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WO2010015148A1 - Oligodésoxynucléotides ayant une fonction d’immunosuppression - Google Patents

Oligodésoxynucléotides ayant une fonction d’immunosuppression Download PDF

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Publication number
WO2010015148A1
WO2010015148A1 PCT/CN2009/000878 CN2009000878W WO2010015148A1 WO 2010015148 A1 WO2010015148 A1 WO 2010015148A1 CN 2009000878 W CN2009000878 W CN 2009000878W WO 2010015148 A1 WO2010015148 A1 WO 2010015148A1
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Prior art keywords
oligonucleotide
virus
peripheral blood
cpg
cells
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Chinese (zh)
Inventor
于永利
杨光
王丽颖
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Changchun Huapu Biotechnology Co Ltd
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Changchun Huapu Biotechnology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents

Definitions

  • the present invention relates to an oligonucleotide having an immunosuppressive function which has an inhibitory effect on Toll-like receptor activation, interferon production, and an abnormally enhanced immune response caused by microorganisms.
  • the present invention also relates to the use of such a oligodeoxynucleotide as a medicament for the prevention and treatment of a disease associated with activation of a Toll-like receptor, a microorganism which is excessively stimulating a disease caused by a host immune system and an autoimmune disease. Background technique: .
  • Halpern MD et al. found that oligodeoxyguanosine inhibited bacterial DNA production of IFN- ⁇ in mouse spleen cells in vitro [Halpem MD, et al. Immunopharmacology. 1995 Feb;29(l):47 -52].
  • Pisetsjiy DS et al found that genomic DNA fragments of calf thymus and human placenta inhibited the stimulation of bacterial DNA and synthetic ODN to IL-12 secretion in mouse immune cells [Pisetsky DS, et al. Clin Immunol. 2000 Sep; 96(3): 198-204].
  • TLRs Toll like receptors
  • PAMP PAMP-activated mediated immune responses by TLRs can effectively control pathogenic microbial infections in the body (Arthur M. Krieg. Nature Reviews Drug Discovery, Vol. June 2006, 471-484).
  • TLRs-mediated immune response is out of regulation, causing damage to the body and causing disease.
  • Such diseases associated with TLRs activation are known as Toll-like receptor activation-related diseases.
  • SLE Systemic Lupus Erythematosus
  • IFN- ⁇ / ⁇ type I interferon
  • TLR7/9 Toll-like receptor 7/9
  • bacterial and viral infections are a cause of autoimmune diseases such as SLE (Glinda S. et al. Environ Health Perspect. 2008 June; 116(6): 695-702).
  • Sepsis is a Toll-like receptor-associated disease, a systemic inflammatory response caused by bacterial and/or viral infections. Patients can develop systemic inflammatory response syndrome (SIRS) and multiple organs. Multiple organ dysfunction syndromes (MODS). Hyperc tokinemia or cytokine storm caused by infection with viruses and other microorganisms is an important cause of SIRS and MODS.
  • SIRS systemic inflammatory response syndrome
  • MODS Multiple organ dysfunction syndromes
  • Hyperc tokinemia or cytokine storm caused by infection with viruses and other microorganisms is an important cause of SIRS and MODS.
  • WHO World Health Organization
  • N Engl can be detected in the plasma of patients infected with the highly pathogenic avian influenza virus H5N1. J Med 2005; 353: 1374-85) o
  • bacterial DNA can be used to induce sepsis by activating TLR9 (Sparwasser T. et al. Nature. 1997 386:336-337). Inhibition of TLR9 activation reduces the
  • TLR9 Toll-like receptor 9
  • IFN type I interferon
  • pDC plasma cell dendritic cells
  • Rheumatoid arthritis is a disease associated with Toll-like receptor activation.
  • IgG2a, .DNA, RNA, or DNA/RA immune complexes activate autoreactive B cells via the TLR7/9 receptor-dependent pathway and promote the development of rheumatoid arthritis (Lau CM, et al. J Exp Med. 2005 Nov 7; 202(9): 1171-7).
