G-quadruplicate-containing oligonucleotides for prevention and treatment

Abstract

The present invention provides oligonucleotides for treating diseases caused by viral or bacterial infections, pharmaceutical compositions containing the oligonucleotides, and kits containing the oligonucleotides. [Solution] Provided are oligonucleotides selected from the group comprising one of a specific sequence group and / or one of the specific sequence group in which at least one nucleotide has been truncated, for use in the treatment of viral infections caused by viruses selected from the group consisting of monkeypox virus, HCN virus, Zika virus, and coronaviruses such as SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

Description

Technical Field

[0001] The present invention relates to oligonucleotide molecules as described in the subject matter of claims 1 and 2. The present invention also relates to a pharmaceutical composition as described in the subject matter of claim 10, a kit comprising the oligonucleotide molecule of the present invention as described in the subject matter of claim 11, and the therapeutic use of the oligonucleotide molecule of the present invention.

Background Art

[0002] Tumor cells often evade the body's immune defenses by expressing programmed death ligand 1 (PD-L1). When it binds to a receptor (PD1) present on the surface of T cells, the immune response is attenuated. Inhibition of this interaction by blocking antibodies (e.g., pembrolizumab, Keytruda®; nivulomab, Opdivo®; avelumab, Bavencio®) activates T cells. The activated T cells mediate an increase in autoimmunity and are particularly effective against tumor cells. The clinical efficacy of this approach has been significantly demonstrated in large-scale trials, particularly in metastatic melanoma (Robert C, Schachter J, Long GV et al., (2015) Pembrolizumab versus Ipilimumab in Advanced Melanoma. N Engl J Med 372:2521 - 32; Schachter J, Ribas A, Long GV et al., (2017) Pembrolizumab versus ipilimumab for advanced melanoma: final overall survival results of a multicentre, randomised, open-label phase 3 study (KEYNOTE-006). Lancet 390:1853 - 62).

[0003] Infectious diseases, such as those caused by viruses and bacteria, pose significant health problems for patients and healthcare systems. For example, seasonal respiratory viral diseases have been known for thousands of years, and annual outbreaks of colds and influenza affect people living in temperate regions during the winter season. Newly emerging viral infections continue to pose a major threat to global public health. Recently, the discovery of highly pathogenic avian influenza A (H5N1) virus and other avian influenza A virus subtypes (H7N9, H9N2, H7N3) linked to human disease has raised concerns about pandemics due to the potential transmission of influenza A virus subtypes circulating in poultry and livestock to humans. Severe acute respiratory syndrome coronavirus (SARS-CoV) is associated with atypical pneumonia, first appearing in Guangdong Province, China, between 2002 and 2003, and killing 800 people worldwide in 2003. Bats have been identified as a natural reservoir of SARS-CoV-like viruses. In 2012, Middle East Respiratory Syndrome coronavirus, belonging to the same coronavirus group, was identified in Saudi Arabia. In the latter half of 2019, pneumonia associated with SARS-CoV-2 infection emerged in patients in Wuhan, China, leading to the COVID-19 epidemic, infecting millions and killing millions worldwide.

[0004] The SARS-CoV virus belongs to the beta-coronavirus family and is a positive-sound single-stranded (ss)RNA virus with a large RNA genome of approximately 30 kb. Like other coronaviruses, the SARS-CoV2 genome contains 14 open reading frames (ORFs) that encode 27 proteins. ORF1 and ORF2 in the 5' terminal region of the genome encode 15 non-structural proteins that are important for viral replication. The 3' terminal region of the genome encodes structural proteins, particularly the spike protein (S), envelope protein (E), membrane protein (M), nucleocapsid protein (N), and eight accessory proteins.

[0005] There is a need for effective drugs and vaccines to treat emerging viral diseases, particularly coronavirus infections. Small molecule inhibitors can be readily identified from compound libraries. However, because the action of small molecule inhibitors is limited to small surface regions of the target, a single amino acid change in the target can significantly reduce the effectiveness of the small molecule inhibitor. Oligonucleotide molecules that bind to specific target molecules ("aptamers") can be isolated using SELEX (Systematic Evolution of Ligands by Exponential Enrichment) and function as affinity probes or molecular recognition elements for diagnostic and therapeutic purposes. These aptamers are synthetic single-stranded DNA or RNA, bind to target molecules with high affinity in a three-dimensional shape, and have widespread applications in analytical, bioanalysis, imaging, diagnostic, and therapeutic fields. This invention addresses the need for effective drugs to treat diseases caused by viral or bacterial infections using oligonucleotide molecules. [Overview of the Initiative]

[0006] In a first aspect, the present invention relates to an oligonucleotide molecule having 10 to 50 nucleotides, comprising at least one G-quartet forming motif comprising 10 to 20 nucleotide residues, wherein at least 60% of the residues are guanosine residues or deoxyguanosine residues, and the molecule inhibits the replication of viruses or bacteria in mammalian cells and / or exerts an anti-inflammatory effect.

[0007] According to the present invention, the term “oligonucleotide molecule” is understood to refer to a short DNA or RNA molecule, also known as an oligomer. Most commonly, DNA oligonucleotides are synthesized as single-stranded molecules by solid-phase chemical synthesis and are used for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, molecular cloning, and as molecular probes. RNA oligonucleotides exist in vivo as small RNA molecules and are involved in the regulation of gene expression (e.g., microRNAs), or are degradation intermediates derived from the degradation of larger nucleic acid molecules. Oligonucleotides that bind to a specific target molecule are called “aptamers,” and aptamers may be oligonucleotide molecules or peptide molecules. In the context of the present invention, the term “aptamer” refers to an oligonucleotide molecule, and the terms “oligonucleotide molecule” and “aptamer” are used interchangeably and synonymously throughout this application.

[0008] In the context of this invention, a "G-quartet forming motif" is a higher-order nucleic acid structure rich in guanine residues. A G-quartet is formed by four G-bases linked via Hougsteen H-bonds, forming a square planar structure in which each G-base forms two H-bonds with an adjacent G-base. To form a G-quadrilateral (G4), two or more G-quartets overlap to form a polymorphic structure. Thus, a folded intramolecular G-quadrilateral consists of two main elements: a core and a loop. The core contains a layer of one or more stacked GGGG tetramolecules (or G-quartets), and the loop is a linker sequence that connects the chains of the G-quadrilateral core. Therefore, G-quadrilateral structures are highly polymorphic depending on the relative orientation of the chains and the type of loop. The stability of these G-quartets depends on several factors, including the presence of monovalent cations such as K+ and Na+, the concentration of G-rich oligonucleotides present, and the sequence of G-rich oligonucleotides used, although this also depends on the sequence of the G-rich oligonucleotides.

[0009] The oligonucleotide molecules of the present invention can inhibit the replication of viruses or bacteria, and the replication of viral or bacterial genomes results in the production of multiple copies of the virus or bacteria in infected cells. The oligonucleotide molecules of the present invention preferably target viral or bacterial helicases. Helicases are enzymes that separate the strands of double-stranded nucleic acids, typically utilizing ATP hydrolysis to obtain the necessary energy. In addition to helicases that act on double-stranded DNA, there are also helicases that unwind DNA-RNA or RNA-RNA double helicases. Regarding bacterial replication, it is also preferable that the oligonucleotide molecules of the present invention target bacterial DNA polymerase III holoenzyme, a major enzyme complex involved in prokaryotic DNA replication. Since viral replication primarily depends on the metabolic function of the host cell, the oligonucleotide molecules of the present invention advantageously target virus-specific steps in the viral replication mechanism while preserving the function of the host cell. Different viral polymerases, namely RNA-dependent RNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, and DNA-dependent DNA polymerase, play central roles in viral replication and transcription of the viral genome and are generally active as a single protein capable of performing multiple functions related to viral replication. When used to inhibit viral replication, the oligonucleotide molecules of the present invention may also preferably target one or more viral polymerases.

