Antisense oligonucleotide for suppressing proliferation of coronavirus

Abstract

One problem to be addressed by the present invention is to provide a drug which is capable of suppressing the proliferation of SARS-CoV-2 with high efficiency. Another problem to be addressed by the present invention is to provide a drug which exhibits an inhibitory effect on a wide range of coronaviruses and thereby can immediately respond to the pandemic of unknown SARS-CoV-2 mutants and new types of coronaviruses which could happen in the future.　Provided is an oligonucleotide or a pharmaceutically acceptable salt thereof, wherein: the oligonucleotide comprises an oligonucleotide that is composed of 17-30 nucleotides and comprises a nucleotide sequence and is complementary to a region lying between a nucleotide located at position-13520 to a nucleotide located at position-13550 in SARS-CoV-2 RNA genome comprising the nucleotide sequence represented by SEQ ID NO: 1; and the oligonucleotide is a mixmer and is capable of inhibiting the viral proliferation of coronaviruses, wherein the 5'-end and / or the 3'-end of the oligonucleotide may be chemically modified. Also provided is a drug comprising the oligonucleotide or a pharmaceutically acceptable salt thereof.

Description

Antisense oligonucleotides that inhibit coronavirus proliferation

[0001] The present invention relates to antisense oligonucleotides that inhibit the proliferation of coronaviruses.

[0002] There are four known coronaviruses capable of infecting humans, including those that cause common cold symptoms and three that cause severe pneumonia. All of these infect and multiply primarily in the respiratory tract, resulting in various health problems. The three species that cause severe pneumonia—SARS-CoV, the pathogen of Severe Acute Respiratory Syndrome (SARS), MERS-CoV, the pathogen of Middle East Respiratory Syndrome (MERS), and SARS-CoV-2, the pathogen of novel coronavirus disease (COVID-19)—pose a public health threat due to their high infectiousness and lethality. SARS-CoV-2 became a global pandemic at the end of 2019, infecting more than 700 million people and killing more than 6 million by the time the public health emergency ended in May 2023. During this time, the effectiveness of mRNA vaccines has been confirmed, and new treatments and therapeutic approaches have been discovered. However, infections remain, and vigilance is needed to prevent resurgence and the emergence of mutant strains. For this reason, the development of preventive measures other than vaccines and new therapeutic drugs against coronaviruses remains an important challenge. Coronaviruses possess an extremely long, single-stranded, positive-sense RNA genome of 26 kb to 32 kb. Genomic RNA is prone to mutations in various regions due to incomplete replication during proliferation, leading to the emergence and spread of mutant viruses in a short period of time, making prevention and treatment difficult. Nucleic acid drugs can control the function of targeted RNA by binding to it in a sequence-dependent manner, and are therefore expected to be an effective means of inhibiting coronavirus infection and proliferation. In fact, it has been reported that the use of nucleic acid sequences complementary to SARS-CoV and SARS-CoV-2 inhibits viral proliferation (Patent Documents 1 and 2). Because the coronavirus genome is so long, it is difficult to conduct comprehensive screening of the entire genome. Therefore, targeting sequences are being investigated, focusing on regions encoding proteins responsible for coronavirus functions, regions that inhibit coronavirus replication, or regions with little reported mutation information.These studies have led to the discovery of suitable sequences in each region, such as the 5'-UTR region and TRS sequence (Non-Patent Document 10), the 5'-AUG sequence (Non-Patent Documents 1, 2, and 3), the ORF1ab region (Non-Patent Documents 4 and 5), the FSP sequence (Non-Patent Documents 7, 8, and 9), the ORF7a and N protein region (Non-Patent Document 6), and gapmers for frameshift pseudoknot structures (Non-Patent Document 11).

[0003] International Publication No. 2005 / 023083 International Publication No. 2021 / 211928

[0004] Journal of Virology, 2004, pp. 5891-5899Journal of Antimicrobial Chemotherapy, 2021, Vol. 76, pp. 413-417Biomedicines, 2021, Vol. 9, pp. 1018-1027Nucleic Acids Research, 2022, Vol. 50, No. 1, pp. 333-349bioRxiv, 2022, doi. org / 10.1101 / 2022.03.20.485044 Proceedings of the National Academy of Sciences, 2023, Vol. 120, No. 11, e2219523120 Journal of Virology, 2005, pp. 9665-9676 Journal of Virology, 2007, pp. 5637-5648 Antiviral Research, 2011, Vol. 91, p. 1-10Nature Communications, 2022, 13:4503 Published at the 16th Annual Meeting of the Oligonucleotide Therapeutics Society (September 30, 2020) (Lisa Henderson et al., NINDS, 10:20-10:40, title: Antisense oligonucleotides as therapeutic agents for SARS-CoV-2) https: / / www.oligotherapeutics.org / wp-content / uploads / 2021 / 06 / OTS20-Agenda-Virtual-Meeting-25-SEPT.pdf

[0005] An object of the present invention is to provide a pharmaceutical product that can highly efficiently inhibit the proliferation of SARS-CoV-2. Furthermore, the inventors' research evaluated previously reported antisense nucleic acids targeting coronaviruses, and found that none of them exhibited an inhibitory effect against all of MERS-CoV, SARS-CoV-1, and SARS-CoV-2. In other words, known antisense nucleic acids can only be expected to be effective against specific coronaviruses. Therefore, an object of the present invention is to provide a pharmaceutical product that exhibits an inhibitory effect against a wide range of coronaviruses, thereby enabling a rapid response to potential future pandemics of unknown SARS-CoV-2 mutants and new coronaviruses.

[0006] The present inventors conducted extensive research and found that antisense oligonucleotides (ASOs) targeting a specific base sequence in the FSP sequence in the SARS-CoV-2 RNA genome exhibit extremely high viral growth inhibitory activity within cells. Furthermore, they found that those with particularly high activity also exhibit high growth inhibitory effects against other coronaviruses, leading to the completion of the present invention.

[0007] The gist of the present invention is as follows: [1] An oligonucleotide comprising an oligonucleotide of 17 to 30 bases, the nucleotide sequence of which is substantially complementary to the region of nucleotide numbers 13520 to 13550 of the SARS-CoV-2 RNA genome consisting of the nucleotide sequence of SEQ ID NO: 1, and which may be chemically modified at the 5' end and / or 3' end, wherein the oligonucleotide is a mixmer that does not recruit RNase H and is capable of inhibiting coronavirus proliferation, or a pharmaceutically acceptable salt thereof. The mixmer structure that does not recruit RNase H preferably does not contain a region of five or more consecutive DNAs, more preferably does not contain a region of four or more consecutive DNAs. [2] The oligonucleotide according to [1], or a pharmaceutically acceptable salt thereof, which binds to a frameshift pseudoknot (FSP) structure of coronavirus-derived RNA. [3] The oligonucleotide according to [1] or [2], or a pharmaceutically acceptable salt thereof, comprising at least 17 consecutive bases in the base sequence of any one of SEQ ID NOS: 2, 5 to 8, and 11 to 13 (provided that t in the sequence may be u, and each base may be a natural base or a modified base). Here, the oligonucleotide preferably comprises the same base sequence. [4] The oligonucleotide according to any one of [1] to [3], or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has 18 to 24 bases. [5] The oligonucleotide according to [4], or a pharmaceutically acceptable salt thereof, wherein the oligonucleotide has 21 bases. [6] The oligonucleotide according to any one of [1] to [5], or a pharmaceutically acceptable salt thereof, wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified. [7] The oligonucleotide according to [6], or a pharmaceutically acceptable salt thereof, wherein the sugar constituting the oligonucleotide is D-ribofuranose, and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose. [8] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [7], wherein the sugar modification is 2'-O-alkylation and / or 2'-O, 4'-C-alkylenation of D-ribofuranose.[9] The oligonucleotide or a pharmaceutically acceptable salt thereof according to [7], wherein the sugar modification is 2'-O-methylation and / or 2'-O,4'-C-ethylation of D-ribofuranose.

[10] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [6] to [9], wherein the phosphodiester bond modification is phosphorothioate.

[11] The oligonucleotide or a pharmaceutically acceptable salt thereof consisting of any of the following sequences: -A. m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t - (SEQ ID NO: 24) -G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1ts -A m1s -C e2s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t -(SEQ ID NO: 27) -A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s-T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A e2s -G m1t -(SEQ ID NO:28) -A m1s -A e2s -A m1s -A m1s -G​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​m1s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G e2s -U m1t -(SEQ ID NO: 33) -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -(SEQ ID NO: 34) -A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t - (SEQ ID NO: 35) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA nucleoside (the base position of C is 5-methylcytosine) linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1s , G m1s , C m1s , U m1s, represents the corresponding 2'-OMe-RNA nucleoside linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1t , G m1t , C m1t , U m1t , represent the corresponding 2'-OMe-RNA nucleoside in which the 3'-position is bonded to the adjacent atom via an oxygen atom. The 5'-position carbon of the 5'-terminal nucleoside in each sequence is bonded to a hydroxyl group or to another structural unit via a phosphate group, and the 3'-position carbon of the 3'-terminal nucleoside is bonded to a hydroxyl group or to another structural unit via a phosphate group.]

[12] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[11] , characterized in that a phosphate group linked to a fatty acid is further bonded to the 5'- or 3'-end of the oligonucleotide (preferably the 5'-position carbon of the 5'-end). Here, preferred examples of the "phosphate group-containing linker" include an aminoalkylphosphate group bonded to a fatty acid via an amino group, and a compound in which the alkylphosphate group containing the fatty acid is bonded to an oxygen atom corresponding to the hydroxyl group contained in another amidite compound.