  • TLR9 Activation of the TLR9 signaling pathway is associated with the development of autoimmune diabetes, and TLR9 antagonists (such as chloroquine) can reduce the incidence of diabetes in model animals (Danny Zipris, et al. The Journal of
  • Toll-like receptors can recognize many sources of pathogenic microorganisms, all of which are Toll-like receptor agonists.
  • TLR4 recognizes bacterial lipopolysaccharide (LPS); TLR2 and TLR6 dimers recognize lipoteichoic acid and bisacetyl peptide; TLR2 and TLR1 dimers recognize triacyl peptide; TLR9 recognizes synthetic or source BpG ODNs for bacteria and viruses; TLR5 recognizes bacterial flagellin; TLR2 and TLR6 dimers also recognize zymosan; TLR4 recognizes respiratory syncytial virus F; TLR3 recognizes polyinosine Acid cytosine nucleotides (poly IC), synthetic or viral-derived double-stranded RNA; TLR7 and TLR8 recognize single-stranded viral RNA; TLR4 recognizes protozoal products, such as GPI-anchored proteins; TLR4 also recognizes inflamed tissues Products such as HSP60 and fibrin fragments
  • TLR9 IL-12 receptor-a/beta
  • IFN-a/beta type I interferons
  • Inhibition of Toll-like receptor agonists on Toll-like receptors and their signal transduction pathways may have therapeutic effects on Toll-like receptor activation-related diseases by inhibiting the activation of Toll-like receptors.
  • Microbial invasion can cause microbial over-stimulation of diseases and autoimmune diseases caused by the host's immune system.
  • Microorganisms that are too strong to stimulate the host's immune system can occur after influenza virus infection and after SARS virus infection.
  • High levels of TNF-a, IL-1, IL-6, IL-12, IFN-a, IFN_p may occur in the blood of individuals infected with influenza viruses such as H5N1 influenza virus or SARS virus.
  • IFN- ⁇ chemical factor interferon-inducible protein 10, monocyte chemoattractant protein 1, IL-8 and IL-1-P.
  • This level of cytokine in the blood caused by microbial infection is significantly elevated, known as cytokineemia or cytokine storms.
  • Microorganisms Excessively stimulate the disease caused by the host's immune system can be expressed as sepsis syndrome, acute respiratory distress syndrome (ARDS) and multiple organ failure (The Writing Committee of the World Health Organization (WHO) Consultation on Human Influenza A/H5. N Engl J Med 2005;353:1374-85). Microorganisms causing such diseases include viruses, bacteria, parasites and fungi and spongiform encephalopathy. .
  • Viral infection stimulates interferon production by somatic cells to promote SLE (Ronnblom, L. et al. 2003. Arthritis Res. Ther. 5:68-75), Psoriasis (Frank O. et al. JEM, Volume 202, Number 1 , 135-143, 2005) and the development and progression of rheumatoid arthritis (Bokarewa M, et al Scandinavian Journal of Immunology, 2007, 66 (2/3): P.192-198).
  • the preparation for inhibiting the inducing effect of the virus on the interferon has a therapeutic effect on the disease caused by the excessive immunity of the host and the autoimmune diseases including SLE, psoriasis and rheumatoid arthritis.
  • OBJECTIVE To clarify an oligonucleotide that has an inhibitory function on Toll-like receptor activation and interferon production, and to use it for activation of Toll-like receptors. Microorganisms are too strong to stimulate diseases caused by host immune system. And treatment and prevention of autoimmune diseases. Technical solutions and effects:
  • the present invention provides a synthetic oligonucleotide having an oligonucleotide sequence characterized by one or more of the basic sequences as follows: 5'- ACCCCCTCT-3', including but not limited to
  • the oligonucleotide represented by the sequence of SEQ NO. 1 or SEQ NO. 2 or SEQ N0.3, the oligonucleotide described above may be chemically modified, and the chemically modified site may be a phosphate backbone, a ribose, a base, Modifications include, but are not limited to, thio modifications. 2.
