Antiviral oligonucleotide

Abstract

[Problem] To provide an antiviral oligonucleotide that acts on cells infected by viruses having multiple RNA genomes, exhibiting an antiviral effect, and does not tend to the emergence of resistant viruses, and a pharmacological composition thereof. [Solution] An oligonucleotide that takes on a G-quadruplex structure that inhibits the binding of the genomic RNA and nucleocapsid (N) protein of a virus having an RNA genome, the antiviral oligonucleotide suppressing proliferation of the virus by contact with virus-infected cells, and a pharmacological composition thereof are used.

Description

Antiviral Oligonucleotides

[0001] The present invention relates to antiviral oligonucleotides that act on virus-infected cells, inhibiting the interaction between the viral nucleocapsid (N) protein and genomic RNA, thereby inhibiting proliferation, and to the antiviral oligonucleotides and their use as therapeutic agents for viral infections caused by human and animal viruses, and other diseases whose etiology is viral.

[0002] The following discussion is provided solely to aid the reader's understanding and is not an admission that any of the information discussed or documents cited constitute prior art to the present invention.

[0003] Smallpox, which had plagued people around the world since the dawn of history, was declared eradicated in 1977, and there was a time when it was believed that viral infectious diseases could be overcome. However, new infectious diseases such as Ebola virus disease, new strains of influenza, and COVID-19 infection have been discovered one after another, causing suffering to humanity. Many of the new infectious diseases that cause illness in humans are zoonotic diseases that originated from animals in the natural world.

[0004] Influenza A viruses have a genome divided into eight segments and infect humans, pigs, and birds. When multiple different types of influenza A viruses simultaneously infect the respiratory epithelial cells of pigs, the segmented genomes recombine. If this process of repeated mutation leads to infection with humans and then human-to-human transmission, this can lead to the emergence and spread of a new influenza virus that humanity has never experienced before (to which there is no immunity).

[0005] Coronaviruses exist endemic to many animals. Human coronaviruses (HCoVs) include HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, which account for 10-15% of common colds (35% during epidemics). In addition to these cold coronaviruses, other viruses have emerged, including the severe acute respiratory syndrome (SARS) coronavirus (2002), which causes severe pneumonia; the Middle East respiratory syndrome (MERS) coronavirus (2012); and the novel coronavirus (SARS coronavirus type 2, 2019). SARS infected 8,096 people and had a high case fatality rate of 9.7%, but was contained within less than a year. Meanwhile, MERS infected more than 22,500 people and had a high case fatality rate of 35%, and infections continue. Four years have passed since the outbreak of the coronavirus disease (COVID-19) in December 2019, and more than 760 million people have been infected, with 6.9 million deaths. This new infectious disease has caused confusion in medical care and the stagnation and disruption of socioeconomic activity due to infection control measures. Most of these coronaviruses are thought to have originally originated from coronaviruses carried by bats and then become infectious to humans (Non-Patent Document 1).

[0006] Given these circumstances, it will be difficult to prevent the outbreak of new infectious diseases such as new strains of influenza and COVID-19 in the future, and it is therefore necessary to develop effective preventive and treatment methods to respond when an outbreak occurs.

[0007] The only infectious disease that humanity has successfully eradicated to date is smallpox. Smallpox is caused by contact and droplet infection of the smallpox virus. This was achieved by early detection of infected individuals, isolation and treatment, and widespread vaccination (vaccination). Humans are the only animals that can contract the smallpox virus in the wild, and because symptoms appear when infected with smallpox, and because smallpox vaccination is highly effective in preventing infection, it was successfully eradicated in 1980.

[0008] On the other hand, it is difficult to prevent the outbreak of new influenza and COVID-19, which are zoonotic diseases derived from animals other than humans in the natural world. Furthermore, the spread of these infectious diseases is due to the movement of people who do not show symptoms even after infection, or during the incubation period of several days before symptoms appear.

[0009] The mRNA vaccines put into practical use in response to the outbreak of COVID-19 have been shown to be effective in preventing infection and preventing the disease from becoming severe (Non-Patent Document 2). However, the immunity that prevents infection against COVID-19 weakens within several months. Furthermore, as mutations in the viral genome accumulate, the antigenicity of the S protein responsible for antigenicity changes, resulting in the emergence of mutant strains that escape human immunity, leading to repeated epidemics (Non-Patent Document 3). Therefore, in order to suppress the spread of infectious diseases, it is desirable to put into practical use antiviral drugs along with effective vaccines.

[0010] Influenza, other than pandemic influenza, commonly known as seasonal influenza, is an infectious disease that recurs annually, infecting approximately 30,000 to 50,000 people worldwide and causing an estimated 290,000 to 650,000 respiratory deaths each year (Non-Patent Document 4). It spreads somewhere on the planet and undergoes repeated mutations, changing its antigenicity and shifting epidemic areas. Therefore, a planned approach is taken to predict the antigenic types that may be prevalent each year and prepare vaccines tailored to those types. Furthermore, because the effectiveness of vaccines weakens within a few months, annual vaccination is required for prevention.

[0011] In order to prevent influenza, new influenza, and COVID-19 infection from becoming severe, in addition to prevention through vaccination, antiviral drugs that suppress viral proliferation during infection are effective in alleviating the pathology caused by the viral infection and preventing the disease from becoming severe.

[0012] Antiviral drugs act specifically on limited structures of viral proteins to inhibit their activity. To date, several types of antiviral drugs specific to each virus species have been developed, targeting several proteins that function in viral replication. Since these target proteins have different three-dimensional structures for each virus, it is desirable to develop antiviral drugs for each target virus.