[13] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[12] , wherein the fatty acid is myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid.

[14] An oligonucleotide or a pharmaceutically acceptable salt thereof having any of the following sequences: HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO20) (SEQ ID NO: 24) HO-G m1s -C e2s -Cm1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1ts -A m1s -C e2s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t [[ID=)37]]-H(ASO39) (SEQ ID NO: 27) HO-A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U[[ID=)54]] m1s -A m1s -T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A e2s -G m1t -H(ASO40) (SEQ ID NO: 28) HO-A m1s [[ID=8)1]]-A[[ID=)82]] e2s -A m1s -A m1s -G e2s -C m1s [[ID=)91]]-C[[ID=9))2]] m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s ] -C m1t -H(ASO41) (SEQ ID NO: 29) HO-U m1s -C e2s It should be noted that there seem to be some inconsistent or incorrect tags in the original text (such as "))" in some lines), which may affect the accurate understanding and translation. The above translation is based on the best attempt to follow the rules while dealing with the existing text.-A m1s -A m1s -A e2s -A m1s -G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1t -H(ASO42) (SEQ ID NO: 30) HO-A m1s -G e2s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -U m1s -A m1s [[ID=​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO76) (SEQ ID NO: 35) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA nucleoside (the base position of C is 5-methylcytosine) linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1s , G m1s , C m1s , U m1s , represents the corresponding 2'-OMe-RNA nucleoside linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1t , G m1t , C m1t , U m1t, represents the corresponding 2'-OMe-RNA nucleoside in which the 3'-position is linked to the adjacent atom via an oxygen atom.]

[15] A coronavirus proliferation inhibitor comprising the oligonucleotide according to any one of [1] to

[14] or a pharmaceutically acceptable salt thereof.

[16] A medicine comprising the oligonucleotide according to any one of [1] to

[14] or a pharmaceutically acceptable salt thereof.

[17] A therapeutic and / or prophylactic agent for coronavirus infection comprising the oligonucleotide according to any one of [1] to

[14] or a pharmaceutically acceptable salt thereof.

[18] The therapeutic and / or prophylactic agent according to

[17] , wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[19] A method for treating and / or preventing coronavirus infection in a subject by administering the oligonucleotide according to any one of [1] to

[14] or a pharmaceutically acceptable salt thereof to the subject.

[20] The method for treatment and / or prevention according to

[19] , wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[21] The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[14] , for use in the treatment and / or prevention of a coronavirus infection.

[22] The oligonucleotide or a pharmaceutically acceptable salt thereof according to

[21] , wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[23] Use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of [1] to

[14] , for the manufacture of a medicament for the treatment and / or prevention of a coronavirus infection.

[24] The use according to

[22] , wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

[0008] The antisense oligonucleotides (ASOs) of the present invention exhibit a high growth inhibitory effect against a wide range of coronaviruses and can inhibit the proliferation of these viruses. It is assumed that the oligonucleotides of the present invention can inhibit viral proliferation by binding to a frameshift pseudoknot (FSP) structure present in coronavirus-derived RNA.

[0009] 1 is a graph showing the viral growth inhibitory activity of each ASO against SARS-CoV-2 in Experiment 1 of Test Example 1. The Y axis represents the activity of Lipofectamine. TM The control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The cells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. This figure shows the viral growth inhibitory activity of each ASO against SARS-CoV-2 (BA.1 strain) in Test Example 3. The Y-axis represents the percentage of Lipofectamine. TMThe control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The control wells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. TM The cells were treated with a culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated.

[0010] Hereinafter, embodiments of the present invention will be described in more detail.

[0011] The present invention provides an oligonucleotide having 17 to 30 bases and a nucleotide sequence complementary to the region of nucleotide numbers 13520 to 13550 (preferably the region of 13522 to 13542) of the SARS-CoV-2 RNA genome consisting of the nucleotide sequence of SEQ ID NO: 1, wherein the oligonucleotide is a mixmer and capable of inhibiting coronavirus proliferation, and a pharmaceutically acceptable salt thereof. The oligonucleotide of the present invention is presumed to be able to inhibit viral proliferation by binding to a frameshift pseudoknot (FSP) structure present in coronavirus-derived RNA.

[0012] As used herein, nucleic acid or nucleic acid molecule is a general term for nucleosides, nucleotides, oligonucleotides, and polynucleotides.

[0013] "Nucleoside" refers to a chemical structural moiety that combines a base moiety that is important for genetic information with a sugar moiety that is important for the physicochemical properties of the molecule.

[0014] The term "nucleotide" refers to a compound in which the hydroxyl group at the 3' position of the sugar moiety of a nucleoside forms an ester with a phosphate group, or a partial structure thereof.

[0015] An "oligonucleotide" refers to an oligomer composed of two or more nucleotides linked by a phosphate group moiety of one nucleotide to the 5'-hydroxyl group of the sugar moiety of another nucleotide (phosphodiester bond). However, the 3'-end and / or 5'-end may be a hydroxyl group or a phosphate group, or may be chemically modified. Furthermore, the phosphodiester bond between nucleosides may be chemically modified. In this specification, the terms oligonucleotide and polynucleotide are used interchangeably.

[0016] The base moieties of nucleic acids are adenine (A), guanine (G), cytosine (C), uracil (U), and thymine (T). A and U or T, and G and C, respectively, form Watson-Crick base pairs through hydrogen bonds, and the relationship between bases that form a base pair is called "complementary." Each base moiety may be chemically modified, but even modified bases may usually exhibit the same base pairing ability as the original base. Representative modified bases include 5-methylcytosine (5meC) and 5-methyluracil (5meU), and 5meU has the same structure as T. In the sequence listing attached to this specification, 5meC is represented as C, and 5meU is represented as T or U.

[0017] As used herein, the term "substantially identical base sequence" refers to a base sequence that is entirely identical to the target base sequence, and also includes a base sequence that has 70% or more (preferably 80% or more, more preferably 90% or more, and even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity with the target base sequence, as well as a base sequence that contains one or several (preferably 10 or less, 8 or less, 5 or less, 4, 3 or less, or 2) non-identical bases but that can exhibit the desired function as an oligonucleotide. Here, "identical base" includes not only bases that are completely identical to the original base, but also bases that have base-pairing ability equal to or greater than that of the original base. Examples of such bases include chemically modified bases that have base-pairing ability equal to or greater than that of the original base (e.g., cytosine and 5-methylcytosine, uracil and 5-methyluracil), which are each considered to be identical bases.

[0018] As used herein, a "substantially complementary base sequence" refers to a base sequence in which all bases are complementary to the target base sequence, and in which 70% or more (preferably 80% or more, more preferably 90% or more, and even more preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the bases are complementary to the bases of the target sequence, as well as a base sequence in which one or several (preferably 10 or less, 8 or less, 5 or less, 4, 3 or less, or 2) bases are not complementary, and in which oligonucleotides can associate with each other through base pairing. Furthermore, as used herein, a "complementary base" refers to a base that can form a Watson-Crick base pair with the target base, and may be an unmodified base or a chemically modified base. For example, 5-methyluracil and adenine are complementary bases, and 5-methylcytosine and guanine are complementary bases. The identity or complementarity of the base sequences can be calculated using known gene analysis software such as BLAST (registered trademark).

[0019] Furthermore, as used herein, a wobble base pair can also be used as a "substantially complementary base sequence." A wobble base pair is a base pair that exhibits thermodynamic stability comparable to that of Watson-Crick base pairs, which are observed in sites that form higher-order structures of RNA and in codon-anticodon interactions between mRNA and tRNA, and refers to combinations of guanine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthine-cytosine. Furthermore, bases that can form the above-mentioned "substantially complementary base sequences" that are considered to exhibit similar stability to these base pairs can also be used.

[0020] The sequence information of the SARS-CoV-2 RNA genome has been registered in GenBank with the accession number NC_045512.2. The nucleotide sequence is shown in SEQ ID NO: 1 in the Sequence Listing. The sequence of the SARS-CoV-2 RNA genome is the sequence of nucleotides 1 to 29903 of the nucleotide sequence of SEQ ID NO: 1.

[0021] The frameshift pseudoknot (FSP) structure is a common structure among coronaviruses. It inhibits ribosomal translation through the steric effect of a nucleic acid structure (pseudoknot) formed by the combination of two stem-loop regions in mRNA, thereby controlling the -1 ribosomal frameshifting, a mechanism unique to viral replication. -1 ribosomal frameshifting is one of the mechanisms by which a single mRNA can produce two different proteins. During translation, the ribosome substitutes a -1 ribosomal frame, i.e., a frame shifted one frame in the 5' direction, for the normal translation frame at a specific position during translation, producing two different proteins. Coronaviruses use the -1 ribosomal frameshifting induced by the frameshift pseudoknot (FSP) structure in host cells to synthesize two or more proteins required for their own replication. Therefore, targeting the frameshift pseudoknot (FSP) structure to control the efficiency of -1 ribosomal frameshifting can control viral replication.

[0022] In the present invention, "RNA derived from coronavirus" means any form of RNA produced in association with coronavirus proliferation, such as coronavirus genomic RNA, RNA synthesized during viral replication, and replicated mRNA.