  • the present invention provides the use of the above oligonucleotide for the preparation of a preparation for the treatment of a Toll-like receptor-associated disease, a microbial over-stimulation of a disease caused by the host immune system and an autoimmune disease.
  • the oligonucleotide provided by the present invention can inhibit CpG 2216 (a CpG dinucleotide-containing oligonucleotide, which is a TLR-9 agonist) to produce an interferon-stimulating effect on mouse spleen cells; inhibit CpG 2006, CpG 684, CpG C274 (CpG 2006, CpG 684, CpG C274 (both oligonucleotides containing CpG dinucleotide, an agonist of TLR-9) stimulated the proliferation of mouse spleen cells.
  • CpG 2216 a CpG dinucleotide-containing oligonucleotide, which is a TLR-9 agonist
  • the oligonucleotide provided by the present invention can inhibit the stimulation of interferon production by human peripheral blood mononuclear cells by CpG 2216; and inhibit the stimulation of proliferation of human peripheral blood mononuclear cells by CpG 2006, CpG 684, and CpG C274.
  • the oligonucleotide provided by the present invention can inhibit the stimulation effect of interferon on human peripheral blood mononuclear cells by HSV-K herpes simplex virus or PR8, FMl-Flu (two influenza viruses).
  • the oligonucleotide provided by the present invention can inhibit the stimulating effect of serum of SLE patients on interferon production by human peripheral blood mononuclear cells.
  • the present invention provides the use of the above oligonucleotide for the preparation of a preparation for preventing or treating a Toll-like receptor, a microorganism-excessive stimulation of a disease caused by a host immune system, and an autoimmune disease.
  • CpG 2216 Dominique De Wit, et al. Blood. 2004; 103: 1030-1032
  • TLR-9 a Toll-like receptor agonist that activates TLR-9, which in turn causes peripherin in human peripheral blood mononuclear cells.
  • the oligonucleotide provided by the present invention can inhibit the production of interferon by a type CpG ODN (CpG 2216) stimulating human peripheral blood mononuclear cells, and the inhibitory effect is enhanced as the concentration of the oligonucleotide increases. Therefore, the oligonucleotide provided by the present invention can inhibit the activation of TLR-9 and inhibit the production of interferon.
  • CpG 2006 Dominique De Wit, et al. Blood. 2004; 103: 1030-1032
  • the oligonucleotide provided by the present invention can inhibit proliferation of human peripheral blood mononuclear cells induced by type B CpG ODN (CpG 2006), and the inhibitory effect is enhanced as the concentration of the oligonucleotide increases.
  • CpG 684 is a Toll-like receptor agonist that activates TLR-9 (Wang X, et al.
  • the oligonucleotide provided by the present invention can inhibit proliferation of human peripheral blood mononuclear cells induced by type B CpG ODN (CpG 684 ), and the inhibitory effect is enhanced as the concentration of the oligonucleotide increases.
  • the oligonucleotides provided herein can inhibit the activation of TLR-9.
  • CpG C274 (Marshall JD. J Leukoc Biol 2003; 73(6): 781-92) is a Toll-like receptor agonist that activates TLR-9, which in turn causes proliferation of human peripheral blood mononuclear cells.
  • the oligonucleotides provided by the present invention inhibit the proliferation of human peripheral blood mononuclear cells induced by C-type CpG ODN (CpG C274), and the inhibition is enhanced as the concentration of the oligonucleotide increases.
  • CpG C274 C-type CpG ODN
  • HSV-1 virus (Hochrein H, et al. Proc Natl Acad Sci US A. 2004 Aug 3; 101(31): 11416-21) is a Toll-like receptor agonist that activates TLR-9 and causes Peripheral blood mononuclear cells produce interferon.