[0010] The term "inflammation" refers to a complex biological response of one or more tissues to a harmful stimulus, such as a viral or bacterial pathogen. This response plays a role in eliminating the initial cause of cellular damage in order to protect tissue cells. In mammals, acute inflammatory responses are generally considered beneficial, immediate adaptive responses with limited specificity. However, they can be harmful if left uncontrolled, as seen in septic shock. Inflammatory pathways consist of a series of events involving inducers, sensors, mediators, and effectors. The "anti-inflammatory effect" of a molecule or substance refers to its property of reducing inflammation, for example, through a specific interaction between one or more of the inducers, sensors, mediators, and effectors and the molecule.

[0011] Surprisingly, the oligonucleotide molecules of the present invention were found to exhibit strong antiviral or antibacterial activity through inhibition of replication, with at least 80% of the residues being guanosine or deoxyguanosine residues. Furthermore, these molecules were also found to exhibit anti-inflammatory effects in mammalian cells. Advantageously, the oligonucleotide molecules of the present invention are short molecules that can be synthesized relatively easily and inexpensively by chemical synthesis. Compared to antibodies, oligonucleotide molecules are characterized by low immunogenicity and high stability. Moreover, oligonucleotide molecules can bind to a variety of targets, including organic molecules, proteins, viruses, bacteria, whole cells, and tissues.

[0012] In further embodiments, the oligonucleotide molecules described in the present invention may be oligonucleotide molecules in which more than 70% of the residues are guanosine residues or deoxyguanosine residues. Increasing the number of guanosine residues results in significantly increased antiviral or antibacterial activity of the oligonucleotides of the present invention compared to scrambled or mixed sequences. It is particularly preferable that all residues of the oligonucleotide molecule are guanosine residues.

[0013] In alternative embodiments, oligonucleotide molecules are synthesized from deoxyribonucleotides. Each nucleotide consists of three subunit molecules: a nucleic acid base, a five-carbon sugar (ribose or deoxyribose), and a phosphate group consisting of one to three phosphate groups. The nucleic acid base and sugar moiety together form a nucleoside. In DNA, guanine, adenine, cytosine, and thymine are used as nucleic acid bases, while in RNA, uracil is used instead of thymine. RNA is a nucleic acid polymer characterized by the presence of ribose as the sugar in the sugar-phosphate backbone, while DNA has deoxyribose instead. Advantageously, oligonucleotide molecules are DNA molecules that are far less susceptible to hydrolysis.

[0014] In further advantageous embodiments of the oligonucleotide molecules of the present invention, at least one guanosine residue or deoxyguanosine residue may be chemically modified. The oligonucleotide molecule is a DNA or RNA molecule and may be chemically modified at the backbone or 2' sugar position to achieve different effects such as higher binding affinity and / or specificity, lower sensitivity to nuclease degradation, enhanced in vivo stability, longer in vivo half-life, and lower sensitivity to excretion via renal filtration. Common chemical modifications include modifications of nucleic acid ends, such as 3' end capping or PEGylation with inverted thymidine, to improve resistance to nucleases (which initially bind to each end) and renal clearance, respectively. Further modifications include phosphodiester bonds, sugar rings (e.g., substitution of the 2'O position of the ribofuranose ring with a fluoro(-F), amino(-NH2), azide(-N3), or methoxy / OMe(-OCH3) group), and nucleic acid bases (e.g., purine modifications, 2,6-diaminopurine, 3-deaza-adenine, 7-deaza-guanine, and 8-azide-adenine, or pyrimidine modifications, 2-thio-thymidine, 5-carboxamide-uracil, 5-methyl-cytosine, and 5-ethynyl-uracil).

[0015] In particularly preferred embodiments of oligonucleotide molecules, the chemical modification may be a modification of the phosphate skeleton. Modification of the phosphate skeleton affects the phosphodiester bond by definition, and the phosphate group is altered by atomic substitution, resulting in neutral, anionic, or cationic modifications. For example, substituting one or two oxygen atoms with one or two sulfur atoms each yields a phosphorothioate group or a phosphorodithioate group. Substituting one oxygen atom of the phosphate group with an uncharged methyl group yields a methyl phosphate skeleton. Cationic modifications include substituting one oxygen atom with a positively charged group such as guanidinopropyl phosphoramidate. Preferably, the phosphodiester bond of the oligonucleotide molecule is substituted with a methylphosphonate or phosphorothioate analog such that the skeletal oxygen atom is substituted with a methyl group, or one or more skeletal oxygen atoms are substituted with one or more sulfur atoms. Advantageously, this modification results in improved resistance to extracellular or intracellular nucleases, higher thermal stability, enhanced target binding affinity, and / or improved delivery into the cell via the plasma membrane.

[0016] It is particularly preferable that the thiophosphoryl substitution is selected from phosphorothioates or phosphorodithioates, such that the thiophosphoryl substitution replaces at least 35% of the phosphodiester bonds in the sugar-phosphate backbone of the oligonucleotide molecule. Substituting all phosphodiester bonds of the oligonucleotide with a thiophosphoryl group significantly improves resistance to nucleases. To obtain an oligonucleotide molecule with improved target binding specificity, the thiophosphoryl substitution of phosphodiester bonds can be titrated in the range of 35% to complete substitution. Furthermore, by preserving some phosphodiester bonds in the molecule through partial substitution, the enhanced toxicity that may be found in association with complete substitution can be avoided. In oligonucleotide molecules in which the thiophosphoryl substitution replaces at least 35% of the phosphodiester bonds in the sugar-phosphate backbone, if the number of guanosine residues increases, the oligonucleotide molecules of the present invention exhibit significantly increased antiviral or antibacterial activity compared to scrambled or mixed sequences. It is particularly preferable that all residues of the PTO oligonucleotide molecule are guanosine residues.

[0017] In further embodiments, the oligonucleotide molecule may contain a triplet of at least four consecutive guanosine or deoxyguanosine residues. Advantageously, oligonucleotide molecules containing a triplet of at least four consecutive guanosine residues were found to exhibit a stronger antiviral effect compared to shorter oligonucleotide molecules, suggesting that the effectiveness in inhibiting viral replication depends on the length of the molecule.

[0018] The oligonucleotides of the present invention advantageously comprise the sequences described in SEQ ID NOs: 1 to 4, where SEQ ID NO: 1: 5'-GGG GGG GGG GGG GGG GGG GGG -3', SEQ ID NO: 2: 5'-GGG GGG GGG GGG GGG -3', SEQ ID NO: 3: 5' GGg gtc aag ctt gaG GGG Gg, and SEQ ID NO: 4: GGT GGT GGT GGT TGT GGT GGT GGT GG. Uppercase letters represent phosphorothioate bonds (PTOs), and lowercase letters represent classical phosphodiester bonds. Shorter sequences are often associated with slightly reduced replication inhibition. Oligonucleotides containing these sequences spontaneously form G-quartets or G-quadruplexes (G4).