[0023] The oligonucleotides of the present invention have high binding ability to target RNA and can bind to the frameshift pseudoknot (FSP) structure of coronavirus-derived RNA and change its structure, and are therefore expected to be able to inhibit viral proliferation.

[0024] Examples of oligonucleotides of the present invention include those containing at least 17 consecutive bases in any of the sequences of SEQ ID NOs: 2, 5 to 8, and 11 to 13 (however, t in the sequence may be u, and u may be t).

[0025] The number of bases in the oligonucleotide of the present invention is suitably 17 to 30, preferably 18 to 24, and more preferably 21.

[0026] The oligonucleotide (antisense oligonucleotide) of the present invention may contain natural DNA nucleotides, natural RNA nucleotides, or modified forms thereof, but it is preferable that the oligonucleotide is a mixmer and at least one of the nucleotides constituting the oligonucleotide is a modified nucleotide.

[0027] The modified nucleotides of the present invention include those in which the sugar is modified (for example, those in which the hydroxyl group at the 2'-position of D-ribofuranose is modified (D-ribofuranose is 2'-O-alkylated), those in which D-ribofuranose is 2'-,4'-bridged (D-ribofuranose is 2'-O, Examples of such modified nucleosides include those in which the phosphodiester bond is modified (e.g., thioated, modified bases, and combinations thereof). Modified nucleosides in which at least one D-ribofuranose constituting the antisense oligonucleotide is 2'-O-alkylated (e.g., a 2'-O-methylated modified nucleoside is referred to as a "2'-OMe-RNA nucleoside") and 2'-O,4'-C-alkylenated bridged or other modified nucleosides (e.g., a 2'-O,4'-C-methylenated nucleoside is referred to as an "LNA nucleoside" and a 2'-O,4'-C-ethylenated nucleoside is referred to as an "ENA nucleoside") are also known. Because of their high binding strength to RNA and high resistance to nucleases, they can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligo-DNA, oligo-RNA). Furthermore, oligonucleotides having an internucleotide bond (phosphorothioate bond) in which at least one phosphodiester bond constituting the oligonucleotide is thioated also have high resistance to nucleases, and therefore can be expected to have a higher therapeutic effect than natural nucleotides (i.e., oligo-DNA, oligo-RNA). Because oligonucleotides containing both the above-mentioned modified sugar and modified phosphate have higher resistance to nucleases, they can be expected to have an even higher therapeutic effect.

[0028] With respect to the oligonucleotides (antisense oligonucleotides) of the present invention, examples of sugar modifications include 2'-O-alkylation (methylation, ethylation, propylation, isopropylation, butylation, etc.), 2'-O-alkoxyalkylation (methoxyethylation, methoxypropylation, methoxybutylation, etc.), 2'-halogenation (chlorination, fluorination, etc.), 2'-O,4'-C-bridges (alkylenation bridges, etc.), etc. Furthermore, as used herein, the term "sugar-modified nucleoside" refers to a nucleoside in which the sugar moiety of the nucleoside has been modified.

[0029] Among sugar-modified nucleosides, an example of 2'-O-alkylation is 2'-O-methylnucleoside (sometimes referred to as "2'-OMe-RNA nucleoside"). The 2'-O-methylnucleoside corresponding to each base is prepared by converting 2'-O-methyladenosine to A m1 , 2'-O-methylguanosine to G m1 , 2'-O-methylcytidine to C m1 , 2'-O-methyl-5-methylcytidine to 5meC m1 , 2'-O-methyluridine to U m1 , 2'-O-methyl-5-methyluridine to T m1 , and may also be written as.

[0030] Among sugar-modified nucleosides, an example of 2'-O-alkoxyalkylation is 2'-O-methoxyethyl nucleoside (sometimes referred to as "2'-MOE-RNA nucleoside"). The 2'-O-methoxyethyl nucleoside corresponding to each base is prepared by converting 2'-O-methoxyethyl adenosine to A. m2 , 2'-O-methoxyethylguanosine to G m2 , 2'-O-methoxyethyl-cytidine to C m2 2'-O-methoxyethyl-5-methylcytidine 5meC m2 , 2'-O-methoxyethyluridine to U m2 , 2'-O-methoxyethyl-5-methyluridine to T m2 It may also be written as:

[0031] Among sugar-modified nucleosides, an example of the 2'-O,4'-C-bridged modification is, for example, 2'-O,4'-C-ethylene-bridged nucleoside (sometimes referred to as "ENA nucleoside" or "ENA unit"). The 2'-O,4'-C-ethylene-bridged nucleoside corresponding to each base has a structure represented by the following formula, and 2'-O,4'-C-ethylene-bridged adenosine is A e2 , 2'-O,4'-C-ethylene-bridged guanosine to G e2 , 2'-O,4'-C-ethylene bridged-5-methylcytidine to C e2 (The base structure is 5meC, but it is displayed this way for convenience. It can also be used in place of 2'-O,4'-C-ethylene-bridged cytidine.) 2'-O,4'-C-ethylene-bridged uridine is U e2 , 2'-O,4'-C-ethylene-bridged-5-methyluridine e2 , and may also be written as.

[0032] Another example of the 2'-O,4'-C-bridged modification is a 2'-O,4'-C-methylene-bridged nucleoside (sometimes referred to as an LNA nucleoside or an LNA unit). The 2'-O,4'-C-methylene-bridged nucleoside corresponding to each base is formed by converting a 2'-O,4'-C-methylene-bridged adenosine to A. e1 , 2'-O,4'-C-methylene-bridged guanosine to G e1 , 2'-O,4'-C-methylene bridge-5-methylcytidine to C e1 (The base structure is 5meC, but it is displayed this way for convenience. It can also be used in place of 2'-O,4'-C-methylene-bridged cytidine.) 2'-O,4'-C-methylene-bridged uridine is represented by U e1 , 2'-O,4'-C-methylene-bridged-5-methyluridine to T e1 It can also be expressed as:

[0033] In the oligonucleotides (antisense oligonucleotides) of the present invention, examples of modifications of phosphodiester bonds include phosphorothioate bonds, methylphosphonate bonds, methylthiophosphonate bonds, phosphorodithioate bonds, phosphoramidate bonds, and the like.

[0034] Examples of base modifications for the oligonucleotides (antisense oligonucleotides) of the present invention include 5-methylation (5meC), 5-fluorolation, 5-bromination, 5-iodination, and N4-methylation of cytosine, 5-demethylation (uracil), 5-fluorolation, 5-bromination, and 5-iodination of thymine, N6-methylation and 8-bromination of adenine, and N2-methylation and 8-bromination of guanine. Further examples include trace bases such as pseudouracil, dihydrouracil, and hypoxanthine.

[0035] The nucleotide residues constituting the oligonucleotide (antisense oligonucleotide) of the present invention include A t , G t , 5meC t , C t , T t , U t , A p , G p , 5meC p , C p , T p , U p , A s , G s , 5meC s , C s , T s , U s , A m1t , G m1t , C m1t , 5meC m1t , U m1t , A m1p , G m1p , C m1p , 5meC m1p , U m1p , A m1s , G m1s , C m1s , 5meC m1s , U m1s , A 2t , G 2t , C 2t , T 2t , A e2p , G e2p , C e2p , T e2p , A e2s , G e2s , Ce2s , T e2s , A 1t , G 1t , C 1t , T 1t , A e1p , G e1p , C e1p , T e1p , A e1s , G e1s , C e1s , T e1s , A 3t , G 3t , C 3t , T 3t , A e3p , G e3p , C e3p , T e3p , A e3s , G e3s , C e3s , T e3s , A m2t , G m2t , 5meC m2t , T m2t , A m2p , G m2p , 5meC m2p , T m2p , A m2s , G m2s , 5meC m2s , T m2s and has the structure shown below.

[0036]

[0037] The oligonucleotides (antisense oligonucleotides) of the present invention are mixmers that do not recruit RNase H. A mixmer is an oligonucleotide containing nucleosides with at least two different sugar structures, including DNA nucleosides, RNA nucleosides, and various sugar-modified nucleosides. Antisense oligonucleotides containing a region of more than four or more consecutive DNA nucleosides are generally called gapmers and are known to recruit RNase H and degrade target mRNA. In Test Example 3, oligonucleotides with gapmer structures reported in Non-Patent Document 11 showed almost no inhibitory effect on coronavirus proliferation, whereas oligonucleotides with mixmer structures of the present invention exhibited strong inhibitory activity. Thus, the antisense oligonucleotides of the present invention exhibit strong viral proliferation inhibitory activity by having a mixmer structure (preferably not containing a region of five or more, more preferably four or more, consecutive DNA nucleosides) that does not recruit RNase H.

[0038] The antisense oligonucleotides of the present invention may be mixmers containing both sugar-unmodified nucleosides and sugar-modified nucleosides. The sugar-unmodified nucleosides may be RNA nucleosides and / or DNA nucleosides. When DNA nucleosides are used, the antisense oligonucleotide does not contain a region of five or more (preferably four or more) consecutive DNA nucleosides. Specific examples of such mixmers include oligonucleotides with a design in which one DNA nucleoside (D) and one sugar-modified nucleoside (M) are alternately arranged (e.g., 5'-MDMD-3'), oligonucleotides with a design in which two DNA nucleosides (D) and one sugar-modified nucleoside (M) are alternately arranged (e.g., 5'-MDDMDD-3'), and oligonucleotides with the aforementioned design in their entirety. The sugar-modified nucleoside used here is not particularly limited, but is preferably a 2'-OMe-RNA nucleoside, an LNA nucleoside and / or an ENA nucleoside.