[0013] The effectiveness of compounds that act specifically on viral proteins can be reduced by structural changes in the viral proteins due to viral mutations (Non-Patent Document 5). As viral infections spread, if a specific antiviral drug that is effective against that virus is used frequently, mutations in the viral genome can change the binding site of the antiviral drug, resulting in resistance.

[0014] There are known anti-influenza drugs that have become unusable due to the spread of resistant viruses. Amantadine, a drug that was once used to inhibit the "uncoating" stage in which influenza A viruses invade cells and release genomic RNA from viral particles into the cells, is now unusable due to the spread of amantadine-resistant viruses. Furthermore, viruses resistant to Tamiflu, which inhibits neuraminidase, which functions in viral budding, have also appeared and spread (Non-Patent Document 6). Furthermore, the emergence of viruses resistant to Xofluza, a new anti-influenza drug that inhibits the mRNA synthesis stage, has also been noted (Non-Patent Document 7).

[0015] Nucleozin has been developed as an anti-influenza drug that binds to the N protein and inhibits its function (Non-Patent Document 8). This inhibits the formation of vRNP, which is formed when genomic ribonucleic acid (RNA) binds to the N protein, and inhibits the intracellular transport of vRNP, thereby inhibiting viral proliferation. On the other hand, the Y289H mutation in the N protein is resistant to Nucleozin (Non-Patent Document 9).

[0016] As described above, as the infection spreads, viruses repeatedly mutate to escape the immunity of infected hosts and the antiviral drugs used, and therefore viruses that have acquired resistance to the antiviral drugs tend to spread. Furthermore, in order to prepare for new infectious diseases, it is desirable to create antiviral drugs that are resistant to a wide range of virus species and viral mutations.

[0017] Antisense oligonucleotides (ASOs) are nucleic acid drugs that are approximately 21 nucleotides long and complementary to target RNA. These ASOs hybridize with complementary mRNA in cells, promoting the degradation of that mRNA by intracellular enzymes and suppressing gene expression.

[0018] ALN-RSVO1, an ASO drug developed for the treatment of respiratory syncytial virus infection, inhibits respiratory syncytial virus infection (Non-Patent Documents 10 and 11), and clinical trials using intranasal administration have also shown favorable results (Non-Patent Document 12). Because the effect of ASO is sequence-specific, it is desirable to select a sequence for each virus. Furthermore, it is desirable to prevent the weakening of ASO efficacy due to viral mutations, such as by selecting a sequence that is less susceptible to viral mutation.

[0019] In Japanese Patent No. 5,514,179, SEQ ID NO: 23 (REP 2055) is a 40-nucleobase nucleotide polymer consisting of 20 repeats of A and C, all of which are phosphorothioated, and was shown to function as an antiviral oligonucleotide to inhibit the proliferation of hepatitis B virus (HBV). REP2139 is a nucleotide polymer in which 5'-methylcytosine and 2'-O-methylribose are added to REP 2155. It was revealed that this nucleotide polymer does not inhibit viral replication (synthesis of HBV DNA and RNA) within HBV-infected cells or release of HBV from the cells, but inhibits the release of HBs antigen. In Japanese Patent No. 5,796,024, at least one fully phosphorothioated antiviral nucleotide polymer of 20 and 120 nucleotides in length exhibits antiviral activity against various viruses, including influenza virus and coronavirus. Based on the above-mentioned results with HBV, these nucleotide polymers are expected to be effective in inhibiting extracellular viral release.

[0020] To enhance antiviral efficacy, drugs that can be introduced into cells, inhibit viral replication through a defined mechanism, and remain effective even when the virus mutates are desirable.

[0021] A G-quartet structure is a planar structure formed by hydrogen bonding between oxygen molecules facing the center of four guanine nucleobases (G) and a monovalent cation (preferably a potassium ion) at the center. When a nucleobase sequence contains spacer nucleobases and multiple guanines, the multiple G-quartet structures formed by each guanine form a quadruplex structure called a G-quadruplex (G4 structure) (Non-Patent Document 13). This structure exists in both DNA and RNA, and in DNA, it is present in telomeres and promoter regions, where interactions with specific binding proteins contribute to their functional control. G4 structures are also present in the untranslated regions of mRNA and various small RNAs such as miRNA, and affect translational control and the function of small RNAs through the influence of binding proteins (Non-Patent Document 14). G4 structures can be formed within a single nucleic acid strand or between two or more nucleic acid strands.

[0022] In Japanese Patent No. 2818031 (1999, U.S. Patent No. 5,952,490), it was shown that a DNA oligonucleotide with a phosphorothioate sugar chain bond, TTGGGGTT, forms a G4 structure upon prolonged incubation, and among sequences containing TTGGGGTT, the 24-nucleobase sequence TTGGGGTTGGGGTTGGGGTTGGGG was shown to suppress the viral infectivity of herpesvirus type 1 to approximately one-hundredth at a concentration of 3 μM. Furthermore, in the case of influenza A virus, the sequence GGGGTTGGGG was the most effective example, suppressing the viral infectivity to approximately one-tenth at a concentration of 10 μM.

[0023] The untranslated regions of the mRNAs of the human MTA2 and ADAM10 genes contain RNA sequences with G4 structures, which form intramolecular G4 structures as 13- and 14-nucleobase-long RNA oligos (Non-Patent Document 15).