  • the oligonucleotides provided by the present invention can inhibit the inactivation of virus HSV-1 to stimulate interferon production by human peripheral blood mononuclear cells. Therefore, the oligonucleotide provided by the present invention can inhibit the activation of TLR-9 by the HSV-1 virus and inhibit the stimulating effect of the virus on interferon production.
  • Influenza virus (Sandra S Science 2004, 303 (5663): 1529-1531) is a Toll-like receptor agonist that activates Toll-like receptor 7, which in turn stimulates the production of interferon.
  • the oligonucleotides provided by the present invention inhibit the inactivation of the virus PR8 to stimulate interferon production by human peripheral blood mononuclear cells. Therefore, the oligonucleotide provided by the present invention can inhibit the activation of Toll-like receptor 7 by influenza virus and inhibit the stimulating effect of the virus on interferon production.
  • oligonucleotides provided by the present invention can inhibit the stimulation of interferon production by human peripheral blood mononuclear cells by serum of SLE patients.
  • the oligonucleotide of the present invention may have various chemical modifications, and the modified site may occur in a phosphodiester bond between nucleosides, a ribose unit or/and an organic base.
  • Base A, T, C, G.
  • Modifications can be made during or after synthesis of the oligonucleotide. Chemical modifications during synthesis can be modified inside or at the 5' end of the oligonucleotide.
  • Synthetic Oligonucleotides can be, but are not limited to, chemically modified at a reactive group such as a 5' or: terminal phosphate or hydroxyl group.
  • the chemical modification of the oligonucleotides of the invention may be localized to a phosphodiester bond or/and a local ribose unit or/and a local nucleobase, as compared to a naturally occurring oligonucleotide of the same sequence.
  • Chemical modifications in the present invention include backbone modifications of oligonucleotides.
  • the backbone modification of the oligonucleotide includes, but is not limited to, a phosphate backbone modification.
  • the phosphate backbone modification can be in a stable phosphate backbone of the nucleotide molecule wherein the non-bridged phosphate oxygen atom in at least one internucleotide linkage is replaced by a sulfur atom.
  • the non-bridged phosphate oxygen atom in the internucleotide linkage may be partially or completely substituted by a sulfur atom.
  • the backbone of the oligonucleotide of the present invention may also undergo nonionic DNA analogs, such as alkyl, aromatic-carbonate compounds (charged carbon phosphate compound oxygen atoms are replaced by alkyl groups, aryl groups), and then Part of the oxygen atom in the phosphodiester and decyl phosphate is modified by alkylation.
  • the oligonucleotide of the present invention may be a chimera of a phosphorosulfonyl group and a phosphodiester.
  • chemical modifications also include base substitutions such as C-5 propyne pyrimidine and 7-deaza-7 instead of purine substitution.
  • Alternative purines and pyrimidines include, but are not limited to, adenine, guanine, cytosine, thymine, other natural and non-naturally occurring bases.
  • chemical modifications also include base modifications. Modified bases are chemically distinct from typical natural bases (such as A, T, C, G. :), but have their basic chemical structure.
  • the oligonucleotide of the present invention may also be modified with a cytosine derivative or a thymidine derivative.
  • a cytosine derivative refers to a cytosine-like nucleoside (except cytosine).
  • a thymidine derivative refers to a thymidine-like nucleoside (excluding thymine).
  • the modification of the oligonucleotide in the present invention may be such that a diol such as tetraethyl or hexaethylene glycol is attached to two or one end of the oligonucleotide.
  • a pharmaceutically acceptable carrier refers to one or more solid or liquid fillers, diluents or encapsulating substances which are suitable for the oligonucleotides of the invention. Applied to individuals.
  • the carrier can be organic, inorganic, natural or synthetic.
  • the carrier includes various solutions, diluents, solvents, dispersing agents, liposomes, emulsions, sugar coatings, antibacterial agents, antifungal agents, isotonic and delayed absorption agents, and other oligonucleosides suitable for use in the present invention.
  • Carrier for acid application The choice of a pharmaceutically acceptable carrier will depend on the mode of application of the oligonucleotides in the present invention.