[0039] The oligonucleotide (antisense oligonucleotide) of the present invention may be an antisense oligonucleotide that does not contain DNA or RNA nucleosides, i.e., a totalmer. In this case, the mixmer is designed to contain a mixture of multiple different types of sugar-modified nucleosides, preferably a mixture of bridged and non-bridged sugar-modified nucleosides. Examples of such oligonucleotides include oligonucleotides having a configuration in which nucleosides are arranged alternately (e.g., 5'-BNBNBN-3') with one bridged sugar-modified nucleoside (B) and one non-bridged sugar-modified nucleoside (N) (e.g., 5'-BNNBNN-3'), or oligonucleotides having the above-mentioned design as a whole, and other oligonucleotides having designs in which the frequency of bridged sugar-modified nucleosides varies depending on the region. The bridged sugar-modified nucleoside is not particularly limited, but is preferably an LNA nucleoside or an ENA nucleoside. The non-bridged sugar-modified nucleoside is not particularly limited, as long as it is a sugar-modified nucleoside in which the sugar ring structure is not bridged, but is preferably a 2'-OMe-RNA nucleoside.

[0040] The oligonucleotides shown below are suitable as the oligonucleotides (antisense oligonucleotides) of the present invention. m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t-H(ASO20) (SEQ ID NO: 24) HO-G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t -H(ASO39) (SEQ ID NO: 27) HO-A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s [[ID=6))-T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A<OO00533>-G m1t -H(ASO40) (SEQ ID NO: 28) HO-A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -Cm1t -H(ASO41) (SEQ ID NO: 29) HO-U m1s -C e2s -A m1s -A m1s -A e2s -A m1s -G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1t -H(ASO42) (SEQ ID NO: 30) HO-A m1s -G e2s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -U m1s -A m1s -C m1s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G e2s -U m1t -H(ASO73) (SEQ ID NO: 33) HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -H (ASO75) (SEQ ID NO: 34) HO-Am1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO76) (SEQ ID NO: 35)

[0041] The most preferred oligonucleotide (antisense oligonucleotide) of the present invention is the oligonucleotide shown below: HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO20) (SEQ ID NO: 24)

[0042] The oligonucleotides (antisense oligonucleotides) of the present invention can be synthesized using a commercially available synthesizer (e.g., Perkin-Elmer Model 392) or the like, using the phosphoramidite method in accordance with the method described in the literature (Nucleic Acids Research, 12, 4539 (1984)). Commercially available phosphoramidite reagents can be used for natural nucleosides and 2'-O-methyl nucleosides (i.e., 2'-O-methylguanosine, 2'-O-methyladenosine, 2'-O-methylcytidine, and 2'-O-methyluridine). The phosphoramidite reagents used for 2'-O-alkylguanosine, adenosine, cytidine, and uridine, each having an alkyl group of 2 to 6 carbon atoms, are as follows:

[0043] 2'-O-aminoethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Blommers et al., Biochemistry (1998), 37, 17714-17725).

[0044] 2'-O-propylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0045] Commercially available reagents can be used for 2'-O-allylguanosine, adenosine, cytidine, and uridine.

[0046] 2'-O-Methoxyethylguanosine, adenosine, cytidine, and uridine can be synthesized according to the patent (US Pat. No. 6,261,840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504).

[0047] 2'-O-butylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0048] 2'-O-pentylguanosine, adenosine, cytidine, and uridine can be synthesized according to the literature (Lesnik, EA et al., Biochemistry (1993), 32, 7832-7838).

[0049] Commercially available reagents can be used for 2'-O-propargylguanosine, adenosine, cytidine, and uridine.

[0050] 2'-O,4'-C-methyleneguanosine, adenosine, cytidine, 5-methylcytidine, and thymidine can be produced according to the method described in WO99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, cytidine, 5-methylcytidine, and thymidine having an alkylene group having 2 to 5 carbon atoms can be produced according to the method described in WO00 / 47599.

[0051] The 2'-deoxy-2'-C,4'-C-methyleneoxymethylenated nucleoside of D-ribofuranose can be synthesized according to the literature (Wang, G. et al. Tetrahedron (1999), 55, 7707-7724).

[0052] S-cEt (constrained ethyl) can be synthesized according to the literature (Seth, P.P. et al. J. Org. Chem (2010), 75, 1569-1581.).

[0053] AmNA can be synthesized according to the literature (Yahara, A. et al. ChemBioChem (2012), 13, 2513-2516.) or WO2014 / 109384.

[0054] In the present invention, nucleic acid base sequences can be represented as adenine (A) or (a), guanine (G) or (g), cytosine (C) or (c), thymine (T) or (t), and uracil (U) or (u), respectively. 5-methylcytosine can be used in place of cytosine. Among nucleic acid bases, uracil (U) or (u) and thymine (T) or (t) are interchangeable. Either uracil (U) or (u) or thymine (T) or (t) can be used to form a base pair with adenine (A) or (a) in a complementary strand. Furthermore, wobble base pairs can also be used as base pairs. Specifically, either uracil (U) or (u) and thymine (T) or (t) can be used to form a base pair with guanine (G) or (g) in a complementary strand. Additionally, any of adenine (A) or (a), cytosine (C) or (c), 5-methylcytosine, uracil (U) or (u), and thymine (T) or (t) can be used to form base pairs with inosine in the complementary strand.

[0055] After coupling with a phosphoramidite reagent, antisense oligonucleotides having phosphorothioate bonds can be synthesized by reacting with a reagent such as sulfur, tetraethylthiuram disulfide (TETD, Applied Biosystems), Beaucage reagent (Glen Research), or xanthan hydride (Tetrahedron Letters, 32, 3005 (1991), J. Am. Chem. Soc. 112, 1253 (1990), PCT / WO98 / 54198).

[0056] As the controlled pore glass (CPG) used in the synthesizer, commercially available products can be used for 2'-O-methylnucleosides. 2'-O,4'-C-methyleneguanosine, adenosine, 5-methylcytidine, and thymidine can be bound to CPG according to the method described in WO 99 / 14226, and 2'-O,4'-C-alkyleneguanosine, adenosine, 5-methylcytidine, and thymidine, each of which has an alkylene group containing 2 to 5 carbon atoms, can be bound to CPG according to the method described in WO 00 / 47599 (Oligonucleotide Synthesis, Edited by M.J. Gait, Oxford University Press, 1984). Modified CPG (described in Example 12b of JP-A-7-87982) can be used to synthesize oligonucleotides with a 2-hydroxyethyl phosphate group attached to the 3'-terminus. Furthermore, 3'-amino-Modifier C3 CPG, 3'-amino-Modifier C7 CPG, Glyceryl CPG (Glen Research), 3'-spacer C3 SynBase CPG 1000, or 3'-spacer C9 SynBase CPG 1000 (Link Technologies) can be used to synthesize oligonucleotides with a hydroxyalkyl phosphate group or an aminoalkyl phosphate group attached to the 3'-terminus.

[0057] The oligonucleotides (antisense oligonucleotides) of the present invention can be chemically modified by linking their 3' and / or 5' ends to a non-nucleotide molecular structure via a phosphodiester bond or a phosphorothioate bond. The present invention also provides such chemically modified oligonucleotides.

[0058] Preferred examples of such chemically modified oligonucleotides are those in which a phosphate group linked to a hydrophobic group is attached to the 5' or 3' end, either directly or via a linker. The linking structure between the hydrophobic group and the phosphate group refers to an alkyl phosphate group having a linking group. Here, the alkyl is a linear alkyl having 3 to 9 carbon atoms, preferably C6 or C3. The "linking group" refers to a functional group capable of binding to a functional group contained in the hydrophobic moiety, such as an amino group capable of binding to a carboxyl group in the case of a fatty acid. When the phosphate group linked to the hydrophobic group at the end of the oligonucleotide is attached via a linker, the linker structure is not particularly limited, but various known phosphoramidite compounds can be used to enable conjugation in succession with the oligonucleotide elongation reaction.

[0059] After chain elongation of an oligonucleotide having a target sequence is completed, an oligonucleotide having an aminoalkyl phosphate group bound to the 5' end can be synthesized by using 5'-Amino-Modifier C6 (Glen Research), 5'-TFA-Amino-Modifier C6-CE Phosphoramidite, 5'-TFA-Amino-Modifier-C5-CE Phosphoramidite (Link Technologies), or the like.

[0060] The oligonucleotide (antisense oligonucleotide) of the present invention may have a hydrophobic group at the 5' or 3' end. After chain elongation of an oligonucleotide having a target sequence is completed, an amidite unit corresponding to a fatty acid such as 2-cyanoethyl (6-palmitamidohexyl) diisopropylphosphoramidite (Nucleic Acids Res. (2020) 47, 6029-6044, Link Technologies) can be synthesized by coupling.

[0061] It can be synthesized by reacting an oligonucleotide having an aminoalkyl phosphate group (e.g., the alkyl has 3 to 9 carbon atoms) at the 5' or 3' end and having a target sequence with an activated ester such as a pentafluorophenyl ester of a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid (Nucleic Acids Res. (2020) 47, 6029-6044). Alternatively, the oligonucleotide can be synthesized by solid-phase synthesis of an oligonucleotide having a target sequence on a solid support having an aminoalkyl phosphate group (e.g., the alkyl has 3 to 9 carbon atoms) at the 5' end, condensing the oligonucleotide with a fatty acid such as myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid using a condensing agent such as HATU, followed by deprotection and purification (PCT / WO2017 / 192679).