[0019] In a second aspect, the present invention relates to a pharmaceutical composition comprising at least one oligonucleotide molecule in combination with at least one pharmaceutically acceptable additive, carrier, adjuvant, or combination thereof.

[0020] The pharmaceutically acceptable additives described in this invention include any and all solvents, dispersions, diluents, or other liquid vehicles, dispersions or suspension aids, surfactants, isotonic agents, preservatives, solid binders, etc., suitable for the desired specific dosage form. Remington's The Science and Practice of Pharmacy, 23rd edition, A. Adejare (Lippincott, Williams & Wilkins, Baltimore, Md., 2020) discloses various additives used in the formulation of pharmaceutical compositions and known techniques for their preparation. Pharmaceutically acceptable additives may be of at least 95%, 96%, 97%, 98%, 99%, or 100% purity. Examples of pharmaceutically acceptable additives used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and / or granulators, surfactants and / or emulsifiers, disintegrants, binders, preservatives, buffers, lubricants, and / or oils.

[0021] In a third aspect, the present invention relates to a kit comprising at least one oligonucleotide molecule as described above.

[0022] In a fourth aspect, the present invention relates to a pharmaceutical composition or kit comprising an oligonucleotide molecule, such as described, for use in the treatment of diseases caused by viral or bacterial infections and / or inflammation associated therewith.

[0023] In preferred embodiments, oligonucleotide molecules can induce inhibition of viral replication in mammalian cells.

[0024] In a more preferred embodiment, the viral infection may be caused by a virus selected from the group including HS-1 virus, HCN virus, adenovirus, Zika virus, hepatitis B or C virus, West Nile virus, influenza virus, RSV virus, paramyxovirus, HIV virus, coronavirus, e.g., SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

[0025] In an alternative embodiment, the disease is a viral infection of the respiratory tract.

[0026] In another embodiment, the treatment of inflammation associated with viral or bacterial diseases may result from interference with the type I interferon (IFN) and / or type II IFN pathways, or from suppression of interleukin-mediated signaling. In the context of the present invention, type I interferon (IFN) refers to polypeptides secreted by infected cells. The following functions are associated with type I IFN: induction of a cell-autonomous antibacterial state in infected and neighboring cells to limit the spread of infectious pathogens, particularly viral pathogens; regulation of a balanced innate immune response to promote antigen presentation and natural killer cell function while suppressing pro-inflammatory pathways and cytokine production; activation of the adaptive immune system to promote the development of high-affinity antigen-specific T and B cell responses and immune memory. Type I IFN consists of a group of structurally similar cytokines and includes 13-14 subtypes of IFN-α, along with IFN-β, IFN-ε, IFN-κ, IFN-ω, IFN-δ, IFN-ζ, IFN-τ. Type II IFN, known as IFN-γ, signals through a different receptor and has an independent action from type I IFN. IFN-γ signaling plays an important role in host defense by promoting macrophage activation, upregulating the expression of antigen processing and presentation molecules, promoting the development and activation of Th1 cells, enhancing the activity of natural killer cells, regulating B cell function, and inducing the production of chemokines that promote the transport of effector cells to the site of inflammation. Advantageously, the oligonucleotide molecules can be used for the treatment of different stages of viral diseases, such as the subacute state of SARS-Cov2 infection ("long COVID").

[0027] In a further embodiment, in the oligonucleotide molecules used for therapeutic purposes, at least one guanosine residue or deoxyguanosine residue may be chemically modified, the chemical modification is a phosphate backbone modification, and the oligonucleotide contains at least four consecutive triplets of guanosine residues or deoxyguanosine residues.

[0028] In alternative embodiments, the molecule may be administered by a pharmaceutically acceptable route selected from the group consisting of oral, parenteral, enteral, ocular or nasal routes, or topical and combinations thereof. Topical applications include creams, foams, gels, lotions, ointments, pastes, powders, shakable lotions, solids, sponges, tapes, tinctures, topical solutions, manicures, transdermal patches, application as a vapor.

[0029] It should be noted that as used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "oligonucleotide molecule" refers to one or more oligonucleotide molecules, i.e., a single oligonucleotide molecule and multiple oligonucleotide molecules. Further, it should be noted that the claims can be drafted to exclude any element. Accordingly, this statement is intended to serve as a basis for using exclusive terms such as "solely" and "only" in connection with the recitation of elements of the claims, or for using "negative" limitations.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the preferred methods and materials are described below. In addition, the materials, methods, and examples described herein are illustrative only and not intended to be limiting.

[0031] Other features and advantages of the present invention will be apparent from and encompassed by the following detailed description and claims.