  • Injectable carriers include water, physiological saline, balanced salt solutions, dextrose solutions, glycerol, and the like.
  • non-toxic solid carriers include: mannitol having a pharmacological purity, lactose, starch, magnesium stearate, and the like.
  • the pharmacological component of the application may contain non-toxic auxiliary substances including moisturizing or emulsifying agents, preservatives, pH buffering agents, sodium acetate and monolaurate.
  • the dose of the oligonucleotide of the present invention is from 1 g to 100 mg per application to an individual.
  • those skilled in the art can adjust the dosage of the application, such as the dosage adjustment made by the attending physician based on reliable medical judgment, and the dosage range can be 10 times to 1000 of the aforementioned range. Times.
  • a pharmaceutical composition refers to a therapeutically effective amount of an oligonucleotide of the invention or a mixture thereof with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may comprise one or more oligonucleotides of the invention.
  • the pharmaceutical composition may be, but is not limited to, an aqueous solution, a saline solution, a granule, an aerosol, a tablet, a powder, a sugar-coated tablet, a capsule, a suppository, a syrup, an emulsion, a suspension, a cream, a drop, and the like. Substance.
  • the pharmaceutical composition can be applied by: parenteral, oral, rectal, transvaginal, intraperitoneal, topical (powder, ointment, gel, drop, dialysis), oral or oral cavity. And nasal sprays, etc.
  • the pharmaceutical composition must be sterile and stable.
  • the pharmaceutical composition for injection of the present invention comprises a pharmaceutically acceptable aqueous solution, a nonaqueous solution, a dispersing agent, a suspension, an emulsion or a powder, which is dissolved with sterile water for injection or a dispersing agent before injection.
  • the oligonucleotide of the present invention can be dissolved in an aqueous phase carrier, such as an isotonic buffer of pH 3.0 to pH 8.0, and the pH range can also be pH 3.5-pH 7.4, pH 3.5-pH 6.0, pH 3.5- pH 5.0.
  • Buffers include: citrate buffer, phosphate buffer, acetate buffer.
  • the pharmaceutical composition for oral administration can be formulated into a powder with an edible carrier, a tablet, a pill, a troche, a capsule, a liquid, a gel, a syrup, a 'paste or a suspension, and the like.
  • Conventional non-toxic solid carriers as solid components may be pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, and the like.
  • the pharmaceutical pharmaceutical composition for oral administration can be a tablet and a tablet in a conventional manner.
  • the pharmaceutical pharmaceutical composition as an inhalation method is a spray, and those skilled in the relevant art can choose to use a pressure capsule, a spray or a dry powder.
  • the pharmaceutical composition can be applied after treatment with a suitable sustained release system.
  • the oligonucleotide or pharmaceutical composition of the invention can be treated to a suspension with crystals or a non-crystalline material with less moisture to stably delay the release.
  • the delayed release of an oligonucleotide of the invention for injection can be achieved by dissolving the oligonucleotide with a hydrophobic material such as an acceptable oily carrier.
  • a pharmaceutical composition for injection form which may be a liposome-coated oligo Nucleotide or lactose, or a biodegradable semipermeable polymer such as a polylactic acid compound, a polyorthoester or a polyanhydride.
  • Active substance includes non-steroidal anti-inflammatory agents, steroids, non-specific immunosuppressive agents, biological response modifiers, compounds, small molecules, nucleic acid molecules, TLR antagonists and the like. Active substances also refer to agents that inhibit immunological activity by antagonizing chemokines, inducing regulatory T cells (CD4+CD25+ T cells), inhibiting the complement system, inhibiting matrix metalloproteinases, inhibiting NO synthase, and blocking costimulatory molecules. , inhibits signal transduction of immune cells.
  • Non-steroidal anti-inflammatory agents include, but are not limited to, diclofenac, diflunisal, acetoacetic acid, flupirtine, ibuprofen, indomethacin, ketoprofen, painkiller, naproxen , naproxen, piroxicam, sulindac, tohnetiru celecoxib, rofecoxib.