[0024] Aptamer AS1411 is a 26-nucleobase deoxyoligonucleotide rich in guanine nucleotides that forms a G4 structure, binds specifically to intracellular nucleolin in cancer cells, and suppresses the proliferation of acute myeloid leukemia (Non-Patent Document 16).

[0025] On the other hand, the cell growth inhibitory effect of extracellular treatment with guanine nucleotide-rich DNA that forms G4 structures in vitro has been suggested to be uncorrelated with the formation of G4 structures and to be due to degradation products of deoxyoligonucleotides such as deoxyguanosine monophosphate and guanine (Non-Patent Document 17), and the site and mechanism of action may be unclear.

[0026] Patent No. 2818031 Patent No. 5514179 Patent No. 5796024

[0027] Cui, J., F. Li, and Z.L. Shi, Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol, 2019. 17(3): p. 181-192.Zeng, B., et al., Effectiveness of COVID-19 vaccines against SARS-CoV-2 variants of concern: a systematic review and meta-analysis. BMC Med, 2022. 20(1): p. 200.Araf, Y., et al., Omicron variant of SARS-CoV-2: Genomics, transmissibility, and responses to current COVID-19 vaccines. J Med Virol, 2022. 94(5): p. 1825-1832.Iuliano, A.D., et al., Estimates of global seasonal influenza-associated respiratory mortality: a modelling study. Lancet, 2018. 391(10127): p. 1285-1300.Hampton, T., New Flu Antiviral Candidate May Thwart Drug Resistance. JAMA, 2020. 323(1): p. 17.Hussain, M., et al., Drug resistance in influenza A virus: the epidemiology and management. Infect Drug Resist, 2017. 10: p. 121-134.Imai, M., et al., Influenza A variants with reduced susceptibility to baloxavir isolated from Japanese patients are fit and transmit through respiratory droplets.Nat Microbiol, 2020. 5(1): p. 27-33.Cheng, H., et al., Design, synthesis, and in vitro biological evaluation of 1H-1,2,3-triazole-4-carboxamide derivatives as new anti-influenza A agents targeting virus nucleoprotein. J Med Chem, 2012. 55(5): p. 2144-53.Amorim, M.J., R.Y. Kao, and P. Digard, Nucleozin targets cytoplasmic trafficking of viral ribonucleoprotein-Rab11 complexes in influenza A virus infection. J Virol, 2013. 87(8): p. 4694-703.Alvarez, R., et al., RNA interference-mediated silencing of the respiratory syncytial virus nucleocapsid defines a potent antiviral strategy. Antimicrob Agents Chemother, 2009. 53(9): p. 3952-62.Bitko, V., et al., Inhibition of respiratory viruses by nasally administered siRNA. Nat Med, 2005. 11(1): p. 50-5.DeVincenzo, J., et al., Evaluation of the safety, tolerability and pharmacokinetics of ALN-RSV01, a novel RNAi antiviral therapeutic directed against respiratory syncytial virus (RSV). Antiviral Res, 2008. 77(3): p. 225-31.Harkness, R.W.t. and A.K. Mittermaier, G-quadruplex dynamics. Biochim Biophys Acta Proteins Proteom, 2017. 1865(11 Pt B): p. 1544-1554.Dumas, L., et al., G-Quadruplexes in RNA Biology: Recent Advances and Future Directions. Trends Biochem Sci, 2021. 46(4): p. 270-283.Binas. O., et al., Structure validation of G-rich RNAs in noncodingregions of the human genome. Chembiochem, 2020. 21, p. 1656-1663.Bates, P.J., et al., G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochim Biophys Acta Gen Subj, 2017. 1861(5 Pt B): p. 1414-1428.Zhang, N., et al., Cytotoxicity of guanine-based degradation products contributes to the antiproliferative activity of guanine-rich oligonucleotides. Chem Sci, 2015. 6(7): p. 3831-3838.Sekine, R., et al., Inhibition of SARS-CoV-2 nucleocapsid protein-RNA interaction by guanosine oligomeric RNA. J. Biochem. 2023. 173(6): p. 447-457.Chung, W.J., et al., Structure of left-handed DNA G-quadruplex. Proc Natl Acad Sci, 2015. 112: p. 2729-2733.Bakalar, B., et al., A minimal sequence for left-handed DNA G-quadruplex. Angew Chem Int Ed Engl, 2019, 58: p. 2331-235.

[0028] The problem to be solved is to provide an antiviral oligonucleotide and a pharmaceutical composition thereof that exhibit antiviral effects against a wider range of virus species than conventional antiviral drugs and that are less likely to develop resistant viruses.

[0029] The present invention is an extension of the invention of the "Non-genomic sequence antiviral oligonucleotide" (Patent Application No. 2022-181458) filed by the inventors on November 11, 2022.

[0030] The present invention relates to an RNA oligonucleotide selected from a sequence of approximately 12 nucleic acid bases in length characterized by G4 structure formation, or an oligonucleotide of approximately 28 nucleic acid bases in length containing such a sequence, which inhibits the interaction between viral genomic RNA and its binding protein (nucleocapsid protein (N protein)), thereby suppressing viral replication. The details of the oligonucleotide are described below.

[0031] Generally, the binding of N protein and RNA contributes to viral protein synthesis and genomic RNA replication. Ultimately, the entire genomic RNA forms an N protein-RNA (viral ribonucleoprotein, vRNP) polymer, which forms the core of the viral particle, independent of the RNA sequence. Proper formation of vRNP polymers is essential for efficient viral particle formation, infection of other cells, viral gene expression, viral gene replication, and maintaining viral infectivity.