[0062] Furthermore, by using 5'-tocopherol-CE phosphoramidite, 5'-cholesterol-CE phosphoramidite, 5'-cholesterol-TEG-CE phosphoramidite (Link Technologies), or the like, it is possible to synthesize oligonucleotides having cholesterol or tocopherol bound to the 5' end.

[0063] The oligonucleotides (antisense oligonucleotides) of the present invention can be used as pharmaceuticals, specifically for the treatment of coronavirus infections. Treatment may be administered before, simultaneously with, or after the onset of symptoms. In the present invention, coronaviruses include four types that cause common cold symptoms, namely, human coronavirus 229E, human coronavirus NL63, human coronavirus HKU1, and human coronavirus OC43, and three types that cause severe pneumonia, namely, SARS-CoV, the pathogen of Severe Acute Respiratory Syndrome (SARS), MERS-CoV, the pathogen of Middle East Respiratory Syndrome (MERS), and SARS-CoV-2, the pathogen of novel coronavirus disease (COVID-19), as well as their mutant viruses.

[0064] The oligonucleotide (antisense oligonucleotide) of the present invention may be used in the form of a pharmaceutically acceptable salt thereof. "A pharmaceutically acceptable salt thereof" refers to a salt of the oligonucleotide (antisense oligonucleotide), and examples of such salts include metal salts such as alkali metal salts (e.g., sodium salt, potassium salt, and lithium salt), alkaline earth metal salts (e.g., calcium salt and magnesium salt), aluminum salt, iron salt, zinc salt, copper salt, nickel salt, and cobalt salt; inorganic salts (e.g., ammonium salt), t-octylamine salt, dibenzylamine salt, morpholine salt, glucosamine salt, phenylglycine alkyl ester salt, ethylenediamine salt, N-methylglucamine salt, guanidine salt, diethylamine salt, triethylamine salt, dicyclohexylamine salt, N,N'-dibenzylethylenediamine salt, chloroprocaine salt, procaine salt, diethanolamine salt, N-benzo[b]thiazolinone salt, ... Examples of suitable salts include amine salts, such as organic salts like di-phenethylamine salts, piperazine salts, tetramethylammonium salts, and tris(hydroxymethyl)aminomethane salts; hydrohalides like hydrofluoride, hydrochloride, hydrobromide, and hydroiodide; inorganic acid salts like nitrate, perchlorate, sulfate, and phosphate; lower alkanesulfonates like methanesulfonate, trifluoromethanesulfonate, and ethanesulfonate; arylsulfonates like benzenesulfonate and p-toluenesulfonate; organic acid salts like acetate, malate, fumarate, succinate, citrate, tartrate, oxalate, and maleate; and amino acid salts like glycine salt, lysine salt, arginine salt, ornithine salt, glutamate, and aspartate. These salts can be prepared by known methods.

[0065] Furthermore, oligonucleotides (antisense oligonucleotides) may exist as solvates (e.g., hydrates), and such solvates are also included in the pharmaceutically acceptable salts of the oligonucleotides of the present invention.

[0066] Thus, the present invention provides a pharmaceutical comprising the above-described oligonucleotide or a pharmaceutically acceptable salt thereof. The present invention also provides a therapeutic and / or prophylactic agent for coronavirus infections, particularly SARS-CoV-1, SARS-CoV-2, or MERS-CoV infections, comprising the above-described oligonucleotide or a pharmaceutically acceptable salt thereof.

[0067] The oligonucleotides of the present invention bind to the frameshift pseudoknot (FSP) structure common to coronaviruses and inhibit viral proliferation, and are therefore effective in treating and / or preventing coronavirus infections.

[0068] When the oligonucleotide (antisense oligonucleotide) of the present invention or a pharmaceutically acceptable salt thereof is used to treat coronavirus infection, it can be administered orally in the form of tablets, capsules, granules, powders, syrups, etc., or parenterally in the form of injections, suppositories, patches, topical preparations, etc., either by itself or mixed with an appropriate pharmaceutically acceptable excipient, diluent, etc.

[0069] These preparations contain excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol, and sorbitol; starch derivatives such as corn starch, potato starch, alpha starch, and dextrin; cellulose derivatives such as crystalline cellulose; gum arabic; dextran; organic excipients such as pullulan; silicate derivatives such as light anhydrous silicic acid, synthetic aluminum silicate, calcium silicate, and magnesium aluminometasilicate; phosphates such as calcium hydrogen phosphate; carbonates such as calcium carbonate; and sulfates such as calcium sulfate). lubricants (e.g., stearic acid; metal stearates such as calcium stearate and magnesium stearate; talc; colloidal silica; waxes such as beeswax and Gay's wax; boric acid; adipic acid; sulfates such as sodium sulfate; glycol; fumaric acid; sodium benzoate; DL-leucine; lauryl sulfates such as sodium lauryl sulfate and magnesium lauryl sulfate; silicic acids such as silicic anhydride and silicic acid hydrate; the above-mentioned starch derivatives, etc.), binders (e.g., hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinyl alcohol, Examples of the excipients include: disintegrants (e.g., cellulose derivatives such as low-substituted hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, and internally crosslinked sodium carboxymethyl cellulose; chemically modified starches and celluloses such as carboxymethyl starch, sodium carboxymethyl starch, and crosslinked polyvinylpyrrolidone); emulsifiers (e.g., colloidal clays such as bentonite and Veegum; metal hydroxides such as magnesium hydroxide and aluminum hydroxide; anionic surfactants such as sodium lauryl sulfate and calcium stearate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, and sucrose fatty acid esters); stabilizers (parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol, benzyl alcohol, and phenylethyl alcohol; benzalkonium chloride; phenols such as phenol and cresol;The pharmaceutical composition is prepared by a known method using additives such as thimerosal, dehydroacetic acid, sorbic acid, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants, flavoring agents, etc.), diluents, etc.

[0070] The therapeutic and / or prophylactic agent of the present invention may contain 0.1 to 250 μmoles / mL of an oligonucleotide (antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, and preferably 1 to 50 μmoles / mL of an oligonucleotide (antisense oligonucleotide) or a pharmaceutically acceptable salt thereof, 0.02 to 10% w / v of a carbohydrate or polyhydric alcohol, and 0.01 to 0.4% w / v of a pharmaceutically acceptable surfactant.

[0071] The carbohydrate is preferably a monosaccharide or disaccharide. Examples of the carbohydrate and polyhydric alcohol include glucose, galactose, mannose, lactose, maltose, mannitol, and sorbitol. These may be used alone or in combination.

[0072] Preferred examples of surfactants in the present invention include polyoxyethylene sorbitan mono- to tri-esters, alkylphenyl polyoxyethylene, sodium taurocholate, sodium cholate, and polyhydric alcohol esters. Of these, polyoxyethylene sorbitan mono- to tri-esters are particularly preferred, and oleate, laurate, stearate, and palmitate are particularly preferred esters. These may be used alone or in combination.

[0073] Furthermore, the therapeutic and / or prophylactic agent of the present invention may further preferably contain 0.03 to 0.09 M of a pharmaceutically acceptable neutral salt, such as sodium chloride, potassium chloride and / or calcium chloride.

[0074] Furthermore, the therapeutic and / or prophylactic agent of the present invention may more preferably contain 0.002 to 0.05 M of a pharmaceutically acceptable buffer. Preferred examples of the buffer include sodium citrate, sodium glycinate, sodium phosphate, and tris(hydroxymethyl)aminomethane. These buffers may be used alone or in combination.

[0075] Furthermore, the above-mentioned therapeutic and / or prophylactic agent may be supplied in a solution state. However, when it is necessary to store the agent for a certain period of time, it is usually preferable to freeze-dry the oligonucleotide (antisense oligonucleotide) in order to stabilize it and prevent a decrease in its therapeutic effect. In this case, the agent is reconstituted with a solvent (such as distilled water for injection) at the time of use, i.e., in a liquid state for administration. Therefore, the therapeutic and / or prophylactic agent of the present invention also includes a freeze-dried agent that is reconstituted with a solvent so that each component has a predetermined concentration range. In order to promote the solubility of the freeze-dried agent, an amino acid such as albumin or glycine may be further contained.

[0076] When the oligonucleotide (antisense oligonucleotide) of the present invention or a pharmaceutically acceptable salt thereof is administered to a human, it may be administered, for example, at a dose of about 0.01 to 100 mg / kg (body weight), preferably 0.1 to 20 mg / kg (body weight) per day for an adult, by subcutaneous injection, intravenous drip injection, or intravenous injection, either once or in several divided doses. However, the dose and number of administrations may be appropriately changed depending on the type of disease, symptoms, age, administration method, etc.

[0077] The oligonucleotide (antisense oligonucleotide) of the present invention or a pharmaceutically acceptable salt thereof can be administered to humans, for example, patients with coronavirus infection or patients for whom coronavirus infection should be prevented, as follows: The oligonucleotide (antisense oligonucleotide) or a pharmaceutically acceptable salt thereof is produced by a method well known to those skilled in the art, sterilized by conventional methods, and an injectable solution of, for example, 125 mg / mL is prepared. This solution is administered intravenously to the patient, for example, in the form of an infusion, so that the dose of the oligonucleotide (antisense oligonucleotide) is, for example, 10 mg per kg of body weight. Administration is carried out, for example, at weekly intervals, and the treatment is then repeated as appropriate while monitoring the therapeutic effect.