  • Steroids include, but are not limited to, cortisol, dexamethasone, hydrocortisone, methylprednisolone, hydrocortisone, prednisone, and hydroxyprednisolone.
  • Non-specific immunosuppressants are agents used to inhibit immune-mediated diseases, including but not limited to: cyclophosphamide, cyclosporine, methotrexate, steroids, FK506, tacrolimus, mycophenolic acid, and Sirolimus.
  • Biological response modifiers include: recombinant IL-1 receptor antagonist (Kineret or anakima), soluble p75, TNF-a receptor gG, fusion protein (etanercept or Enbrel), anti-TOF-a monoclonal antibody (infliximab or RemicadeX) Also included are interferon beta-1, interleukin-10 and TGFp.
  • Delivery Carrier Oligonucleotides in the present invention can be delivered for delivery using a delivery vehicle.
  • Delivery vehicles include, but are not limited to, steroids (such as cholesterol), complexes, emulsions, immunostimulating complexes (ISCOMs), lipids (such as cationic lipids and anionic lipids), liposomes, living bacterial carriers (such as Salmonella, Escherichia coli, Mycobacterium sinensis, Lactobacillus, and live viral vectors (such as vaccinia, adenovirus, herpes simplex virus), virions, virus-like particles, microspheres, nucleic acids Vaccines, polymer materials (such as carboxymethyl cellulose, chitosan), cyclic polymers.
  • the delivery vector can also recognize a targeting ligand molecule of the target cell by a specific receptor.
  • FIG. 1 Oligonucleotides mt01a, mt01b, mt01 inhibit human peripheral blood mononuclear cells (PBMC) to produce interferon under CpG 2216 stimulation.
  • PBMC peripheral blood mononuclear cells
  • the abscissa indicates the culture supernatant of human PBMC under the action of culture medium and various oligonucleotides
  • the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • the oligonucleotides mt01a, mt01b, and mtOl inhibited the production of interferon in human peripheral blood mononuclear cells stimulated by CpG 2216, and increased the number of deaths of Vero cells under VSV virus challenge.
  • Figure 2 Oligonucleotides mt01a, mt01b, mtOl inhibit the proliferation of human peripheral blood mononuclear cells stimulated by CpG 684.
  • oligonucleotides mt01a, mtOlb, mtOl or control oligonucleotide msl9 8 g/ml
  • CpG 684 1 g/ml
  • the abscissa indicates the type of oligonucleotide acting on human PBMC
  • the ordinate indicates the cpm value (pulse per minute)
  • cpm ⁇ SD indicates the proliferation of cells under the action of each oligonucleotide.
  • Oligonucleotides mt01a, mt01b, mtO1 inhibited proliferation of human peripheral blood mononuclear cells under CpG 684 stimuli compared to the control oligonucleotide msl9.
  • Figure 3 Different concentrations of oligonucleotides mtO1 inhibits the production of interferon by human peripheral blood mononuclear cells under CpG 2216 ⁇ stimulation.
  • the abscissa indicates the culture supernatant of human PBMC under the action of culture medium and different concentrations of oligonucleotide mtOl
  • the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • the oligonucleotide mtOl can inhibit the production of antiviral substances in human peripheral blood mononuclear cells stimulated by CpG 2216, and the inhibitory effect is enhanced with the increase of mtOl concentration.
  • Oligonucleotide mt01 (8 g/ml) or control oligonucleotide msl9 (8 g/ml) was co-operated with inactivated virus HSV-1 on human peripheral blood mononuclear cells for 48 hours, and the culture supernatant was collected. The supernatant protects the Vero E6 against the effects of the 10xTCID50 VSV virus attack.
  • the abscissa indicates the culture supernatant of the human PBMC under the action of the culture medium and the two oligonucleotides
  • the ordinate indicates the A value of the Vero cells detected by the crystal violet staining method at 578 nm.