[0032] Viral replication enzymes mistakenly incorporate nucleic acid bases during the replication process in which RNA viral genomes make copies. If a sequence unfavorable to viral replication is formed during viral evolution, the growth of viruses containing that sequence is inhibited, resulting in their elimination. Conversely, mutations that do not adversely affect the function of the encoded protein are thought to accumulate in genomic RNA. Assuming vRNP polymer formation, the presence of RNA sequences that bind extremely strongly to the N protein or that affect the structure of vRNPs will adversely affect replication by inhibiting the binding of normal genomic RNA to the N protein, and such sequences will be eliminated from the genomic RNA through mutation. This insight led to extensive research and resulted in the present invention.

[0033] That is, the present invention relates to the following 1) to 6). 1) RNA of approximately 12 nucleobases in length that forms a G4 structure in vivo and in vitro, and oligonucleotides containing this sequence, which have the activity of inhibiting binding of viral N protein to RNA. 2) The oligonucleotide described in 1) above forms a G4 structure within a single molecule. 3) Representative sequences of 1) and 2) above include RNA with 12 consecutive guanines (RNA of SEQ ID NO: 1, GGGGGGGGGGGGGG) and RNA oligonucleotides with a phosphorochiate backbone and a sequence in which GG is sandwiched between Us (RNA of SEQ ID NO: 7, UGGUGGUGGUGG), and 2'-substituted with OH. 4) RNA of approximately 12 nucleobases in length containing 1) to 3) above, and modified oligonucleotides thereof, or 28 nucleobases in length oligonucleotides containing these sequences. 5) A method for inhibiting viral proliferation in virus-infected cells by contacting the oligonucleotides described in 1) to 4) above with the cells. 6) A method in which the viral infection described in 5) above is coronavirus infection or influenza A virus infection. 7) An antiviral pharmaceutical composition comprising the oligonucleotides 1) to 4) above. 8) An antiviral pharmaceutical composition comprising the oligonucleotides 1) to 4) above in combination with other antiviral agents.

[0034] The present invention provides an oligonucleotide that exhibits antiviral effects against multiple viruses with RNA genomes, and an antiviral pharmaceutical composition containing the same. Furthermore, the oligonucleotide and antiviral pharmaceutical composition are preferably useful as antiviral oligonucleotides and antiviral pharmaceutical compositions that are less susceptible to a decrease in antiviral effect due to genetic mutation.

[0035] Analysis of SEQ ID NO: 1 by circular dichroism (CD) spectroscopy. CD spectra of phosphorothioated (PS) RNA (G12(S)) of SEQ ID NO: 1 in Tris buffer after the addition of NaCl or KCl (A), and CD spectra under physiological conditions (PBS) (B). Electrophoretic analysis of the similarity in G4 structure formation between PS-modified RNA of SEQ ID NO: 1 (G12(S)) and PS-modified RNA of SEQ ID NO: 10 (rLHG4(S)). CD spectral analysis of Gq(S) (PS-modified RNA, UGGGUUUGGGUU) and PS-modified RNA of known sequences (MTA2) of SEQ ID NO: 11 and PS-modified RNA of SEQ ID NO: 12 (ADAM10). Analysis of intracellular G4 structures using the G4-specific probe QUMA-1. Confocal laser microscope images (A) and quantification of QUMA-1 spots (B) of fixed A549 cells cultured with each PS-modified oligo, stained with DAPI and QUMA-1. TAMRA-labeled G12(S) was added to droplets formed by liquid-liquid phase separation in a mixed aqueous solution of SARS-CoV-2 N protein and FITC-labeled 32-nucleobase RNA. Differential interference contrast (DIC) images and FITC and TAMRA fluorescence images were then observed over time. TAMRA-labeled G12(S) was added to A549 cell culture medium, and its entry into the cells was analyzed over time. Reporter gene analysis of G12(S) cytotoxicity (A), effects on protein synthesis (B), and innate immune induction (C, D) are shown. G12(S) reduces the viral infectious titer (A) released from cells infected with human coronavirus OC43, but has little effect in suppressing released RNA (B). G12(S) suppresses the viral infectious titer (A) released from cells infected with human coronavirus 229E, but has little effect in suppressing released genomic RNA (B). Suppression of the infectious titer of virus released from influenza A virus (H3N2 and H1N1) infected cells by G12(S). Time-dependent changes in the ratio of influenza A virus RNA levels in the culture medium and cells of influenza A virus (H3N2) infected cells after addition of G12(S). Comparison of the anti-influenza A virus activity of G12(S) with oseltamivir and naniramivir. Inhibition of intracellular transport of influenza A virus vRNP by G12(S).Comparison of anti-influenza virus activity among G12(S), propagated SEQ ID NO:10 (rLHG4(S)), SEQ ID NO:11 (MTA2(S)), and SEQ ID NO:12 (ADAM10(S)) (A, B). Anti-influenza virus effect of G12(S) treated with KCl or NaCl (A). Comparison of anti-influenza A virus effect of G12(S) with rLHG4(S), rLHG4(S), and propagated SEQ ID NO:7 (BL1(S). In vivo effect of G12(S). Antiviral effect of G12(S) on OC43-infected newborn mice. G12(S) and OC43 were administered intranasally, and changes in pathogenicity were observed over time. G12(S) was administered once (A) and three times (B).

[0036] Preferred embodiments of the present invention will be described below, but the present invention is not limited to the following embodiments.

