect on viral replication, multiple logio reductions were observed with our anti-miR LNAs designed to sequester miR-191. The extent of inhibition with the anti-miR LNAs was greater than the EIDD positive control. Moreover, this degree of inhibition was observed with LNAs at 25 nanomolar concentration, whereas the EIDD positive control was used at 5 micromolar concentration. Other microRNA-targeting LNAs with anti-respiratory virus activity have been similarly identified and include but not limited to: LNA-602.1; LNA-602.6; LNA-6769-5P.3; LNA-6769-5P.6; LNA- 942.3P.4; LNA-376c.3; LNA-4433b.l; 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 to have potent antiviral activity, all having inhibition of viral replication about lloglO or more.

Example 10: Determining the in vitro antiviral efficacy of LNA combinations against respiratory viruses:

We hypothesized that combining an LNA targeting a conserved SARS-CoV-2 RNA secondary structure with an LNA designed to sequester miR-191 would lead to profound inhibition of viral replication. Using the assay described in example 9 above, individual LNAs were combined to show anti-respiratory virus activity (Figure 4).

Example 11: In vivo efficacy of LNA combination against respiratory viruses.

Mice transgenic for human ACE2 were treated with vehicle, small molecule A, or a combination of LNAs administered intranasally once 5 days prior to infection with a lethal inoculum of SARS-Cov-2. Following infection, animals were monitored by clinical score daily where 1= asymptomatic and higher score indicates worsening clinical status (Figure 5). Combination of LNAs inhibit respiratory virus infection in vivo.

Example 12: Anti-viral activity of single microRNA-targeting LNAs, or LNAs targeting either negative or positive strand of respiratory viruses.

Using assay described in example 9 individual LNAs were shown to have anti-respiratory virus activity (Figure 6). Other LNAs with anti-respiratory virus activity have been similarly identified and include but not limit to: Neg 3.1; Neg 8.2; Neg 8.4; Neg 10.1; Neg 10.1; Neg 12.2; Neg 18.2; Neg 18.3; Neg 18.4; Cov 8.5; Cov 10.1; Cov 11.3; Cov 12.8; Cov 13.9; Cov 14.3; Cov 16.3; Cov 18.1; Covl.l; Cov 1.2; Cov 1.4; Cov 2.1; Cov 2.2; Cov 3.2; Cov 3.2-2; Cov 3.2-3; Cov 3.2-4; Cov 3.2-5; Cov 3.2-6; Cov 3.2-7; Cov 4.1; Cov 4.2; Cov 6.4; Cov 6.5; Cov 6.6; Cov 6.7; Cov 6.8; Cov 6.9; Cov 6.7-1; Cov 6.7-2; Cov 6.8; Cov 6.9; Cov 6.7-1; Cov 6.7-2; Cov 7.2; Cov 7.7; Cov 7.9; Cov 8.1; Cov 8.2; Cov 8.3; Cov 8.4; Cov 8.5; Cov 8.6; Cov 8.2-1; Cov 10.1; Cov 10.4; Cov 11.1; Cov 11.2; Cov 11.3; Cov 12.4; Cov 12.5; Cov 12.7; Cov 12.8; Cov 13.1; Cov

13.4; Cov 13.5; Cov 13.6; Cov 13.8; Cov 13.9; Cov 14.1; Cov 14.2; Cov 14.3; Cov 14.4; Cov

16.1; Cov 16.2; Cov 16.3; Cov 18.1; Cov 18.5; IBV-LNA0.2; IBV-LNA4.4; IBV-LNA3.4; IBV-

LNA2.5; IBV-LNA 1.5 to have potent antiviral activity, all having inhibition of viral replication about llog 10 or more

Summary

The examples disclosed herein allow for: 1) determination of LNAs that target SARS- CoV-2; 2) determination of LNAs that sequester miR191 to target SARS-CoV-2; 3) determination of optimal formulation for in vivo delivery of SARS-CoV-2-targeting LNAs; and 4) determination of LNAs that exhibit cytotoxic effects against lung cancer cells.