Brief Description of the Drawings

[0032] [Figure 1]This figure compares different nucleotide sequences regarding the antiviral effects in SARS-CoV-2 infected Calu-3 human lung cancer cells. It shows that nCpG-6-PTO inhibits SARS-CoV-2 infection. [Figure 2] This figure shows that nCpG-6-PTO inhibits SARS-CoV-2 replication but does not affect viral entry into target cells. [Figure 3] This diagram shows that antiviral effectiveness depends on the length of the molecule, emphasizing that longer molecules are more effective against viral infection than shorter molecules. [Figure 4] This figure shows that the antiviral effect depends on the ODN skeleton, based on a comparison of oligonucleotides of the same length that have either a phosphodiester skeleton (nCpG-6-PDE) or a phosphorothioate skeleton (nCpG-6-PTO). [Figure 5] This figure compares the efficacy of nCpG-6-PTO with AS1411, a well-known aptamer used in anticancer therapy. When used in a viral inhibition assay, AS1411-PDE showed no efficacy against SARS-CoV-2, but AS1411-PTO was found to be as effective as CpG-6-PTO. [Figure 6] This figure shows that nCpG-6-PTO completely inhibits the cytopathogenic effects of HSV-1 at concentrations ranging from 0.5, 1, 2, and 4 μM. [Figure 7] This figure shows the formation of a quadruple-chain (G4) structure from nCpG-6-PTO using BG4, a G4-specific antibody. [Figure 8] This figure shows the results of a helicase assay demonstrating that nCpG-6-PTO inhibits SARS-CoV-2 helicase. [Figure 9]This figure shows the potential for G4-forming PTO-ODNs (nCpG-6-PTO and AS1411-PTO) to inhibit IFNβ-mediated signaling molecules. Figure 9A schematically shows the type I interferon signaling pathway (from Gonzalez-Cao et al., 2018). Western blot analysis (Figure 9B) shows strong downregulation of tyrosine phosphorylation of canonical signaling molecules p-Stat1 and pStat2 by nCpG-6-PTO and AS1411-PTO. [Figure 10] This figure demonstrates that G4-forming PTO-ODNs (nCpG-6-PTO and AS1411-PTO) inhibit IFNy-mediated signaling molecules. Figure 10A schematically shows the type II interferon signaling pathway (from Gonzalez-Cao et al., 2018). Western blot analysis (Figure 10B) shows strong downregulation of tyrosine phosphorylation of canonical signaling molecules p-JAK2 and pStat2 by nCpG-6-PTO and AS1411-PTO. [Figure 11] This figure shows the potential of G4-forming PTO-ODNs (nCpG-6-PTO and AS1411-PTO) to inhibit interleukin-6 (IL-6)-mediated STAT3 phosphorylation. Figure 11A schematically shows the IL-6 signaling pathway (from Jin et al., 2017). Western blot analysis (Fig. 9B) shows strong downregulation of Stat3 tyrosine phosphorylation by nCpG-6-PTO and AS1411-PTO. Furthermore, an inhibitory effect on p-Stat1 was also observed. [Figure 12]Exemplary data shows that nCpG-6-PTO suppresses HSV-1 infection in a fragmented skin model. In addition to Example 6 (Figure 6), in which nCpG-6-PTO suppressed HSV-1 infection in in vitro cultured foreskin fibroblasts, as indicated by CPE reduction, antiviral efficacy was also tested in fragmented skin samples. Excess fragmented skin from the thigh that was not used for wound dressing was provided by the surgical unit. Skin samples were placed in PBS and subjected to dermarolling using a dermaroller (Segminismart®, Nicosia, Cyprus) as described in (Tajpara P, Mildner M, Schmidt R et al., A Preclinical Model for Studying Herpes Simplex Virus Infection. The Journal of investigative Dermatology 139:673~82, 2019). The skin was continuously cut into 3 × 3 mm sections. Each fragment was placed in one well of a 24-well MS and covered with 500 μl of DMEM (10% FBS, 1% P / S). Skin samples were treated with 10⁶ HSV-1 copies / ml + / - 4 μM nCpG-6-PTO (GQ20-PTO) for 2 days (37°C). Tissue samples were then fixed and sectioned into 4 μm thick sections using a standard protocol. HSV-1 was detected using anti-HSV-1 (1:10, Invitrogen: PA1-29210) and HistoGreen (Histo Green Kit, Linaris, E109) ​​as a peroxidase substrate. [Figure 13] This figure shows that nCpG-6-PTO is effective against monkeypox virus. [Figure 14]This diagram schematically illustrates the action of checkpoint molecules in tumorigenesis. Top diagram: Antigen-presenting cells activate T cells by presenting tumor antigens and B7 molecules. When T cells recognize a tumor, IFN-γ is produced, and PD-1 ligands on tumor cells and antigen-presenting cells are upregulated. Binding of PD-1 on T cells to PD-1 ligands inhibits T cell activation, resulting in a weakening of the antitumor immune response. Bottom diagram: Blocking PD-1 with nivolumab (or another antibody such as pembrolizumab) reverses T cell inhibition and reactivates the antitumor immune response. (Excerpted from Brahmer JR, Hammers H, Lipson EJ (2015) Nivolumab: targeting PD-1 to bolster antitumor immunity. Future Oncology 11:1307~26). This invention introduces an alternative concept to checkpoint blockade. The addition of G4 inhibits the IFNγ signaling cascade in tumor cells, resulting in the inhibition of PD-L1. [Figure 15]This diagram illustrates the relationship between IFNγ signaling and PD-L1 expression. IFNγ signaling triggers "immune evasion" in tumor cells. This diagram shows that IFNγ activates a cascade of inflammatory mediators (Jak-1, Jak-2, Stat-1), which subsequently stimulates the expression of IRF-1 (interferon regulator-1). The transcription factor IRF1 is a central component of the PD-L1 promoter. The interaction between PD-L1 and PD-1 leads to the inhibition of the immune response (Garcia-Diaz A, Shin DS, Moreno BH et al., (2017) Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell reports 19:1189~201). SARS-CoV-2, like many other viruses (e.g., Hanta and Ebola), induces massive IFNγ induction, leading to T cell depletion and destruction via the expression of checkpoint molecules such as PD-L1 and PD-L2 (Aghbash PS, Eslami N, Shamekh A et al., (2021) SARS-CoV-2 infection: The role of PD-1 / PD-L1 and CTLA-4 axis. Life Sci 270:119124~). This subsequently leads to a weakened immune response to the virus. Therefore, the use of the aforementioned checkpoint inhibitors in combination with other treatments for COVID-19 has been considered. However, it has been observed that cancer patients treated with checkpoint inhibitors after SARS-CoV-2 infection develop more severe disease (Robilotti EV, Babady NE, Mead PA et al., (2020) Determinants of COVID-19 disease severity in patients with cancer. Nat Med 26:1218~23). This could be due to side effects (steroids), or it could be caused by a cytokine cascade triggered by the inhibitor.Therefore, a 2021 study by Aghbash et al. called for the need for PD-1-based therapies for COVID-19: "A key point in the treatment of COVID-19 and PD-1-targeted therapies is to employ methods that can simultaneously reduce and eliminate the inflammatory cascade and the formation of exhausted T cells." The results of this invention regarding the efficacy of G4 suggest that there are both antiviral, anti-inflammatory, and immunostimulatory mechanisms at play here. [Figure 16] This figure shows that oligonucleotides suppress the expression of PD-L1 and PD-L2 proteins. [Figure 17] This figure shows the inhibition of PD-L1 and PD-L2 by nCpG-6-PTO and CpG-1-PTO, and the effects of concentration and molecular length. [Figure 18] This figure shows the expression analysis of PD-L1 and PD-L2 using real-time RT-PCR. [Figure 19] This is a diagram showing the promoter function analysis. [Figure 20] This figure shows the Western blot analysis of interferon receptor signaling proteins. [Figure 21] This figure shows that nCpG-6-PTO forms a G-quadrilateral (G4) and binds to IFNGR2. [Modes for carrying out the invention]

[0033] Example 1 G-rich PTO-ODN exhibits antiviral activity, with nCpG-6-PTO being particularly potent. Virus preparation Virus preparation was performed as described (Bojkova et al., 2020). Briefly, SARS-CoV-2 variants were isolated using the human colon cancer cell line Caco-2. The SARS-CoV-2 stock used in the experiment had undergone up to 3 passages on Caco-2 cells and was stored at -80°C. Viral titers were measured in TCID in confluent cells in 96-well microtiter plates. 50 It was decided to use / ml.

[0034] Antiviral assay The confluent layer of Calu-3 cells in a 96-well plate was infected with SARS-CoV-2 at an MOI of 0.01. "MOI" stands for "infection multiplicity," referring to the ratio of pathogens (e.g., viruses, bacteria) to infection targets (e.g., cells). The virus was added simultaneously with oligonucleotides and incubated in MEM containing 1% FBS. Antiviral efficacy was evaluated after 2 days by immunohistochemical detection of virus-specific antigens using an antibody against SARS-CoV-2S (1:1500, Sino Biological, Eschborn, Germany). Quantitative detection was performed using Bioreader® 7000-FZ-Imicro (Biosys).