  • Oligonucleotide mtOl inhibited the production of interferon by human peripheral blood mononuclear cells stimulated by HSV-1 compared to the control oligonucleotide msl9.
  • Figure 5 mtOl inhibits human peripheral blood mononuclear cells from producing interferon under the stimulation of inactivated virus PRS-Flu.
  • the oligonucleotide mt01 (8 g/ml) or the control oligonucleotide msl9 (8 g/ml) was co-operated with the inactivated virus PRS-Flu on human peripheral blood mononuclear cells for 48 hours, and the culture supernatant was collected. The supernatant protects Vero E6 against the effects of 10> TCID50 VSV virus attack.
  • the abscissa indicates the culture supernatant of the human PBMC under the action of the culture medium and the two oligonucleotides
  • the ordinate indicates the A value of the Vero cells detected by the crystal violet staining method at 578 nm.
  • Oligonucleotide mtOl inhibits the production of interferon by human peripheral blood mononuclear cells stimulated by PRS-Flu compared to the control oligonucleotide msl9.
  • Figure 6 mtOl inhibits the production of interferon by human peripheral blood mononuclear cells stimulated by inactivated virus FM1.
  • the oligonucleotide mt01 (8 g/ml) or the control oligonucleotide msl9 (8 g/ml) was co-operated with the inactivated virus FM1 on human peripheral blood mononuclear cells for 48 hours, and the culture supernatant was collected and observed. Clear protects Vero E6 against the effects of the 10xTCiD50 VSV virus attack.
  • the abscissa indicates the culture supernatant and the culture supernatant of human PBMC under the action of two kinds of oligonucleotides
  • the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • the oligonucleotide mtOl can inhibit the production of interferon by human peripheral blood mononuclear cells stimulated by inactivated virus FM1.
  • Oligonucleotide mtO1 inhibits the production of interferon by mouse spleen cells under CpG 2216 stimulation.
  • Oligonucleotide mt01 (8 g/ml) or control oligonucleotide msl9 (8 g/ml) and CpG 684 (1 g/ml) were co-operated on mouse spleen cells for 48 hours, and the culture supernatant was taken and observed. The supernatant protects L929 cells against the effects of 10XTCID50 VSV virus challenge.
  • the abscissa indicates the culture supernatant and the human PBMC culture supernatant under the action of CpG 2216, and the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • Oligonucleotide mtO1 inhibited the production of interferon by mouse spleen cells stimulated by CpG 2216 compared to the control oligonucleotide msl9.
  • Figure 8 Different concentrations of oligonucleotides mtOl inhibits proliferation of human peripheral blood mononuclear cells stimulated by CpG 2006.
  • Oligonucleotide mtOl or control oligonucleotides of different concentrations interact with CpG 2006 (1 g/ml) in human peripheral blood alone After 48 hours of nucleation, the cells were cultured for 16 hours after incorporation of [ 3 H]thymidine for cell proliferation.
  • the abscissa indicates the different concentrations of oligonucleotide mtOl acting on human PBMC
  • the ordinate indicates cpm value (pulse per minute)
  • cpm ⁇ SD indicates the proliferation of cells under the action of oligonucleotide mtOl.
  • the oligonucleotide mtOl inhibited the proliferation of human peripheral blood mononuclear cells stimulated by CpG 2006, and the inhibitory effect increased with the increase of mtOl concentration.
  • Figure 9 Different concentrations of oligonucleotides mtOl inhibits the proliferation of human peripheral blood mononuclear cells stimulated by CpG C274. ⁇ ⁇ ⁇ ⁇ '
  • the abscissa indicates the different concentrations of the oligonucleotide mtOl acting on the human PBMC
  • the ordinate indicates the cpm value (pulse per minute)
  • the proliferation of the cells under the action of the oligonucleotide mtO1 was expressed by cpm ⁇ SD.
  • the oligonucleotide mtOl can inhibit the proliferation of human peripheral blood mononuclear cells stimulated by CpG C274, and the inhibition is enhanced with the increase of mtOl concentration.