These experiments demonstrate the potential of exemplary LNAs (e.g., as described herein) to be used for the development of broad, natural functional immunity as a result of exposure to SARS-CoV-2 under conditions of protective attenuation provided by exemplary anti- SARS-CoV-2 LNAs. Such LNAs can also provide a critical window of immediate protection for other candidate vaccines that need at least two weeks before beginning to provide protective immunity. Demonstration that exemplary LNAs have in vivo efficacy can then enable their subsequent rapid development via IND-enabling and clinical proof-of concept studies, which in turn can provide a valuable countermeasure to the SARS-CoV-2 outbreak. General Methods

For general methods such as antiviral assays; in vitro transcription of vRNA; sf-SHAPE analysis of vRNA; construct design, RNA synthesis and chemical modification for mutate-and- map experiments, and SHAPE analysis of LNA-targeted vRNA, see the corresponding methods disclosed in Glenn et al, U.S. Patent Publication No. 2019/0136242, entitled “Pan-genotypic agents against influenza virus and methods of using the same”, the disclosure of which is incorporated herein by reference.

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

Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited 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. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended embodiments.

Claims

What is claimed is:

1. An oligonucleotide compound or salt thereof, comprising an oligonucleotide sequence complementary to an RNA secondary structure of a coronavirus (CoV) or mutant thereof, wherein the oligonucleotide compound inhibits virus production.

2. The compound of claim 1 , wherein the oligonucleotide inhibits a microRNA (miRNA) interaction with CoV.

3. The compound of claim 2, wherein the iRNA is miR191.

4. The compound of any one of claims 1 to 3, wherein the oligonucleotide comprises an internucleoside linkage selected from: phosphorothioate, phosphorodithioate, phosphoramidate and thiophosphoramidate linkages.

5. The compound of claim 4, wherein the oligonucleotide comprises one or more chyral internucleoside linkages.

6. The compound of any one of claims 1 to 5, wherein the oligonucleotide comprises a bridged nucleic acid (BNA) nucleotide.

7. The compound of claim 6, wherein the BNA nucleotide is selected from the group consisting of, locked nucleic acid (LNA) nucleotides, ethylene-bridged nucleic acid (ENA) nucleotides, and constrained ethyl (cEt) nucleotides.

8. The compound of any one of claims 1 to 5, wherein the oligonucleotide comprises one or more 2’-modified nucleotides.

9. The compound of any one of claims 1 to 8, wherein the oligonucleotide comprises 8 or more nucleoside subunits.

10. The compound of any one of claims 1 to 9, wherein the oligonucleotide comprises a sequence selected from: 5’ GACGTGATATATGTGG 3’ (SEQ ID NO:1);

5’ CGTGATATATGTGGTA 3’ (SEQ ID NO:2);

5’ GATATGTGGTACCAT 3’ (SEQ ID NO:3);

5’ TGGTACCATGTCAC 3’ (SEQ ID NO:4);

5’ CCTCAGCAGCAGATTT 3’ (SEQ ID NO:5);

5’ TCAGCAGCAGATTTC 3’ (SEQ ID NO:6);

5’ CAGCAGCAGATTTC 3’ (SEQ ID NO:7);

5’ CAGATTTCTTAGTGAC 3’ (SEQ ID NO:8);

5’ TGCAACACGGACGAAA 3’ (SEQ ID NO:9);

5’ GCAACACGGACGAAAC 3’ (SEQ ID NO: 10); 5’ ACGA A ACCCGT A AGC A 3’ (SEQ ID NO: 11); 5’ AACATGTCTGGACCTA 3’ (SEQ ID NO:12);

5’ ACATGTCTGGACCTAT 3’ (SEQ ID NO:13);

5’ TGAATATGACATAGT 3’ (SEQ ID NO: 14);

5’ AATATGACATAGTC 3’ (SEQ ID NO:15);

5’ CACAGATTTTAAAGTT 3’ (SEQ ID NO:16);

5’ GATTTTAAAGTTCGT 3’ (SEQ ID NO:17);

5’ TAAAGTTCGTTTAGA 3’ (SEQ ID NO:18);

5’ AAAGTTCGTTTAGA 3’ (SEQ ID NO: 19);

5’ TCGTTTAGAGAACAGAT 3’ (SEQ ID NO:20); 5’ AGAGAACAGATCTACA 3’ (SEQ ID NO:21); 5’ AGATCTACAAGAGA 3’ (SEQ ID NO:22);

5’ CAGGCAAACTGAGTTG 3’ (SEQ ID NO:23); 5’ CAGGCAAACTGAGT 3’ (SEQ ID NO:24);

5’ AAACTGAGTTGGAC 3’ (SEQ ID NO: 25);

5’ GAGTTGGACGTGTGT 3’ (SEQ ID NO: 26);

5’ GGACGTGTGTTTTCTC 3’ (SEQ ID NO: 27);

5’ TTTCGGTCACACCCGG (SEQ ID NO: 28);

5’ CGGTCACACCCGGACG 3’ (SEQ ID NO:29); 5’ TCACACCCGGACGAAA 3’ (SEQ ID NO:30); 5’ CACCCGGACGAAAC 3’ (SEQ ID NO:31);