[0078] The present invention will be specifically described below with reference to examples. Note that these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention. (Example 1) HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1tSynthesis of -H(ASO20) (SEQ ID NO: 24) Synthesis was performed using an automatic nucleic acid synthesizer (nS8-II manufactured by GeneDesign) using the phosphoramidite method (Nucleic Acids Research, 12, 4539 (1984)). Reagents included BMI (0.30 mol / L 5-benzylthio-1H-tetrazole / 0.5% N-methylimidazole / acetonitrile solution, manufactured by Honeywell, product No. BR731-4), CAP A for AKTA (1-methylimidazole / acetonitrile solution, manufactured by Sigma-Aldrich, product No. L040050), and Cap B solution. The following reagents were used: Deblocking Solution-1 (acetic anhydride-2,6-lutidine acetonitrile (2:3:5) solution, Fujifilm Wako Pure Chemical Industries, Ltd., product No. 034-25401), and Deblocking Solution-2 (3 w / v% trichloroacetic acid-dichloromethane solution, Fujifilm Wako Pure Chemical Industries, Ltd., product No. 042-28921). As a thiolation reagent for forming phosphorothioate bonds, phenylacetyl disulfide (CARBOSYNTH, product No. FP07495) was dissolved to a concentration of 0.2 M in acetonitrile (anhydrous, Kanto Chemical Industries, product No. 01837-05), pyridine (anhydrous, Kanto Chemical Industries, product No. The nucleoside was dissolved in a 1:1 (v / v) solution of 11339-05. The amidite reagents used were 2'-O-Me nucleoside phosphoramidites (adenosine product No. ANP-5751, cytidine product No. ANP-5752, guanosine product No. ANP-5753, uridine product No. ANP-5754) manufactured by ChemGenes. 2'-O-Methoxyethyl nucleoside phosphoramidites (adenosine product No. PB59569, 5-methylcytidine product No. PB64062, guanosine product No. PB64066) manufactured by ChemGenes. PD63448, and 5-methyluridine (product No. PD32565) manufactured by BIOSYNTH were used.The phosphoramidites of LNA nucleosides (adenosine product No. PR1-002, 5-methylcytidine product No. PR3-009, guanosine product No. PR2-005, 5-methyluridine product No. PR4-001) manufactured by HONGENE were used. The phosphoramidite of ENA nucleoside is described in JP-A 2000-297097, Example 14 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-6-N-benzoyladenosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), Example 27 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-2-N-isobutyrylguanosine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), Example 22 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-4-N-benzoyl-5-methylcytidine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite), The compounds used were those from Example 1 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite) and Example 9 (5'-O-dimethoxytrityl-2'-O,4'-C-ethylene-5-methyluridine-3'-O-(2-cyanoethyl N,N-diisopropyl)phosphoramidite). The compounds shown were synthesized using High Load Glen UniSupport 79 μmol / g (manufactured by Glen Research) as the solid phase support. The time required for condensation of the amidite was approximately 10 minutes.

[0079] The protected oligonucleotide analogues having the target sequence were treated with 600 μL of concentrated aqueous ammonia at 55°C for 7 hours to cleave the oligomers from the support and remove the cyanoethyl protecting groups on the phosphorus atoms and the nucleobases. The oligomer mixture was mixed with 300 μL of Clarity QSP DNA Loading Buffer (Phenomenex, product No. AL0-8280) and charged onto Clarity 30 μm QSP 60 mg / 3 mL tubes (Phenomenex, product No. 8B-S102-UBJ). After adding 1 mL of a 1:1 Clarity QSP DNA Loading Buffer:water solution, 3 mL of water, 3 mL of a 3% dichloroacetic acid (DCA) solution, and 6 mL of water in that order, the components extracted with a 9:1 solution of 20 mM Tris and acetonitrile were collected. After distilling off the solvent, the target compound was obtained. This compound was analyzed by reverse-phase HPLC (column (YMC-Triart Bio C18, 50 x 2.1 mm. D. S-5 μm, 30 nm, manufactured by YMC Corporation), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution, solution B: methanol, B%: 10% → 25% (4 min, linear gradient) → 40% (2 min, linear gradient); 60°C; 0.5 mL / min; 260 nm), and eluted at 3.413 minutes. The compound was identified by negative ion ESI mass spectrometry (calculated value: 7392.84, found value: 7392.83).

[0080] The base sequence of this compound is complementary to nucleotides 13542-13522 of the SARS-CoV-2 RNA genome, Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome (NCBI-GenBank accession No. NC_045512.2) (SEQ ID NO: 1).

[0081] (Examples 2 to 8, Comparative Examples 1 to 8) The compounds of Examples 2 to 8 and Comparative Examples 1 to 8 shown in Table 1 were synthesized in the same manner as in Example 1. Data for Examples 1 to 8 and Comparative Examples 1 to 8 are shown in Table 1. Note that Comparative Examples 3 to 8 are control antisense oligonucleotides disclosed in prior art documents, etc. Comparative Example 3: Among the ASOs described in Patent Document 1 (WO 2005 / 023083), IO329729, which has a description of actual evaluation, was replaced with the corresponding SARS-CoV-2 sequence instead of the SARS-CoV sequence described therein. Comparative Example 4: Among the ASOs described in Patent Document 1 (WO 2005 / 023083), IO329551, which has a description of actual evaluation, was replaced with the corresponding SARS-CoV-2 sequence instead of the SARS-CoV sequence described therein. Comparative Example 5: Among the ASOs described in Non-Patent Document 10: Nature Communications, 2022, 13:4503, this corresponds to F-ASO #06. Comparative Example 6: Among the ASOs described in Non-Patent Document 10: Nature Communications, 2022, 13:4503, this corresponds to 5'-ASO #26, for which in vivo evaluation results have been shown. Comparative Example 7: The 16th Annual Meeting of the Oligonucleotide Therapeutics Society (September 30, 2020) (Lisa Henderson et al., NINDS, 10:20-10:40, title: Antisense oligonucleotides as therapeutic agents for SARS-CoV-2) ASO disclosed in Comparative Example 8: Patent Document 2: Among the ASOs described in WO 2021 / 211928, an exemplary sequence corresponds to IONIS-1497377, whose in vitro evaluation results are shown. In the sequences in the table, uppercase letters indicate DNA, lowercase letters indicate 2'-OMe-RNA, underlined uppercase letters indicate ENA, underlined lowercase letters indicate 2'-MOE-RNA, and italicized uppercase letters indicate LNA. The bases used for each nucleoside are A, G, 5meC, and T for DNA, ENA, LNA, and 2'-MOE-RNA, and A, G, C, and U for 2'-OMe-RNA. Note that "^" between sequences indicates a phosphorothioate bond, and "." indicates a phosphodiester bond. The start and end nucleotide numbers indicate the 5'- and 3'-terminal nucleotides of the region targeted by each antisense oligonucleotide in the Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1, complete genome (NCBI-GenBank accession No. NC_045512.2). The measured molecular weights in the table are values ​​determined by negative ion ESI mass spectrometry. The retention times shown are those obtained by reverse-phase HPLC (column (YMC-Triart Bio C18, 50 x 2.1 mm. D. S-5 μm, 30 nm, manufactured by YMC Corporation), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution, solution B: methanol, B%: 10% → 25% (4 min, linear gradient) → 40% (2 min, linear gradient); 60°C; 0.5 mL / min; 260 nm). Note that in the sequence listing, SEQ ID NOs: 2 to 17 show only the sequence information without distinguishing between natural and modified nucleosides, while SEQ ID NOs: 24 to 39 also show information on nucleoside modifications and inter-sequence bonds.

[0082] The oligonucleotides prepared in Examples 2 to 8 are as follows: HO-G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -Am1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t -H(ASO39) (SEQ ID NO: 27) HO-A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A e2s -G<s m1t -H(ASO40) (SEQ ID NO: 28) HO-A m1s -A e2s -A<s m1s -A m1s -G e2s -C m1s -C<000s000730> / m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -H(ASO41) (SEQ ID NO: 29) HO-U m1s -C e2s -A m1s -A m1s -A e2s -A m1s -G m1s -C e2s -C m1s -C [[ID=1s0]] m1s / m1s -T e2s -G m1s 说明:原文中 m1s 和 m1s 疑似格式问题,推测应为 m1s 和 m1s ,翻译时保留了原文格式,同时按照要求保留了所有7位标签。你可根据实际情况进行调整。-U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1t -H(ASO42) (SEQ ID NO: 30) HO-A m1s -G e2s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -U m1s -A m1s -C m1s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G e2s -U<x m1t -H(ASO73) (SEQ ID NO: 33) HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -H(ASO75) (SEQ ID NO: 34) HO-A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -Cm1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO76) (SEQ ID NO: 35)

[0083] (Example 9) (C18-C6)-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t Synthesis of —H(LIPID-ASO20) (“(C18-C6)-” is represented by the following formula.) After synthesizing an oligonucleotide of ASO20 by the method described in Example 1, (C18-C6) amidite (Reference Example 1 of WO2021 / 010301A1) was additionally used as the amidite, and instead of a 0.2 M phenylacetyl disulfide solution as a thiolation reagent, a 0.05 M iodine solution (Sigma-Aldrich, product No. L060050) was used for 2 minutes to bind a fatty acid (C18-C6) to the 5' end of ASO20, thereby synthesizing a protected form of LIPID-ASO20.