[0037] The oligonucleotides used in the present invention can be synthesized by FASMAC or Gene Design. The oligonucleotides of the present invention are phosphorothioated RNAs, except for the fluorescent label. The oligonucleotides of the present invention are characterized by the formation of a G4 structure within the same molecule of an oligonucleotide of approximately 12 nucleobases. G4 structures have often been analyzed using DNA oligonucleotides (Non-Patent Document 19, SEQ ID NO: 14; Non-Patent Document 20, SEQ ID NOs: 7, 10, 15, and 16). Therefore, it is desirable to confirm whether RNA of a similar sequence, or the RNA further modified with PS, forms a G4 structure and whether it forms a right- or left-handed structure.

[0038] As a preferred example, the characteristics of the G4 structure can be measured by CD spectrum analysis from wavelengths of approximately 220 nm to 320 nm in a 5 mm cuvette using an approximately 4 μM aqueous solution. The formation of the G4 structure is induced by adding NaCl or KCl at a concentration of approximately 30 mM. The present invention focuses on the KCl-dependent G4 structure. Since the G4 structure is characterized by its absorption of ultraviolet light at 297 nm, the stability (dissociation temperature) of the G4 structure can be estimated using the increase or decrease in this absorption. Another preferred example is electrophoresis using a 15% acrylamide gel (Tris-borate running buffer). A feature of the present invention is that the G4 structure is stabilized when KCl is added to the sample and running buffer, but is destabilized when KCl is not added or when NaCl is added.

[0039] The oligonucleotides of the present invention are characterized by the formation of dimers between G4 structures. This structure is due to π-π bonds between four guanine planes (G tetrads). We have found that this structure is inhibited by N-methylmesoporphyrin IX (NMM), which specifically binds to G tetrads. This method may also be used to confirm G4 structures. Intracellular G4 structures may be detected using commercially available QUMA-1 (Merck) or N-methylmesoporphyrin IX (NMM). Preferably, the oligonucleotides are added to cell culture media, followed by immobilization and the addition of the fluorescent substance, followed by detection of specific fluorescence using a confocal laser microscope.

[0040] A suitable example of a compound that inhibits the binding of RNA to N protein or the like can be selected using droplet formation by liquid-liquid phase separation (LLPS). Simply by way of example, fluorescently labeled genomic RNA and N protein or a portion of N protein undergo LLPS on a glass surface coated with silanized polyethylene glycol, forming small droplets of a few micrometers in size. Observing the morphology of the formed droplets under a fluorescent microscope using a differential interference contrast optical system and fluorescent images allows for the detection of reversible binding between green fluorescently labeled genomic RNA and N protein or the like, as well as LLPS. Adding another red fluorescently labeled oligonucleotide to the solution causes the green fluorescent droplets to change to red fluorescent over time, allowing the effect of the red fluorescent oligonucleotide on the genomic RNA in the N protein to be detected.

[0041] In virus-infected cells, the infected cells receive the viral genomic nucleic acid, inducing the innate immune system, resulting in the inhibition of intracellular protein synthesis, the induction of type I interferon synthesis, and the induction of NFκB transcription factors. As a result, virus production is suppressed and cell proliferation is also inhibited. CpG oligos target this mechanism. It is necessary to determine whether the oligonucleotides of the present invention act directly on viruses or by activating the innate immune system. For this purpose, suitable methods include using Alamar Blue to monitor cellular respiration to assess cytotoxicity, and the SunSET method to assess protein synthesis inhibition. Similarly, a reporter gene assay using luciferase may be used to determine the activation of type I interferon promoters and promoters containing NFκB-binding sequences.

[0042] The binding of the genomic RNA and N protein of coronaviruses and influenza A viruses, which have RNA as their genome, forms a polymer and is not expected to have strong sequence specificity. Furthermore, unlike when low-molecular-weight compounds exert their effects by binding to proteins, the broad molecular surface of the N protein interacts with RNA. Because the oligonucleotides of the present invention have a high affinity for the N protein of SARS-CoV-2, it is possible that they may have a similar effect across coronavirus species. Similarly, they may also be effective against influenza A viruses.

[0043] Among human coronaviruses, OC43, which causes common cold symptoms, grows in HCT-8 cells or MRC5 cells, while 229E grows in LLC-MK2 cells. Cells cultured in RPMI or MEM medium supplemented with 10% fetal bovine serum were infected with the corresponding virus. After washing the cells, the oligonucleotides of the present invention were added to new medium, allowing for time-dependent measurement of intracellular virus-specific proteins, viral genomic RNA, viral RNA released in the culture supernatant, and viral infectious titer. Proteins were quantified by Western blotting using antibodies specific to the N protein of each virus, and viral RNA was quantified using RT-qPCR. Viral infectious titer can be measured by CPE assay or syncytium formation (multinucleated cells in which viral infection induces cell fusion). Influenza A virus, as a preferred example, can be similarly measured using MDCK cells to measure intracellular viral protein synthesis, viral RNA synthesis, viral RNA in the culture supernatant, and viral infectious titer. RNA and protein may be assayed in the same manner as described above, and infectious virus may be quantified by plaque assay or CPE method.

[0044] Oligonucleotides are digested by nucleases both inside and outside the cell. While enzymes that specifically degrade RNA are ubiquitous, there are few RNases capable of determining the sequence of consecutive guanines. Various modifications have been developed to enhance nuclease resistance when adding oligonucleotides directly to culture media. A suitable example is phosphorothioate modification of the 5'-3' bond between riboses. Modifications of candidate oligonucleotides include partial or complete 2'-deoxygenation of the ribose moiety, 2'-O methylation, 2'-O (2-methoxyethyl)ation, 2' fluorolysis, and modification to bridged nucleic acid and / or locked nucleic acid.