5’ CACCCGGACGAAACC 3’ (SEQ ID NO:32);

5’ CACCCGGACGAAACCT 3’ (SEQ ID NO:33); 5’ CCGGACGA A ACCT A 3’ (SEQ ID NO:34); 5’ TCGATCGTACTCCGC 3’ (SEQ ID NO:35);

5’ ACTCGATCGTACTC 3’ (SEQ ID NO:36);

5’ CGTGGCCTCGGTGAA 3’ (SEQ ID NO:37);

5’ GTGGCCTCGGTGAA 3’ (SEQ ID NO:38);

5’ TAAAGATTGCTATGTG 3’ (SEQ ID NO:39);

5’ AAGATTGCTATGTGAG 3’ (SEQ ID NO:40);

5’ TATGTGAGTTAAAGTT 3’ (SEQ ID NO:41);

5’ TGTGAGTTAAAGTTAA 3’ (SEQ ID NO:42);

5’ TCGTAGAAGCCTTTTG 3’ (SEQ ID NO:43);

5’ AAGCCTTTTGGCAATG 3’ (SEQ ID NO: 44)

5’ TTGGCAATGTTGTTCC 3’ (SEQ ID NO:45)

5’ CTTGAGGAAGTTGT 3’ (SEQ ID NO:46);

5’ AGGAAGTTGTAGCACG 3’ (SEQ ID NO:47);

5’ CTACTAAAATTAATT 3’ (SEQ ID. NO:48);

5’ AATTAATTTTACACAT 3’ (SEQ ID NO:49);

5’ ATTTT AC AC ATT AGGG 3’ (SEQ ID NO:50);

5’ TACACATT AGGGCT C 3’ (SEQ ID NO:51);

5’ ACATTAGGGCTCTTC 3’ (SEQ ID NO:52);

5’ GCTCTTCCATATAGG 3’ (SEQ ID NO:53);

5’ AAGGCTCTCC ATCTT A 3’ (SEQ ID NO:54);

5’ AAGGCTCTCCATCT 3’ (SEQ ID NO:55);

5’ GCTCTCCATCTTACCT 3’ (SEQ ID NO:56);

5’ TCCATCTTACCTTTCG 3’ (SEQ ID NO:57);

5’ CCTCCCTAATGTTACA 3’ (SEQ ID NO:58);

5’ CTCCCTAATGTTACAG 3’ (SEQ ID NO:59);

5’ TAAAACCAACACTACC 3’ (SEQ ID NO:60);

5’ AAACCAACACTACCAC 3’ (SEQ ID NO:61);

5’ ACCAACACTACCACAT 3’ (SEQ ID NO:62); and 5’ CAACACTACCACATGA 3’ (SEQ ID NO:63).

11. The compound of any one of claims 1 to 9, comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from (SEQ ID NOs: 1-63).

12. The compound of any one of claims 10 to 11, wherein the oligonucleotide comprises at least 5 deoxyribonucleotide units and are capable of recruiting an RNase.

13. The compound of claim 10, wherein the oligonucleotide comprises a sequence selected from:

LNA 1.1 5’ +G+A+CGTGATATATG+T+G+G 3’ (SEQ ID NO:64);

LNA 1.25’ +C+G+TGATATATGTG+G+T+A 3’ (SEQ ID NO:65);

LNA 1.3 5’ +G+A+TATGTGGTAC+C+A+T 3’ (SEQ ID NO:66);

LNA 1.45’ +T+G+GT ACC ATGT +C+ A+C 3’ (SEQ ID NO:67);

LNA 2.1 5’ +C+C+TCAGCAGCAG+A+T+T+T 3’ (SEQ ID NO:68);

LNA 2.25’ +T +C+ AGC AGC AGAT+T +T+C 3’ (SEQ ID NO:69);

LNA 2.3 5’ +C+ A+G+C AGC AGAT +T+T+C 3’ (SEQ ID NO:70);

LNA 2.45’ +C+ A+GATTTCTT AGT +G+ A+C 3’ (SEQ ID NO:71);

LNA 3.1 5’ +T +G+C AAC ACGGAC+G+ A+ A+ A 3’ (SEQ ID NO:72);

LNA 3.25’ +G+C+AACACGGACG+A+A+A+C 3’ (SEQ ID NO:73);

LNA 3.5 5’ +A+C+GAAACCCGTA+A+G+C+A 3’ (SEQ ID NO:74);