[0084] The protected oligonucleotide analogue having the target sequence was treated with 3 mL of concentrated aqueous ammonia at 55°C for 7.5 hours to cleave the oligomer from the support and remove the cyanoethyl protecting group on the phosphorus atom and the protecting groups on the nucleic acid bases. After removing the support by filtration, the solution was concentrated and purified by reverse-phase HPLC (column (YMC-Triart Bio C18, 150 x 20 mm. D. S-5 μm, 30 nm, manufactured by YMC Corporation), solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution, solution B: methanol, B%: 10% → 100% (6 min, linear gradient) → 10% (2 min, linear gradient); 60°C; 30 mL / min; 260 nm). The target compound was obtained after distilling off the solvent. Compound LIPID-ASO20 was analyzed by reverse-phase HPLC (column (YMC-Triart Bio C18, 50 x 2.1 mm. D. S-5 μm, 30 nm, manufactured by YMC Corporation); Solution A: 100 mM hexafluoroisopropanol (HFIP), 8 mM aqueous triethylamine solution; Solution B: methanol; B%: 10% → 25% (4 min, linear gradient) → 40% (2 min, linear gradient) → 85% (2 min, linear gradient); 60°C; 0.5 mL / min; 260 nm), and was eluted at 7,360 minutes. The compound was identified by negative ion ESI mass spectrometry (calculated value: 7838.09, found value: 7338.17).

[0085] (Test Example 1) Antiviral activity screening of SARS-CoV-2 antisense oligos 2 × 10 4 TMPRSS2-expressing VeroE6 cells (hereinafter referred to as VeroE6 / TMPRSS2 cells) were seeded at 1 cell / well and cultured overnight at 37°C in 5% CO2. TMEach antisense oligo (Experiment 1: ASO11, 17, or 20; Experiment 2: ASO39-42) was mixed with RNAiMAX Transfection Reagent (Thermo Fisher Scientific) to a final concentration of 100 nM and added, followed by incubation at 37°C for 4 hours. Then, SARS-CoV-2 (Wk-521 strain) was inoculated into the cells at a multiplicity of infection (MOI) of 0.003 and incubated at 37°C, 5% CO2 for 1 hour. After incubation, the culture supernatant was removed, the cells were washed with culture medium, and 150 μL / well of culture medium was added. The cells were then incubated at 37°C, 5% CO2 for 24 hours. After incubation, 80 μL of the culture supernatant was collected and the viral RNA was quantified. After further incubation for 24 hours, the cells were observed under a microscope (OLYMPUS) for cytopathic effect (CPE) associated with viral infection. RNA was extracted from the culture supernatant using the MagMax Viral / Pathogen Nucleic Acid Isolation kit (Thermo Fisher Scientific), and quantitative PCR of SARS-CoV-2 RNA was performed using the THUNDERBIRD Probe One-step qRT-PCR kit (TOYOBO) and the following primers and probes according to a previous report (Ohashi et al. Antiviral Res, 205, 105372, 2022).

[0086] Primer probes for quantification of SARS-CoV-2 RNA: Forward primer S17: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 18) Reverse primer R19: 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 19) Probe R6-148 S21FT: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3' (SEQ ID NO: 20)

[0087] Viral RNA was quantified by the ΔCt method. Control wells not treated with antisense oligos were treated with the same amount of Lipofectamine. TM The cells were treated with culture medium containing only RNAiMAX Transfection Reagent, and the quantitative value of the control well was set to 100%, and the relative percentage of viral RNA in each well was calculated. The results are shown in Figures 1 and 2. In the following figures, the negative control with only Lipofectamine RNAiMAX added is shown as RNAi(+), the negative control without addition is shown as RNAi(-), the control using the small molecule antiviral drug remdesivir is shown as RDV, and the control using the small molecule antiviral drug molnupiravir is shown as MPV. ASO20, which has a sequence complementary to the 3' end of the frameshift pseudoknot structure, showed the strongest virus-inhibitory effect. ASO39, ASO40, ASO41, and ASO42, which are sequences adjacent to ASO20, all exhibit high viral growth inhibitory effects. However, it was confirmed that even antisense oligos complementary to sequences with a frameshift pseudoknot structure have a significantly weakened viral growth inhibitory effect when the sequence is complementary to a position distant from the target sequence of ASO20.

[0088] (Test Example 2) Comparison of anti-SARS-CoV-2 activity of ASO20 and control ASO. 4 TMPRSS2-expressing VeroE6 cells (hereinafter referred to as VeroE6 / TMPRSS2 cells) were seeded at 1 cell / well and cultured overnight at 37°C in 5% CO2. TMVarious antisense oligos mixed with RNAiMAX (Thermo Fisher Scientific) were added at final concentrations of 10, 30, or 100 nM and incubated at 37°C for 24 hours. Subsequently, SARS-CoV-2 (Wk-521 strain) was inoculated into the cells at an MOI of 0.003 and incubated at 37°C and 5% CO2 for 1 hour. After incubation, the culture supernatant was removed, the cells were washed with culture medium, and 150 μL / well of culture medium was added. The cells were then incubated at 37°C and 5% CO2 for 24 hours. The culture supernatant was then recovered, and RNA extraction and viral RNA quantification were performed as in Test Example 1. The results of a comparison of activity with sequences reported to have inhibitory effects on SARS-CoV viral growth (ASO68 in Comparative Example 3 and ASO69 in Comparative Example 4) are shown in Figure 3 (Experiment 3). The reported sequence targets SARS-CoV and differs from the sequence of SARS-CoV-2. Therefore, the sequence described in the literature was modified to a sequence complementary to the corresponding sequence of SARS-CoV-2 and then examined. Under these conditions, the sequence described in the literature, modified to a sequence complementary to the corresponding SARS-CoV-2, did not exhibit any inhibitory effect on viral growth. The results of examining sequences with altered modification patterns and lengths of ASO20 are shown in Figure 4 (Experiment 4). Both sequences were found to exhibit inhibitory effect on viral growth at 30 nM.

[0089] (Test Example 3) Antiviral activity of antisense oligo ASO20 and control antisense oligo against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. 4 VeroE6 / TMPRSS2 cells were seeded at 1 / well and cultured overnight at 37°C in 5% CO2. TMVarious antisense oligos mixed with RNAiMAX (Thermo Fisher Scientific) were added at a final concentration of 10-100 nM and incubated at 37°C for 24 hours. Then, SARS-CoV-2 (Wk-521 strain or TW38-873 strain), SARS-CoV (Frankfurt-1 strain), or MERS-CoV (EMC strain) was inoculated into the cells at an MOI of 0.003 and incubated at 37°C, 5% CO2 for 1 hour. After incubation, the culture supernatant was removed, the cells were washed with culture medium, and 150 μL / well of culture medium was added. The cells were then incubated at 37°C, 5% CO2 for 24 hours. Thereafter, the culture supernatant was collected, and RNA was extracted using a MagMax Viral / Pathogen Nucleic Acid Isolation kit (Thermo Fisher Scientific). Quantitative PCR was then performed using a THUNDERBIRD Probe One-step qRT-PCR kit (TOYOBO) and the following primers and probes according to a previous report (Ohashi et al. Antiviral Res, 205, 105372, 2022).

[0090] Primer / probe for quantification of SARS-CoV and SARS-CoV-2: Forward primer S17: 5'-ACAGGTACGTTAATAGTTAATAGCGT-3' (SEQ ID NO: 18) Reverse primer R19: 5'-ATATTGCAGCAGTACGCACACA-3' (SEQ ID NO: 19) Probe R6-148 S21FT: 5'-FAM-ACACTAGCCATCCTTACTGCGCTTCG-TAMRA-3' (SEQ ID NO: 20)

[0091] Primer / probe for quantification of MERS-CoV: Forward primer upE-F: 5'-GCAACGCGCGATTCAGTT-3' (SEQ ID NO: 21) Reverse primer upE-R: 5'-GCCTCTACACGGGACCCATA-3' (SEQ ID NO: 22) Probe upE (FAM-TAM): 5'-FAM-CTCTTCACATAATCGCCCCGAGCTCG-TAMRA-3' (SEQ ID NO: 23)

[0092] Viral RNA was quantified by the ΔCt method. Control wells not treated with antisense oligos were treated with the same amount of Lipofectamine. TMThe relative percentage of viral RNA in each well was calculated by setting the quantitative value of the control well, treated with culture medium containing only RNAiMAX Transfection Reagent, as 100%. The results for the SARS-CoV-2 wild-type strain (Wk-521 strain) are shown in Figure 5, the results for the SARS-CoV-2 Omicron variant strain (BA.1 strain) are shown in Figure 6, the results for SARS-CoV are shown in Figure 7, and the results for MERS-CoV are shown in Figure 8. ASO20 exhibited antiviral activity not only against the SARS-CoV-2 wild-type strain (Wk-521 strain), but also against the SARS-CoV-2 Omicron variant strain (BA.1 strain), SARS-CoV, and MERS-CoV. On the other hand, the control prior art antisense oligos either showed weaker antiviral activity compared to ASO20 or no antiviral activity against any of the viruses. Therefore, ASO20 was demonstrated to be an antisense oligo that exhibits high antiviral activity against a wide range of coronaviruses. These results demonstrate that, compared to previously reported sequences, only ASOs targeting sequences adjacent to the target sequence of ASO20 exhibit a high viral growth inhibitory effect. Furthermore, Comparative Example 7, an ASO targeting a sequence that differs by one base from the target sequence of ASO20, exhibited a significant difference in viral growth inhibitory activity, demonstrating that a mixmer structure design is suitable for expressing activity. Furthermore, while the target sequence of the present invention targets SARS-CoV-2, it is highly homologous to sequences of SARS-CoV, MERS-CoV, and other viruses, and is thought to be a site that is difficult to mutate, and therefore exhibited a high growth inhibitory effect against these viruses as well. This suggests that growth inhibitory effects against a wide range of coronaviruses with homologous sequences can be expected.