[0035] Oligonucleotide molecules (ODNs) used 1.CpG-1-PTO:5'-TCC ATG ACG TTC CTG ACG TT-3' 2.n-CpG-6-PTO:5'-GGG GGG GGG GGG GGG GGG GG-3' 3.n-CpG-3A-PTO:5'-TTT TTT TTT TTT TTT TTT-3' 4.n-CpG-5-PTO:5'-CCC CCC CCC CCC CCC CCC CC-3' 5. Scramble - CpG-1 - PTO: 5' - CTC TAG GAC TCT CTG GAC TT-3' 6. G3139 Genasense (Obrimarsen): 5'-TCTCCCAGCGTGCGCCAT-3' 7.CpG-2118(KonA):5'-GGg gtc aag ctt gaG GGG Gg-3'

[0036] Uppercase letters represent phosphorothioate bonds (PTOs), and lowercase letters represent classical phosphodiester bonds (CpG-2118). As used herein, CpG oligodeoxyribonucleotides are short, single-stranded synthetic DNA molecules containing cytosine triphosphate deoxyribonucleotide ("C") followed by guanine triphosphate deoxyribonucleotide ("G"), where "p" refers to a phosphodiester bond between consecutive nucleotides. The acronym "n-CpG" refers to oligonucleotides that are non-CpG-ODNs. Tested at different concentrations (0.25, 0.5, 1, 2, 4 mM). Oblimarsen (trade name: Genasense) is an antisense oligodeoxyribonucleotide being studied as a potential treatment for several types of cancer, including chronic lymphocytic leukemia, B-cell lymphoma, and breast cancer. CpG-2118(KonA) is a guanine-rich molecule with a PTO-protected terminal, and is a synthetic oligonucleotide that functions as a control for the mouse TLR9 ligand ODN1585.

[0037] The results are shown in Figure 1, demonstrating that nCpG-6-PTO inhibits SARS-CoV-2 infection.

[0038] Example 2 nCpG-6-PTO inhibits the "replication" of SARS-CoV-2, not its "invasion." material and method To determine whether nCpG-6-PTO prevents viral adsorption and internal migration into cells (invasion) or prevents viral replication (replication), we conducted experiments on the duration of addition.

[0039] Experimental setup for addition time Invasion: Different concentrations of nCpG-6-PTO (0.25, 1, 4 mM) were added with SARS-CoV-2 (0.01 MOI) and incubated for 1 hour. After 1 hour, the virus and treatment were washed away, and the medium was replaced with fresh medium.

[0040] Replication: Calu-3 cells were infected with SARS-CoV-2 for 1 hour (0.01 MOI). After 1 hour, the viral inoculum was removed and the cells were washed to ensure that no viral particles that had not penetrated the cells were washed away. Different concentrations of nCpG-6-PTO were added sequentially.

[0041] In both settings, viral protein detection was performed two days later as described above (immunohistochemical detection of virus-specific antigens using antibodies against SARS-CoV-2S).

[0042] The results are shown in Figure 2, which demonstrate that nCpG-6-PTO inhibits SARS-CoV-2 replication (triangle) but does not affect viral entry into target cells (circle).

[0043] Example 3 The antiviral effect depends on the length of time. Comparison of nCpG-6-PTO deletion mutants. Examples 1 and 2 (Figures 1 and 2) demonstrated the antiviral efficacy of nCpG-6-PTO. Example 3 shows a correlation between molecular length and antiviral effect. For this purpose, nCpG-6-PTO deletion mutants were used.

[0044] The procedure was carried out as described in Example 1. The following oligonucleotide molecules of different lengths were used as ODNs at concentrations of 0.5, 1, 2, and 4 μM.

[0045] TIFF2026090248000001.tif20170

[0046] As shown in Figure 3, antiviral efficacy clearly depended on the length of the molecule, with longer molecules showing a stronger effect against viral infection than shorter molecules. The IC50 was lowest when using nCpG-6-PTO (0.5 mM, black circle) and shifted to a lower efficacy when using 6G ODN nCpG-6G-PTO (1.5 mM, light gray circle). Note that concentrations are plotted on a logarithmic scale.

[0047] Example 4 The antiviral effect depends on the ODN skeleton. The effect of the ODN skeleton was tested by comparing nCpG-6 with a phosphodiester skeleton (nCpG-6-PDE, light gray circle) and nCpG-6 with a phosphorothioate skeleton (nCpG-6-PTO, black circle). Treatment: nCpG-6-PTO or nCpG-6-PDE was applied at concentrations of 0.5, 1, 2, and 4 μM, respectively. The procedure was carried out as described in Example 1.

[0048] Figure 4 shows the possibility that PTO binding transmits antiviral effects.

[0049] Example 5 Comparison of viral inhibition by nCpG-6-PTO, AS1411-PTO, and AS1411-PDE Example 5 compares replication inhibition using the nCpG-6-PTO of the present invention with two forms of the well-known aptamer AS1411, namely AS1411-PTO and AS1411-PDE.

[0050] AS1411 (also known as AGRO100) is a G-rich oligonucleotide with a phosphodiester linkage (PDE) and has long been established as a potent anticancer aptamer. Structurally, AS1411 likely exists in multiple different G-quadrilateral conformations, making it an example of a single oligonucleotide capable of multiple G-quadrilateral conformations. Treatment was carried out at the following concentrations: 0.5, 1, 2, and 4 μM nCpG-6-PTO (black circle), AS1411-PTO (gray circle), and AS1411-PDE (light gray circle), respectively. The procedure was carried out as described in Example 1.

[0051] TIFF2026090248000002.tif19170

[0052] As shown in Figure 5, nCpG-6-PTO demonstrated high antiviral efficacy. AS1411-PDE, which is currently being tested as an antiviral drug, did not show efficacy against SARS-CoV-2. Only when the skeleton was replaced with a PTO linkage did it demonstrate antiviral efficacy comparable to nCpG-6-PTO.

[0053] Example 6 nCpG-6-PTO also shows antiviral efficacy against herpes simplex virus 1 (HSV-1). nCpG-6-PTO or nCpG-6-PDE was applied to human foreskin fibroblasts along with HSV-1. After 2 days, the cytopathogenic effect (CPE) of HSV-1 infection was determined by visual scoring. Treatment was performed at the following concentrations: 0.5, 1, 2, and 4 μM.

[0054] Figure 6 shows that nCpG-6-PTO completely suppresses CPE caused by HSV-1 within the tested concentration range (black bars). Notably, nCpG-6-PDE also showed suppression of HSV-1 CPE (gray bars), but only at concentrations of 1 μM or higher.

[0055] Example 7 nCpG-6-PTO forms a quadruple chain (G4). To elucidate possible mechanisms of action, we investigated the secondary structure formation of the oligonucleotide molecules of the present invention in vitro using specific antibodies that recognize the G-quadruplicate structure of DNA and RNA with high selectivity and low nanomolar affinity.

[0056] 5'-Cy5-labeled nCpG-6-PTO (2 μg) was mixed with 200 ng or 400 ng of BG4 (Biozol ABA-AB00174-1.1, Eching, Germany), an antibody specific to the G4 secondary structure, at room temperature for 15 minutes. After separation by 10% non-denaturing native PAGE (100 V, equivalent to 14.7 V / cm) and 0.5 × TBE, fluorescence was captured using the LI-COR Odyssey Gel Documentation System (Bad Homburg, Germany). Titration of BG4 with G4 containing 5'-Cy5-labeled nCpG-6-PTO demonstrated that the antibody binds to G4-forming DNA (Figure 7A, lanes 2 and 3). In the absence of the BG4 antibody, complex formation was not detected (first lane).

[0057] To visualize G4 structure at the cellular level, A375 cells (human malignant melanoma cell line) were cultured on glass coverslips for 24 hours in or without 4 μM nCpG-6-PTO. After fixation with 2% paraformaldehyde / PBS, the cells were permeabilized and blocked with 0.1% triton-X100 / 5% normal goat serum in PBS. Primary antibody BG4 (0.5 μg / ml in BSA) was applied to the cells for 90 minutes at room temperature; mouse IgG1 antibody (Dako) served as a control. After incubation with anti-mouse IgG (Invitrogen) conjugated to Alexa 488, representative images were taken using a Zeiss microscope. All cells examined showed punctate nuclear staining (Figure 7B, right panel, experimental conditions: arrow for 4 μM nCpG-6-PTO), which was not observed in the absence of primary BG4 antibody (Figure 7, left panel, control).