LNA 3.65’ + A+C+G+ A A ACCCGT A+ A+G+C+ A 3’ (SEQ ID NO:75);

LNA 4.1 5’ +A+A+CATGTCTGGAC+C+T+A 3’ (SEQ ID NO:76);

LNA 4.25’ +A+C+ATGTCTGGAC+C+T+A+T 3’ (SEQ ID NO:77);

LNA 5.1 5’ +T +G+ A AT ATGAC AT + A+G+T 3’ (SEQ ID NO:78);

LNA 5.25’ +A+A+TATGACATA+G+T+C 3’ (SEQ ID NO:79);

LNA 6.1 5’ +C+ A+C AGATTTT A A A+G+T +T 3’ (SEQ ID NO:80);

LNA 6.25’ +C+A+C+ AGATTTT A A+ A+G+T +T 3’ (SEQ ID NO:81);

LNA 6.45’ +G+ A+TTTT A A AGT +T+C+G+T 3’ (SEQ ID NO:82);

LNA 6.5 5’ +T+A+A+AGTTCGTT+T+A+G+A 3’ (SEQ ID NO:83);

LNA 6.65’ +A+A+A+GTTCGTTT+A+G+A 3’ (SEQ ID NO:84);

LNA 6.75’ +T +C+GTTT AGAGA AC+ A+G+ A+T 3’ (SEQ ID NO:85);

LNA 6.8 5’ +A+G+AGAACAGATCT+A+C+A 3’ (SEQ ID NO:86);

LNA 6.95’ +A+G+ATCTACAAG+A+G+A 3’ (SEQ ID NO:87);

LNA 7.25’ +C+ A+G+GC A A ACTGAG+T +T+G 3’ (SEQ ID NO:88);

LNA 7.3 5’ +C+ A+G+GC AAACTG+ A+G+T 3’ (SEQ ID NO:89);

LNA 7.5 5’ +A+A+ACTGAGTTG+G+A+C 3’ (SEQ ID NO: 90);

LNA 7.75’ +G+A+GTTGGACGTG+T+G+T 3’ (SEQ ID NO: 91);

LNA 7.95’ +G+G+ ACGTGTGTTTT +C+T+C 3’ (SEQ ID NO: 92); LNA 8.1 5’ +T+T+TCGGTCACACC+C+G+G (SEQ ID NO: 93);

LNA 8.25’ +C+G+GTCACACCCGG+A+C+G 3’ (SEQ ID NO:94);

LNA 8.3 5’ +T +C+ AC ACCCGGACG+ A+ A+ A 3’ (SEQ ID NO:95);

LNA 8.45’ +C+A+CCCGGACGA+A+A+C 3’ (SEQ ID NO:96);

LNA 8.5 5’ +C+A+CCCGGACGAA+A+C+C 3^! (SEQ ID NO:97);

LNA 8.65’ +C+A+CCCGGACGAAA+C+C+T 3’ (SEQ ID NO:98);

LNA 8.75’ +C+C+GGACGA A AC+C+T + A 3’ (SEQ ID NO:99);

LNA 10.1 5’ +T+C+GATCGTACTC+C+G+C 3’ (SEQ ID NO: 100);

LNA 10.45’ +A+C+TCGATCGT+A+C+T+C 3’ (SEQ ID NO: 101);

LNA 10.5 5’ +C+G+TGGCCTCGG+T+G+A+A 3’ (SEQ ID NO: 102); LNA 10.6 5’ +G+T+GGCCTCGG+T+G+A+A 3’ (SEQ ID NO: 103);

LNA 11.1 5’ +G+T+GGCCTCGG+T+G+A+A 3’ (SEQ ID NO:104);

LNA 11.2 5’ +T + A+ A+ AGATTGCT + A+T +G+T+G 3’ (SEQ ID NO:105); LNA 11.3 5’ +A+A+G+ATTGCTATG+T+G+A+G 3’ (SEQ ID NO: 106); LNA 11.5 5’ +T+ A+T +GTGAGTT A A+ A+G+T +T 3’ (SEQ ID NO:107); LNA 11.6 5’ +T+G+T +GAGTT A A AGT+T + A+ A 3’ (SEQ ID NO: 108); LNA 12.1 5’ +T+C+GT AGA AGCCTT +T+T+G 3’ (SEQ ID NO: 109); LNA 12.45’ +A+A+G+CCTTTTGGC+A+A+T+G 3’ (SEQ ID NO: 110); LNA 12.5 5’ +T+T+G+GCAATGTTG+T+T+C+C 3’ (SEQ ID NO:111); LNA 12.7 5’ +C+T+TGAGGA AG+T +T+G+T 3’ (SEQ ID NO: 112);