[0045] Coronaviruses undergo RNA transcription and replication within a membrane called DMV, which is derived from the endoplasmic reticulum membrane. When the replicated genomic RNA is transported to the cytoplasm, it binds to the N protein to form vRNP. The nascent vRNP is inserted into the ERGIC, an organelle located midway between the endoplasmic reticulum and the Golgi apparatus, to form virus particles.

[0046] In influenza A virus, each of the eight genomic RNA strands is independently transcribed and replicated in the nucleus. The vRNPs formed in the nucleus bind to a transporter between the endoplasmic reticulum and the Golgi apparatus called Rab11 in a region of the cytoplasm close to the nucleus and are transported to the cell membrane. Therefore, both viruses share the common feature of passing through the cytoplasm as vRNPs during their replication process. Since the oligonucleotides of the present invention easily enter the cytoplasm from the culture medium, they may be contacted with coronavirus-infected cells and influenza A-infected cells.

[0047] It is necessary to clarify the site of action of the oligonucleotides of the present invention. As a preferred example, the effect of G12(S) on coronavirus OC43 vRNP may be observed by co-staining the N protein with the ER protein or the N protein with an ERGIC-specific protein and detecting co-localization using a fluorescent antibody technique. Alternatively, the N protein of influenza A virus may be fused to GFP, while Rab11 may be fused to mCherry, allowing for real-time detection of the co-localization of influenza A vRNP formed by the green-colored N protein with Rab11 as part of the transport process, and its effects may be observed.

[0048] The antiviral effect of a candidate oligonucleotide can be evaluated using experimental animals. As a preferred example, the effect of the candidate oligonucleotide can be estimated by administering a candidate oligonucleotide to a newborn experimental animal such as a mouse while inoculating the animal with an RNA virus into the nasal cavity, respiratory tract, or lungs, and detecting the level of viral proliferation in the animal by quantitative PCR or fluorescent antibody testing of tissue sections, or by observing changes in the animal's pathological condition.

[0049] The above may be a suitable example for indicating candidate oligonucleotides having antiviral effects as antiviral oligonucleotides.

[0050] The present invention will be specifically explained below with reference to examples, but the present invention is not limited to these examples.

[0051] The present invention is an extension of the "non-genomic sequence antiviral oligonucleotide" (Patent Application No. 2022-181458).

[0052] The oligonucleotides were dissolved in water or 10 mM Tris-HCl buffer at a concentration of 4 μM and left overnight at room temperature. After adding 30 mM NaCl or KCl, CD spectra were analyzed (Figure 1A). CD spectra were also measured at room temperature using a CD spectrometer (5 mm quartz cuvette) immediately after adding 10x PBS buffer to make the solution 1x, and one day later (Figure 1B). As shown in Figure 1, PS-modified SEQ ID NO: 1 (G12(S)) exhibited a positive peak near 265 nm in the presence of NaCl, indicating a right-handed parallel G4 structure. In contrast, in the presence of KCl and PBS, a positive peak at 258 nm and a negative peak near 280 nm were detected. This was characteristic of a left-handed parallel structure (four strands of nucleotides oriented in the same 5'-3' direction), as shown in Non-Patent Document 20. Although not shown in the figure, the PS-RNA of SEQ ID NO: 10 (rLHG4(S)) also showed a left-handed parallel CD spectrum, as shown for the G4 structure of DNA in Non-Patent Document 20.

[0053] Non-Patent Document 20 reports that the DNA of SEQ ID NO: 10 exhibits a left-handed parallel structure, and its structure, consisting of two overlapping G tetrad planes, has been shown to form a dimer through crystal X-ray structural analysis. The inventors used acrylamide gel electrophoresis to investigate whether G12(S) adopts a structure similar to that of rLHG4(S). As shown in Figure 2, the electrophoretic mobility of rLHG4(S) was found to shift from high molecular weight to low molecular weight in the presence of NaCl and in a NaCl- and NMM concentration-dependent manner when NaCl was added to the sample. Since NMM binds to the G tetrad plane, it is believed that the π-π interaction between rLHG4(S) dimers was reversed by NMM binding, converting the dimer to a monomer. Since G12(S) also showed similar behavior under similar conditions, it was suggested that G12(S) does not have a random G4 structure, but rather that its basic structure is a dimer of G4 structures with two G tetrads on both sides, bonded by π-π bonds, similar to rLHG4(S).

[0054] The CD spectra of PS-modified UGGGTTTGGGTT (Gq(S)), PS-modified SEQ ID NO: 11 (MTA2(S)), and PS-modified SEQ ID NO: 12 (ADAM10(S)) were analyzed. When KCl was added from Tris-HCl, all of these oligonucleotides showed spectra with a positive peak around 260 nm, which is characteristic of right-handed parallel oligonucleotides in the presence of KCl (Figure 3).

[0055] We measured whether the above oligonucleotides formed G4 structures in cells and, if so, the level of their formation. When added to the culture medium of A549 cells, intracellular G4 structures were detected using the G4-specific fluorescent dye QUMA-1 (Figure 4A). Quantification by confocal laser scanning microscopy revealed that red QUMA-1 fluorescence was detected as punctate cytoplasmic fluorescence, with the order of fluorescence intensity being G12(S) > ADAM10(S), MTA2(S) > rLHG4(S), but was barely detectable in Gq(S)-treated cells (Figure 4B).