[0093] Test Example 4: Antiviral effect of the ASO of the present invention in a mouse model infected with SARS-CoV-2 Various ASOs dissolved in saline were intranasally administered once daily at 100 or 400 μg / mouse under isoflurane anesthesia to BALB / cCrSlc retired mice (17 weeks or older) that had been acclimated for one week (n=6). The ASOs used may be those prepared in Examples 1 to 8, or the fatty acid conjugate prepared in Example 9. Three days after the start of administration, 100 μg / mouse of a SARS-CoV-2 mouse-adapted strain (QHmusX) was administered to the mice under triple anesthesia. 3 Mice are infected by intranasal inoculation with TCID50 / mouse, and the lungs are removed 2 or 4 days after infection. A group administered only with saline serves as the negative control group. Saline and the test substance are administered until the day before lung removal. Mice are weighed once a day, and mice that show significant weight loss due to virus infection are euthanized. The removed mouse lungs are disrupted with a homogenizer and centrifuged to prepare a homogenate supernatant, and the virus titer of the homogenate supernatant is measured using the following method. 3 x 10 4 TMPRSS2-expressing VeroE6 cells were seeded at 10 cells / well and cultured overnight at 37°C in 5% CO. Serially diluted mouse lung homogenate supernatant was added to the cells and incubated at 37°C in 5% CO for 2 days. Cytopathic effect (CPE) associated with viral infection was observed under a microscope (OLYMPUS), and the viral titer in the homogenate supernatant was calculated according to standard methods.

[0094] The present invention can be an effective means for suppressing coronavirus infection and proliferation.

[0095] <SEQ ID NO: 1> shows the nucleotide sequence of SARS-CoV-2 isolate Wuhan-Hu-1, complete genome NCBI-GenBank accession no. NC_045512.2. <SEQ ID NOs: 2-17> show the sequences of the antisense oligonucleotides synthesized in Examples 1-8 and Comparative Examples 1-8. The antisense oligonucleotides may be natural DNA, natural RNA, DNA / RNA chimeras, or modified versions thereof, and at least one of the nucleotides constituting the antisense oligonucleotide may be a modified nucleotide. <SEQ ID NOs: 18-23> show the sequences of the primers and probes. <SEQ ID NOs: 24-39> show the sequences (including modification information) of the antisense oligonucleotides synthesized in Examples 1-8 and Comparative Examples 1-8.

Claims

1. An oligonucleotide or a pharmaceutically acceptable salt thereof, comprising an oligonucleotide having 17 to 30 bases and a nucleotide sequence substantially complementary to the region of nucleotide numbers 13520 to 13550 of the SARS-CoV-2 RNA genome consisting of the nucleotide sequence of SEQ ID NO: 1, the 5' end and / or 3' end of which may be chemically modified, wherein the oligonucleotide is a mixmer that does not recruit RNase H and is capable of inhibiting coronavirus proliferation.

2. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 1, which binds to a frameshift pseudoknot (FSP) structure of coronavirus-derived RNA.

3. The oligonucleotide according to claim 1 or 2, or a pharmaceutically acceptable salt thereof, comprising at least 17 consecutive bases in any one of the base sequences of SEQ ID NOs: 2, 5 to 8, and 11 to 13 (wherein t in the sequence may be u, and all bases may be natural bases or modified bases).

4. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the number of bases of the oligonucleotide is 18 to 24.

5. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 4, wherein the number of bases of the oligonucleotide is 21.

6. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein at least one of the sugars and / or phosphodiester bonds constituting the oligonucleotide is modified.

7. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 6, wherein the sugar constituting the oligonucleotide is D-ribofuranose and the sugar modification is modification of the hydroxyl group at the 2'-position of D-ribofuranose.

8. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 7, wherein the sugar modification is 2'-O-alkylation and / or 2'-O,4'-C-alkylenation of D-ribofuranose.

9. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 7, wherein the sugar modification is 2'-O-methylation and / or 2'-O,4'-C-ethylation of D-ribofuranose.

10. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 6 to 9, wherein the modification of the phosphodiester bond is phosphorothioate.

11. An oligonucleotide having any of the following sequences or a pharmaceutically acceptable salt thereof: -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t - (SEQ ID NO: 24) -G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t -(SEQ ID NO: 27) -A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A e2s -G m1t -(SEQ ID NO: 28) -A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -(SEQ ID NO: 29) -U m1s -C e2s -A m1s -A m1s -A e2s -A m1s -G m1s -C<000009)2>-C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1t -(SEQ ID NO: 30) -A m1s -G e2s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s ​​​​​​​​​​​​​​​​​​​​​​​​​ m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -(SEQ ID NO: 34) -A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t - (SEQ ID NO: 35) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA nucleoside (the base position of C is 5-methylcytosine) linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1s , G m1s , C m1s , U m1s , represents the corresponding 2'-OMe-RNA nucleoside linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1t , G m1t , C m1t , U m1t , represent the corresponding 2'-OMe-RNA nucleoside in which the 3' position is bonded to the adjacent nucleotide via an oxygen atom. The 5' carbon of the 5'-terminal nucleoside in each sequence is bonded to a hydroxyl group or to another structural unit via a phosphate group, and the 3' carbon of the 3'-terminal nucleoside is bonded to a hydroxyl group or to another structural unit via a phosphate group.

12. The oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, characterized in that a phosphate group linked to a fatty acid is further bound to the 5'-end or 3'-end of the oligonucleotide.

13. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 12, wherein the fatty acid is myristic acid, palmitic acid, stearic acid, arachidic acid, or behenic acid.

14. An oligonucleotide having any of the following sequences or a pharmaceutically acceptable salt thereof: HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO20) (SEQ ID NO: 24) HO-G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G m1s -T e2s -A m1t -H(ASO39) (SEQ ID NO: 27) HO-A m1s -A e2s -G m1s -C m1s -C e2s -C m1s -U m1s -G e2s -U m1s -A m1s -T e2s -A m1s -C m1s -G e2s -A m1s -C m1s -A e2s -U m1s -C m1s -A e2s -G m1t -H(ASO40) (SEQ ID NO: 28) HO-A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -H(ASO41) (SEQ ID NO: 29) HO-U m1s -C e2s -A m1s -A m1s -A e2s -A m1s -G m1s -C e2s -C m1s -C m1s -T e2s -G m1s -U m1s -A e2s -U m1s -A m1s -C e2s -G m1s -A m1s -C e2s -A m1t -H(ASO42) (SEQ ID NO: 30) HO-A m1s -G e2s -C m1s -C e2s -C m1s -U m1s ] -G e2s -U m1s -A m1s -U m1s -A m1s -C m1s -G m1s [[ID=十一四]]-A m1s -C e2s -A m1s -U m1s -C e2s -A m1s -G e2s -U m1t -H(ASO73) (SEQ ID NO: 33) HO-A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1t -H (ASO75) (SEQ ID NO: 34) HO-A m1s -A e2s -A m1s -A m1s -G e2s -C m1s -C m1s -C e2s -U m1s -G m1s -T e2s -A m1s -U m1s -A e2s -C m1s -G m1s -A e2s -C m1s -A m1s -T e2s -C m1s -A m1s -G e2s -U m1t -H(ASO76) (SEQ ID NO: 35) [In the above sequence, A e2s , G e2s , C e2s and T e2s represents the corresponding ENA nucleoside (the base position of C is 5-methylcytosine) linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1s , G m1s , C m1s , U m1s , represents the corresponding 2'-OMe-RNA nucleoside linked to the adjacent structure on the 3' side via a phosphorothioate bond. m1t , G m1t , C m1t , U m1t , represents the corresponding 2'-OMe-RNA nucleoside in which the 3' position is linked to the adjacent nucleotide via an oxygen atom.] 15. A coronavirus proliferation inhibitor comprising the oligonucleotide according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

16. A medicine comprising the oligonucleotide according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

17. A therapeutic and / or prophylactic agent for coronavirus infections, comprising the oligonucleotide according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

18. The therapeutic and / or prophylactic agent according to claim 17, wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

19. A method for treating and / or preventing a coronavirus infection in a subject by administering to the subject an oligonucleotide according to any one of claims 1 to 14 or a pharmaceutically acceptable salt thereof.

20. The method for treatment and / or prevention according to claim 19, wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

21. An oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 for use in the treatment and / or prevention of coronavirus infections.

22. The oligonucleotide or a pharmaceutically acceptable salt thereof according to claim 21, wherein the coronavirus is SARS-CoV-1, SARS-CoV-2, or MERS-CoV.

23. Use of the oligonucleotide or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 14 for the manufacture of a drug for the treatment and / or prophylaxis of coronavirus infections.

24. The use of claim 23, wherein the coronavirus is SARS-CoV-1 or SARS-CoV-2 or MERS-CoV.