[0058] Example 8 nCpG-6-PTO inhibits SARS-CoV-2 helicase. The helicase assay was performed according to the method of Adedeji et al. (Adedeji et al., 2012). Briefly, nsp13, a helicase derived from SARS-CoV-2, was incubated with hybridization primers consisting of Quench2:5'-CGCAGTCTTCTCCTGGTGCTCGAACAGTGAC-3'-BHQ1 and Flu2:Cy3-5'-GTCACTGTTCGAGCACCA-3'. Helicase activity is separated by preventing further quenching of the fluorescence of primer Flu2. Adding an excess of CaptureQ2:5'-GTCACTGTGTGTG as a capture probe prevents Flu2 from re-annealing to Quench2. The upper panel of Figure 8 is a diagram illustrating the principle of the assay. For comparison, the helicase assay was performed using gradually increasing concentrations of nCpG-6-PTO or nCpG-6-PDE (0.2, 1, 4 mM). The products were separated by 6% non-denaturing PAGE (polyacrylamide gel electrophoresis) (Figure 8, bottom panel). Gradual increase in the concentration of nCpG-6-PTO inhibited nsp13, as indicated by an increase in the amount of quenched hybrid (see lanes 5 and 6 compared to lane 2 for control). Addition of the same concentration of nCpG-6-PDE did not similarly inhibit helicase activity, as indicated by the presence of a fluorescent single-strand comparable to the control.

[0059] Example 9 nCpG-6-PTO suppresses STAT phosphorylation induced by IFN type I (IFNβ). A schematic diagram of the type I interferon signaling pathway is shown in Figure 9A (Gonzalez-Cao et al., 2018). Calu-3 cells were treated with 4 μM nCpG-6-PDE, nCpG-6-PTO, AS1411-PTO, and AS1411-PDE for 1 hour, and then stimulated with IFNβ (20 ng / ml). Baricitinib, a rheumatoid arthritis drug and a well-known inhibitor of Janus kinase subtypes, JAK1 and JAK2, was used as a control (1 mM). After 10 minutes, the cells were sonicated in Strawn Buffer (20 mM HEPES [pH 7.5], 150 mM NaCl, 0.2% Triton X 100, 10% glycerol) supplemented with protease inhibitors and phosphatase inhibitors (Roche, Mannheim, Germany), boiled for 5 minutes, and then separated on SDS-polyacrylamide gel. The proteins were immunoblotted onto PVDF membranes. The membranes were blocked in blocking buffer (TBS [pH 7.6], 0.1% Tween-20, 5% skimmed milk) for at least 1 hour in RT mode, and then incubated with the following primary antibodies: p-Stat-1 (Tyr701), p-Stat-2 (Tyr690), p-Stat-3 (Tyr705), all from CST (Frankfurt, Germany), and antibactin (SantaCruz, Biotechnology, Heidelberg, Germany) as an equal loading control. The conjugated primary antibodies were detected using rabbit anti-goat IgG-horseradish peroxidase conjugate (Dako, Frankfurt, Germany) and visualized using the LumiGlo detection system (CST). The results shown in Figure 9B clearly demonstrate inhibition of Stat1 and Stat2 phosphorylation by nCpG-6-PTO, but not by nCpG-6-PDE; similarly, the PTO form of AS1411, which forms a G-rich quadruplex, sufficiently suppressed tyrosine phosphorylation of both Stat1 and Stat2, while the PDE form could not.

[0060] Example 10 nCpG-6-PTO suppresses JAK2 and STAT phosphorylation induced by IFN type II (IFNg). A schematic diagram of the type II interferon signaling pathway is shown in Figure 10A (Gonzalez-Cao et al., 2018). For experimental setup, please refer to Example 9 above: Calu-3 cells were treated with 4 μM nCpG-6-PDE, nCpG-6-PTO, AS1411-PTO, and AS1411-PDE for 1 hour, and then stimulated with IFNg (20 ng / ml). After separation using SDS pages, phosphorylation was detected using p-Stat-1 (Tyr701), p-Stat-2 (Tyr690), p-Stat-3 (Tyr705), and p-JAK2 (Tyr1007 / Tyr1008) antibodies, all from CST (Frankfurt, Germany), and antibactin (SantaCruz, Biotechnology, Heidelberg, Germany) as an equivalent loading control.

[0061] Western blot analysis, Figure 10B, shows strong downregulation of tyrosine phosphorylation of canonical signaling molecules p-JAK2 and pStat1. Furthermore, an inhibitory effect on pStat2 / 3 was observed, confirming partial overlap between type I and type II signaling pathways (Garcia-Diaz A, Shin DS, Moreno BH et al., (2017) Interferon Receptor Signaling Pathways Regulating PD-L1 and PD-L2 Expression. Cell reports 19:1189~201).

[0062] Example 11 nCpG-6-PTO inhibits the phosphorylation of STAT-3 mediated by interleukin-6 (IL-6). Figure 11A shows a schematic of the interleukin-6 (IL-6) signaling pathway (Jin et al., 2017). For experimental setup, please refer to Example 9 above: Calu-3 cells were treated with 4 μM nCpG-6-PDE, nCpG-6-PTO, AS1411-PTO, and AS1411-PDE for 1 hour and stimulated with IL-6 (20 ng / ml). After separation using SDS-PAGE, phosphorylation was detected using p-Stat-1 (Tyr701), p-Stat-2 (Tyr690), and p-Stat-3 (Tyr705) antibodies, all from CST (Frankfurt, Germany), and antibactin (SantaCruz, Biotechnology, Heidelberg, Germany) as an equal loading control.

[0063] Western blot analysis, Figure 11B, shows strong downregulation of tyrosine phosphorylation. Western blot analysis also shows downregulation of pStat3. Furthermore, an inhibitory effect on pStat1 was observed.

[0064] Example 12 The following demonstrates that nCpG-6-PTO, an oligonucleotide (ODN) consisting of 20 guanosine molecules linked via a phosphorothioate, inhibits the expression of PD-L1 and PD-L2 in melanoma cells.

[0065] The following ODNs were used in the experiment. Uppercase letters represent phophorothioate bonds (PTOs), and lowercase letters represent classical phosphodiester bonds (CpG-2118). Table 1 TIFF2026090248000003.tif82170

[0066] Figure 16 shows that oligonucleotides suppress the protein expression of PD-L1 and PD-L2.

[0067] A375 melanoma cells pre-stimulated with IFNγ (20 ng / ml) for 1 hour were exposed to different oligonucleotides (4 μM). Oligogenic features regarding the skeletal structure and sequence are shown in Table 1. (A) Total protein was extracted after 24 hours and separated by SDS-PAGE. Blotted proteins were searched for anti-PD-L1 and anti-PD-L2. Searching with anti-beta-actin served as a loading control. Images show representative results. (B) An example of FACS scan results for PD-L1 and PD-L2 after treatment of A375 cells with 4 μM CpG-1-PTO and 4 μM nCpG-6-PTO. Summary of 9 independent FACS experiments using (C) A375 cells and (D) SK-Mel-28 cells treated with 4 μM CpG-1-PTO or nCpG-6-PTO. Standard deviations are shown. Data were related to the reference positive control (IFNγ). *p<0.05.