LNA 12.8 5’ +A+G+G+AAGTTGTAG+C+A+C+G 3’ (SEQ ID NO: 113); LNA 13.1 5’ +C+T+A+CTAAAATTA+A+T+T 3’ (SEQ ID. NO: 114); LNA 13.45’ + A+ A+T +T AATTTT AC+ A+C+ A+T 3’ (SEQ ID NO: 115); LNA 13.5 5’ +A+T+T+TTACACATT+A+G+G+G 3’ (SEQ ID NO:116); LNA 13.6 5’ +T + A+C AC ATT AGGG+C+T +C 3’ (SEQ ID NO: 117);

LNA 13.7 5’ + A+C+ ATT AGGGCTC+T +T+C 3’ (SEQ ID NO:118);

LNA 13.9 5’ +G+C+T+CTTCCATAT+A+G+G 3’ (SEQ ID NO: 119); LNA 14.1 5’ +A+A+GGCTCTCCATC+T+T+A 3^! (SEQ ID NO: 120); LNA 14.2 5’ +A+A+G+GCTCTCC+A+T+C+T 3’ (SEQ ID NO: 121); LNA 14.3 5’ +G+C+TCTCCATCTTA+C+C+T 3’ (SEQ ID NO: 122); LNA 14.45’ +T +C+C ATCTT ACCTT +T+C+G 3’ (SEQ ID NO:123);

LNA 16.1 5’ +C+C+T+CCCTAATGT+T+A+C+A 3’ (SEQ ID NO: 124); LNA 16.2 5’ +C+T+C+CCTAATGTT+A+C+A+G 3’ (SEQ ID NO: 125); LNA 16.3 5’ +C+T+CCCTAATGTTA+C+A+G 3’ (SEQ ID NO: 126); LNA 18.1 5’ +T + A+ A+ A ACC A AC AC+T + A+C+C 3’ (SEQ ID NO: 127);

LNA 18.2 5’ + A+ A+ A+CC A AC ACT A+C+C+ A+C 3’ (SEQ ID NO:128);

LNA 18.3 5’ + A+ A+ ACC A AC ACT AC+C+ A+C 3’ (SEQ ID NO:129);

LNA 18.45’ + A+C+C+ A AC ACT ACC A+C+ A+T 3’ (SEQ ID NO:130); and LNA 18.5 5’ +C+ A+ A+C ACT ACC AC+ A+T +G+ A 3’ (SEQ ID NO: 131). wherein a “+” before the letter denotes LNA nucleotides, and other letters denote DNA nucleotides.

14. The compound of claim 13, comprising an oligonucleotide sequence having at least 70% sequence identity with a sequence selected from LNA1.1 - LNA18.5 (SEQ ID NOs: 64-131).

15. A method of inhibiting a coronavirus (CoV) in a cell, the method comprising: contacting a sample comprising viral RNA (vRNA) having a conserved RNA secondary structure of CoV with an effective amount of an oligonucleotide compound according to any one of claims 1 to 14.

16. The method of claim 15, wherein contacting the sample with the oligonucleotide compound disrupts the RNA secondary structure of CoV.

17. The method of claim 16, wherein contacting the sample with the oligonucleotide compound inhibits a host miRNA interaction with CoV.

18. The method of claim 17, wherein the miRNA is miR191.

19. The method of any one of claims 15-18, wherein the vRNA is isolated from a virion or a cell.

20. A method of treating or preventing a coronavirus (CoV) infection in a subject, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the oligonucleotide compound according to any one of claims 1-14.

21. The method of claim 20, wherein the subject is at risk of coronavirus (CoV) infection and the administering of the oligonucleotide compound protects the subject against infection for 1 week or more.

22. The method of claim 21, wherein the administering comprises weekly, biweekly, or monthly administration of an effective dose of the oligonucleotide compound.

23. The method of claim 21 or 22, wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and an antiviral drug.

24. The method of any one of claims 21-23, wherein the subject has been diagnosed with or suspected of having a coronavirus (CoY) infection.

25. A method of treating or preventing a neoplastic condition in a subject, the method comprising: administering to a subject in need thereof a pharmaceutical composition comprising an effective amount of the oligonucleotide compound according to any one of claims 1 to 14.

26. The method of claim 25, wherein the neoplastic condition comprises lung cancer.

27. The method of claims 25 or 26, wherein the pharmaceutical composition further comprises an additional active agent selected from a second oligonucleotide active agent and a chemotherapeutic agent.