[0056] As shown in Figure 5, when SARS-CoV-2 N protein synthesized and purified using E. coli was mixed with FITC-labeled 32-nucleobase RNA, liquid-liquid phase separation occurred, forming droplets. When TAMRA-labeled G12(S) was added, the green fluorescent droplets quickly turned into red fluorescent droplets. Since the FITC-labeled genomic RNA and TAMRA-labeled G12(S) in the initially formed droplets (RNPs) were replaced by each other when mixed at similar concentrations on a nucleobase basis, G12(S) is thought to have a higher affinity for the N protein than the genomic RNA.

[0057] It was found that G12(S) labeled with TAMRA fluorescent dye added to the culture medium of A549 cells was taken up into the cells within a few seconds (FIG. 6).

[0058] As shown in Figure 7, G12(S) did not affect cell proliferation even at a concentration of 10 μM (A). Furthermore, using the SunSET method, which quantifies the level of protein synthesis by incorporating puromycin into nascent proteins and detecting it with a puromycin antibody, G12(S) had no effect on cellular protein synthesis (B). The activity of the interferon-β promoter (C) and the NFκB promoter (D) was not affected by G12(S).

[0059] Exposure of β-coronavirus OC43-infected cells to G12(S) significantly reduced the levels of intracellular OC43N protein and OC43 RNA (data not shown), and also significantly reduced the amount of infectious virus particles released into the culture supernatant (Fig. 8A), but did not significantly affect viral RNA (Fig. 8B).

[0060] Exposure of αcoronavirus 229E-infected cells to G12(S) significantly reduced the levels of intracellular 229E N protein and 229E RNA (data not shown), and also significantly reduced the infectious virus particles released into the culture supernatant (Fig. 9A), but had no significant effect on viral RNA (Fig. 9B).

[0061] The antiviral effect of G12(S) against influenza A virus was examined. As shown in Figure 10, it significantly suppressed the production of H3N2 and H1N1 subtype viruses.

[0062] The RNA in the culture medium of MDCK cells infected with influenza A virus, and the N protein RNA (left) and HA protein RNA (right) in the cells were quantified, and the ratio was observed over time. The value significantly decreased with the addition of G12(S), suggesting that in the case of influenza A virus, the release of synthesized viral RNA outside the cells was suppressed.

[0063] The antiviral effect of G12(S) against influenza A virus was compared with that of laninamivir and oseltamivir, and it was found that G12(S) inhibited the cell division effect at lower concentrations than these existing antiviral drugs (Figure 12).

[0064] Cells expressing both a gene in which the green fluorescent protein (GFP) was fused to the influenza A virus N protein and a gene in which the red fluorescent protein (mCherry) was fused to Rab11 were infected with influenza A virus. In non-infected cells, GFP-N protein accumulated in the nucleus, and Rab11-mCherry was detected in the cytoplasm as a meshwork pattern. In influenza A virus-infected cells, green and red fluorescence were detected as dense, punctate spots. These spots were found to be significantly reduced 85 minutes after the addition of G12(S) compared to 15 minutes (Figure 13).

[0065] The effects of antiviral oligonucleotides on influenza A virus infection were compared. The order of activity was G12(S) > rLHG4(S) (Fig. 14A), followed by G12(S) > ADAM10(S) and MTA2(S). Gq(S) had no effect at all (Fig. 14B). Furthermore, pretreatment of G12(S) with KCl showed a stronger anti-influenza A virus effect than pretreatment with NaCl (Fig. 14A).

[0066] The PS-modified RNA of SEQ ID NO: 7 (BL1(S)) had anti-influenza A activity equivalent to that of G12(S) (FIG. 16).

[0067] An OC43-infected animal model was used to detect the in vivo antiviral activity of G12(S). When CO43 was applied to the nasal cavities of newborn mice, viral infection progressed from the olfactory bulb to the brain, resulting in weight loss and eventual death. Similarly, intranasal administration of G12(S) significantly reduced the weight loss effect induced by OC43 (Figure 16A). Administration three times daily further enhanced the effect (Figure 16B).

[0068] G12(S) was found to rapidly displace RNA in SARS-CoV-2 vRNP. It also entered the cytoplasm within seconds and inhibited the cytoplasmic transport of influenza A virus in the cytoplasm. Regarding coronaviruses, although its inhibitory effect on the release of RNA into the cytoplasm was low, it suppressed infectious virus production by several hundred to several thousand times. Regarding influenza A virus, it also suppressed the release of viral RNA and infectious virus by several hundred to several thousand times. It was found that its anti-influenza A virus effect was similar to that of existing neuraminidase inhibitors at concentrations approximately 10 times lower.

[0069] We invented an RNA oligonucleotide unit of approximately 12 nucleic acid bases that forms a G4 structure within the molecule as a molecule that changes the structure of vRNP due to its high affinity for N protein and is effective against different virus species.

[0070] The antiviral oligonucleotides of the present invention can be used alone or in combination with suitable modifications to the influenza A type that causes the pandemic, increasing resistance to nucleases. Furthermore, because the low-molecular-weight oligonucleotides penetrate cells without the need for a transporter, they can be used in vivo as antiviral oligonucleotides. Furthermore, because they are effective against multiple viruses, they can be used to create antiviral drugs that can be applied to new infectious diseases. Furthermore, because their site of action differs from that of other antiviral drugs, they are expected to be used in combination with pharmaceutically acceptable amounts of other antiviral drugs. Terminology

[0071] 1. "RNA" is ribonucleic acid. 2. "DNA" is deoxyribonucleic acid. 3. "N protein" is a protein that binds to viral genomic RNA to form the virus; in the case of influenza viruses it is called nucleoprotein (NP) and in the case of coronaviruses it is called nucleocapsid (N) protein, but here we will refer to it uniformly as "N protein." 3. "Antiviral oligonucleotide" here refers to an oligonucleotide that affects the binding between the N protein and probe RNA, reducing viral replication and extracellular release.