[0068] Results: nCpG-6-PTO inhibits PD-L1 / 2 expression in A375 and SK-Mel-28 melanoma cells.

[0069] Figure 17 shows the effects of nCpG-6-PTO and CpG-1-PTO on the inhibition of PD-L1 and PD-L2, as well as on concentration and molecular length.

[0070] IFNγ-stimulated A375 cells were treated with escalating concentrations of (A) nCpG-6-PTO (1, 2, 4 μM) or (B) CpG-1-PTO (4, 6, 8 μM). Similarly, SK-Mel-28 cells were treated with escalating concentrations of (C) nCpG-6-PTO or (D) CpG-1-PTO. Furthermore, the effect of ODN length was also investigated. IFNγ-stimulated A375 cells were treated with (E) 4 μM nCpG-6-PTO and reference deletion mutants (nCpG-6B / 6D / 6G-PTO) or (F) 4 μM CpG-1-PTO and reference deletion mutants (CpG-14 / 12 / 9-PTO). Similarly, SK-Mel-28 cells were treated with (G)nCpG-6-PTO or (H)CpG-1-PTO and their deletion mutants. After 24 hours, PD-L1 and PD-L2 expression was measured by FACS. Each bar represents the mean of six independent experiments. Standard deviation is shown. Data were related to the reference positive control (IFNγ). *p<0.05.

[0071] Results: nCpG-6-PTO is effective at low concentrations (1 μM). The effect is length-dependent, and one 6-mer (nCpG-6G-PTO) is already effective.

[0072] Figure 18 shows the expression analysis of PD-L1 and PD-L2 by real-time RT-PCR.

[0073] A375 melanoma cells were stimulated with IFNγ for 1 hour, then incubated with CpG-1-PTO or nCpG-6-PTO for 3, 6, and 24 hours. Total RNA was then extracted and quantitative RT-PCR was performed as described. (A) PD-L1 and (B) PD-L2 results are shown. Each column shows the mean of three independent experiments. Standard deviations are shown. Statistical analysis was performed in relation to a control treated with IFNγ alone. *p<0.05.

[0074] Results: nCpG-6-PTO inhibits PD-L1RNA expression.

[0075] Figure 19 shows the promoter function analysis.

[0076] (A) Transient luciferase reporter assay for PD-L1 and (B) PD-L2 promoters. A375 melanoma cells were transfected with PD-L1 and PD-L2 promoter constructs containing deletions of the relevant transcriptional binding sites. After stimulating with 20 ng / ml IFNγ for 1 hour, cells were treated with 4 μM CpG-1-PTO or nCpG-6-PTO for 16 hours. (C) ChIP assay after pretreatment with 4 μM CpG-1-PTO or nCpG-6-PTO for 1 hour and continuous IFNγ stimulation with IRF1 antibody for 6 hours for precipitation. PCR was performed using PD-L1 or (D) PD-L2 promoter-specific primers. Each column represents the mean of three experiments. Statistical analysis was performed in relation to a control treated with IFNγ alone. *p<0.05.

[0077] Results: nCpG-6-PTO inhibits the PD-L1 / 2 promoter. Action is mediated through IRF-1 (ChIP) and Stat-1 / 3.

[0078] Figure 20 shows the Western blot analysis of interferon receptor signaling proteins.

[0079] A375 cells were stimulated with 4 μM MpG-1-PTO or 4 μM nCpG-6-PTO without further stimulation (basal activation, rows 1-3), or with IFNγ for two additional time intervals (t1 and t2). Protein extracts were subjected to Western blotting to test the expression of IRF1 (t1: 60 min, t2: 3 hours), p-JAK-1 (t1: 10 min; t2: 30 min), p-JAK-2 (t1: 10 min; t2: 30 min), p-STAT-1 (t1: 10 min; t2: 30 min), p-STAT-2 (t1: 10 min; t2: 30 min), and p-STAT-3 (t1: 10 min; t2: 30 min). Equivalent loading was monitored using antibodies against all forms of phosphorylated proteins, or by GAPDH in the case of IRF1 (right-hand blot). The blot shows representative results (n=3). p-, phospho.

[0080] Results: nCpG-6-PTO inhibits the IFNγ signaling pathway.

[0081] Figure 21 shows that nCpG-6-PTO forms a G-quadrilateral (G4) and binds to IFNGR2.

[0082] (A) 5'-Cy5-labeled CpG-1-PTO and nCpG-6-PTO (2 μg) were mixed with 200 ng and 400 ng of BG4, an antibody that specifically recognizes the G4 secondary structure. The image shows fluorescence after PAGE. (B) A375- cells treated with 4 μM CpG-1-PTO, 4 μM nCpG-6-PTO, or 1 μM pyridostatin (to stabilize G4) for 24 hours were fixed and stained with BG4 antibody. BG4 reactivity was also observed in the extracellular lumen of cells treated with nCpG-6-PTO (see arrow). The image shown is a representative section. (C) 5'-Cy5-labeled CpG-1-PTO and nCpG-6-PTO (2 μg) were mixed with 200 ng and 400 ng of IFNGR1 or IFNGR2 and separated by PAGE. The image shows fluorescence after PAGE.

[0083] Results: nCpG-6-PTO forms G4. nCpG-6-PTO binds to the IFNγ receptor signaling subunit (IFNGR2).

[0084] conclusion nCpG-6-PTO forms G4 (Figure 21A, B). nCpG-6-PTO inhibits the expression of PD-L1 and PD-L2 (protein / mRNA / promoter; Figures 16-19). nCpG-6-PTO binds to the IFNγ receptor signaling subunit (IFNGR2) (Figure 21C). nCpG-6-PTO inhibits IFNγ-dependent signaling molecules (JAK-1 / 2, Stat1, 2, 3, and IRF1) (Figure 20).

[0085] Inhibition of IRF1 inhibits the activation of the PD-L1 promoter (Figure 15).

[0086] G4-forming ODNs are useful in immunotherapy, either as monotherapy or in combination therapy, because they activate T cells by inhibiting PD-L1 (and PD-L2) expression, thereby enhancing the endogenous antitumor response. This makes them useful not only for treating tumor diseases but also for virus-related diseases (such as COVID-19). G4-forming ODNs are also useful for inhibiting inflammatory mediators (JAK, Stat). A particularly effective G4-forming ODN is nCpG-6-PTO.

[0087] It has been proven that targeting the interaction of checkpoint molecules such as PD-L1 / PD-1 with antibodies activates T cell function, thereby preventing tumor cells from evading immune recognition. The clinical utility of checkpoint inhibitors has been well established in various tumors, including melanoma and non-small cell lung cancer. Furthermore, they have also been successfully used to treat liver cancer, kidney cancer, Hodgkin's disease, colorectal cancer, and breast cancer.

[0088] Recently, highly pathogenic avian influenza A(H5N1) virus and other avian influenza A virus subtypes (H7N9, H9N2, H7N3) have been found to be linked to human illness, raising concerns about a potential pandemic due to the possibility of influenza A virus subtypes circulating in poultry and livestock being transmitted to humans.

[0089] The therapeutic concept of downregulating PD-L1 / 2 by G4 introduced here can be combined with other therapies that target checkpoint molecules. These include antibody-based drugs that target PD-1 or PD-L1, such as nivolumab, pembrolizumab, atezolizumab, durvalumab, avelumab, semipirimab, and BMS-202. Compounds that target other checkpoint molecules, such as ipilimumab (against CTLA-4), are also possible. Furthermore, G4 oligonucleotides can be combined with other antitumor therapies such as chemotherapy, small molecule drugs, and radiation therapy. Sequence List SEQUENCE LISTING <110> Johann Wolfgang Goethe-Universitaet <120> G- quadruplex- containing oligonucleotides for preventive and therapeutic treatment <130> 6029-1-PCTJP01 <160> 18 <170> PatentIn version 3.5 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> n-CpG-6-PTO <222> (1)...(20) <223> all guanosines are linked by phosphorothioate bonds <400> 1 gggggggggg gggggggggg 20 <210> 2 <211> 14 <212> DNA <213> Artificial Sequence <220> <221> n-CpG-6C-PTO <222> (1)...(14) <223> all guanosines are linked by phosphorothioate bonds <400> 2 gggggggggg gggg 14 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> CpG-2118 (KonA) <222> (1),(2),(15)...(19) <223> n indicates guanosines linked by phosphorothioate bonds <400> 3 nnggtcaagc ttgannnnng 20 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <221> AS1411-PTO <222> (1)... (26) <223> all nucleosides are linked by phosphorothioate bonds <400> 4 ggtggtggtg gttgtggtgg tggtgg 26 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> CpG-1-PTO <222> (1)... (20) <223> all nucleosides are linked by phosphorothioate bonds <400> 5 tccatgacgt tcctgacgtt 20 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> n-CpG-3A-PTO <222> (1)...(18) <223> all thymidines are linked by phosphorothioate bonds <400> 6 tttttttttt tttttttt 18 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> n-CpG-5-PTO <222> (1)...(20) <223> all cytidines are linked by phosphorothioate bonds <400> 7 cccccccccc cccccccccc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <221> Scramb-CpG-1-PTO <222> (1)...(20) <223> all nucleosides are linked by phosphorothioate bonds <400> 8 ctctaggact ctctggactt 20 <210> 9 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> G3139 Genasense <222> (1)...(18) <223> all nucleosides are linked by phosphorothioate bonds <400> 9 tctcccagcg tgcgccat 18 <210> 10 <211> 6 <212> DNA <213> Artificial Sequence <220> <221> nCpG-6G-PTO <222> (1)...(6) <223> all guanosines are linked by phosphorothioate bonds <400> 110 gggggg 6 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <400> 11 ggtggtggtg gttgtggtgg tggtgg 26 <210> 12 <211> 31 <212> DNA <213> Artificial Sequence <220> <221> Quench2 <222> (1)...(31) <223> all guanosines are linked by phosphorothioate bonds <400> 12 cgcagtcttc tcctggtgct cgaacagtga c 31 <210> 13 <211> 18 <212> DNA <213> Artificial Sequence <220> <221> Flu2 <222> (1)...(18) <223> all guanosines are linked by phosphorothioate bonds <400> 13 gtcactgttc gagcacca 18 <210> 14 <211> 13 <212> DNA <213> Artificial Sequence <220> <221> Capture Q2 <222> (1)...(13) <223> all nucleosides are linked by phosphorothioate bonds <400> 14 gctactgtgt gtg 13 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <400> 15 20 gggggggggg gggggggggg

Claims

1. An oligonucleotide molecule having 10 to 50 nucleotides and containing at least one G-quartet forming motif comprising 10 to 20 nucleotide residues, wherein at least 60% of the G-quartet forming motif residues are guanosine residues or deoxyguanosine residues, and the molecule inhibits tumor growth and / or monkeypox virus replication.

2. An oligonucleotide molecule having 10 to 50 nucleotides and containing at least one G-quartet forming motif comprising 10 to 20 nucleotide residues, wherein at least 60% of the G-quartet forming motif residues are guanosine residues or deoxyguanosine residues, and the molecule inhibits the replication of viruses or bacteria in mammalian cells and / or exerts an anti-inflammatory effect.

3. The oligonucleotide molecule according to claim 1 or 2, wherein more than 70% of the residues are guanosine residues or deoxyguanosine residues.

4. An oligonucleotide molecule according to any one of claims 1 to 3, wherein the oligonucleotide is synthesized from a deoxynucleotide.

5. An oligonucleotide molecule according to any one of claims 1 to 4, wherein at least one guanosine residue or deoxyguanosine residue is chemically modified.

6. The oligonucleotide molecule according to claim 5, wherein the chemical modification is a modification of the phosphate backbone.

7. The oligonucleotide molecule according to claim 6, wherein the thiophosphoryl substitution is selected from phosphorothioate or phosphorodithioate, and the thiophosphoryl substitution substitutes for at least 35% of the phosphodiester bonds in the sugar-phosphate backbone of the oligonucleotide molecule.

8. The oligonucleotide molecule according to any one of claims 1 to 7, wherein the oligonucleotide comprises a triplet of at least four consecutive guanosine residues or deoxyguanosine residues.

9. An oligonucleotide molecule according to any one of claims 1 to 8, wherein the molecule comprises one of the sequences described in SEQ ID NOs: 1 to 4, and / or the sequences of SEQ ID NOs: 1 to 4 truncated by at least one nucleotide.

10. A pharmaceutical composition comprising at least one oligonucleotide molecule according to any one of claims 1 to 9, combined with at least one pharmaceutically acceptable additive, carrier, adjuvant, or combination thereof.

11. A kit comprising at least one oligonucleotide molecule according to any one of claims 1 to 9.

12. Oligonucleotide molecules according to any one of claims 1 to 9, the pharmaceutical composition according to claim 10, or the kit according to claim 11, for use in the treatment of diseases and / or inflammation caused by tumors, viral or bacterial infections.

13. The use according to claim 12, wherein the oligonucleotide molecule induces inhibition of tumor growth and / or viral replication in mammalian cells.

14. The use according to claim 13, wherein the viral infection is caused by a virus selected from the group including HS-1 virus, HCN virus, adenovirus, Zika virus, hepatitis C virus, West Nile virus, influenza virus, RSV virus, paramyxovirus, HIV virus, coronavirus, e.g., SARS-CoV-1, SARS-CoV-2, and MERS-CoV.

15. The use according to claim 13, wherein the disease is a viral infection of the respiratory tract.

16. The use according to claim 12, wherein the treatment of inflammation and / or tumor growth associated with a viral or bacterial disease is achieved by interference with or suppression of the type I interferon (IFN) and / or type II IFN pathways via interleukin-mediated signaling.

17. The use according to claim 12, wherein in the oligonucleotide molecule, at least one guanosine residue or deoxyguanosine residue is chemically modified, the chemical modification is a phosphate backbone modification, and the oligonucleotide comprises a triplet of at least four consecutive guanosine residues or deoxyguanosine residues.

18. The use according to claim 12, wherein the molecule is administered by a pharmaceutically acceptable route selected from the group consisting of oral, parenteral, enteral, ocular or nasal routes, or topical and combinations thereof (other routes of application, e.g., as a cream or lotion (for the treatment of HSV infection), as a spray, as a shampoo, as eye drops, as a suppository, as a transdermal patch, as a nail polish, should also be considered).

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