Claims

1. An oligonucleotide consisting of 11 to 28 nucleobase units comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:

16.

2. The oligonucleotide of claim 1, wherein at least one nucleobase unit further comprises a 2'-substitution.

3. The oligonucleotide of claim 2, wherein the 2'-substitution is selected from the group consisting of H, OH, SH, SCH3, F, OCN, O(CH3)nNH2, or O(CH2)nCH3 (wherein n is from 1 to about 10); C1 to R10 lower alkyl, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or NH-alkyl; O-, S-, or NH-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; fluorescein; reporter group; and intercalator.

4. The oligonucleotide of claim 3, wherein said 2'-substitution is a C1 to C10 lower alkyl.

5. The oligonucleotide of claim 1 having at least one phosphorothioate intersugar linkage.

6. The oligonucleotide of claim 3, having at least one phosphorothioate intersugar linkage.

7. The oligonucleotide of claim 6, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:

3.

8. The oligonucleotide of claim 5, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:

7.

9. The oligonucleotide of claim 6, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:

1.

10. The oligonucleotide of claim 6, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:

7.

11. An oligonucleotide consisting of 11 to 28 nucleobase units, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, which is characterized by forming a guanine quadruplex structure within the molecule.

12. The oligonucleotide of claim 11, wherein at least one nucleobase unit further comprises a 2'-substitution.

13. The oligonucleotide of claim 12, wherein the 2'-substitution is selected from the group consisting of H, OH, SH, SCH3, F, OCN, O(CH3)nNH2, or O(CH2)nCH3 (wherein n is from 1 to about 10); C1 to R10 lower alkyl, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or NH-alkyl; O-, S-, or NH-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; fluorescein; reporter group; and intercalator.

14. The oligonucleotide of claim 13, wherein the 2'-substitution is a C1 to C10 lower alkyl.

15. The oligonucleotide of claim 11, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

16. The oligonucleotide of claim 13, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

17. An oligonucleotide consisting of 11 to 28 nucleic acid base units, comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16, characterized by inhibitory activity against the binding of the genomic RNA of a virus having an RNA genome to the nucleocapsid (N) protein of said virus.

18. The oligonucleotide of claim 17, wherein at least one nucleobase unit further comprises a 2'-substitution.

19. The oligonucleotide of claim 18, wherein the 2'-substitution is selected from the group consisting of H, OH, SH, SCH3, F, OCN, O(CH3)nNH2, or O(CH2)nCH3 (wherein n is from 1 to about 10); C1 to R10 lower alkyl, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or NH-alkyl; O-, S-, or NH-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; fluorescein; reporter group; and intercalator.

20. The oligonucleotide of claim 19, wherein the 2'-substitution is a C1 to C10 lower alkyl.

21. The oligonucleotide of claim 17, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

22. The oligonucleotide of claim 19, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

23. A method for inhibiting the activity of a virus, comprising contacting the virus with an oligonucleotide of 11 to 28 nucleobase units in length comprising a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:

16.

24. The method of claim 23, wherein at least one nucleobase unit further comprises a 2'-substitution.

25. The method of claim 24, wherein the 2'-substitution is selected from the group consisting of H, OH, SH, SCH3, F, OCN, O(CH3)nNH2, or O(CH2)nCH3 (wherein n is from 1 to about 10); C1 to R10 lower alkyl, substituted lower alkyl, alkaryl, or aralkyl; Cl; Br; CN; CF3; OCF3; O-, S-, or NH-alkyl; O-, S-, or NH-alkenyl; SOCH3; SO2CH3; ONO2; NO2; N3; NH2; heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; RNA cleaving group; fluorescein; reporter group; and intercalator.

26. The method of claim 25, wherein the 2'-substitution is a C1 to C10 lower alkyl.

27. The method of claim 23, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

28. The method of claim 25, wherein the oligonucleotide has at least one phosphorothioate intersugar linkage.

29. A pharmaceutical composition of an oligonucleotide according to claims 1 to 22 for the prevention or treatment of a viral infection.

30. A pharmaceutical composition of an oligonucleotide according to any one of claims 1 to 22, wherein the viral infection of claim 29 is an infection caused by a virus having an RNA genome.

31. A pharmaceutical composition comprising a therapeutically effective amount of at least one pharmacologically acceptable oligonucleotide according to claims 1 to 22, and a pharmaceutically acceptable carrier.

32. A pharmaceutical composition comprising a therapeutically effective amount of an antiviral agent in the pharmaceutical composition of claim 31.

33. A pharmaceutical composition of an oligonucleotide according to claims 1 to 22, adapted for the treatment, suppression or prevention of a disease of viral etiology.

34. A pharmaceutical composition of an oligonucleotide according to any one of claims 1 to 22, adapted for delivery by a mode selected from the group consisting of intraocular injection, oral ingestion, enteral, nasal, inhalation, dermal injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intrathecal injection, intratracheal injection and intravenous injection.

35. A pharmaceutical composition in combination with at least one other pharmaceutical composition comprising an oligonucleotide according to any one of claims 1 to 22.

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