  • Figure 10 Different concentrations of oligonucleotide mtOl inhibited proliferation of human peripheral blood mononuclear cells stimulated by CpG 684.
  • Oligonucleotide mtOl or control oligonucleotide msl9 at different concentrations (64, 32, 16, 8, 4, 2, 1, 0 g/ml) and CpG 684 (1 g/ml) acted on human peripheral blood Mononuclear cells were cultured for 48 hours, and cells were spiked for 16 hours after incorporation of [ 3 H] thymidine for cell proliferation.
  • the abscissa indicates the different concentrations of oligonucleotide mt01 acting on human PBMC
  • the ordinate indicates cpm value (pulse per minute)
  • cpm ⁇ SD indicates the proliferation of cells under the action of oligonucleotide mtOl.
  • the oligonucleotide mtOl inhibited the proliferation of human peripheral blood mononuclear cells under CpG 684 ⁇ stimulation, and the inhibition increased with the concentration of mtOl.
  • Figure 11 Oligonucleotide mtOl inhibits proliferation of mouse spleen cells stimulated by CpG 2006, CpG C274 or CpG 684.
  • Oligonucleotide mt01 (8 g/ml) or control oligonucleotide (8 g/ml) together with CpG 2006 (1 g/ml), CpG C274 (lg/ml) or CpG 684 (1 g/ml)
  • CpG 2006 (1 g/ml
  • CpG C274 lg/ml
  • CpG 684 1 g/ml
  • the abscissa indicates the different CpG ODN acting on human PBMC
  • the ordinate indicates cpm value (pulse per minute)
  • cpm ⁇ SD indicates the proliferation of cells under the action of oligonucleotide mtOl.
  • Oligonucleotide mtOl inhibited proliferation of mouse spleen cells stimulated by CpG 2006, CpG C274 or CpG 68 compared to the control oligonucleotide msl9.
  • Figure 12 Oligonucleotides mtOl and mt01c, mt01d, mtOlf inhibit human peripheral blood mononuclear cells producing interferon under CpG 2216 stimuli.
  • the abscissa indicates the culture supernatant of human PBMC under the action of culture medium and various oligonucleotides
  • the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • Oligonucleotides mtOl and its mutant sequences mt01c, mtOld, and mtOlf all inhibited the production of interferon by human peripheral blood mononuclear cells under CpG 2216 stimuli.
  • Figure 13 Oligonucleotides mt01, mt01a, mt01b, mt01c, mt01d, mt01e, mtOlf inhibited the production of interferon by human peripheral blood mononuclear cells under inactivated virus FM1.
  • the same concentration (8 g/ml) mt01, mt01c, mt01d, mtOlf or control ODN was combined with inactivated virus FM1 for 48 hours in human peripheral blood mononuclear cells, and the culture supernatant was collected. The supernatant was observed to protect Vero E6 against 10xTCID50 VSV. The role of virus attacks.
  • the abscissa indicates the culture supernatant of human PBMC under the action of culture medium and various oligonucleotides, and the ordinate indicates the A value of Vem cells detected by crystal violet staining at 578 nm.
  • Figure 14 Oligonucleotide mtOl inhibition SLE patient serum stimulates human peripheral blood mononuclear cells to produce interferon.
  • the abscissa indicates the culture supernatant of human PBMC under the action of culture medium and various oligonucleotides, and the ordinate indicates the A value of Vero cells detected by crystal violet staining at 578 nm.
  • the symbols on the way (o, ⁇ , ⁇ , O) each represent a specimen.
  • Oligonucleotide mtOl inhibits the stimulation of interferon by human serum from peripheral blood mononuclear cells in SLE patients.
  • Human peripheral blood mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Vero E6 cells were incubated with IMDM medium containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C, 5% CO 2 cells. Cultivate in the box. The antiviral activity of the treated human peripheral blood mononuclear cell culture supernatant was examined by Vero E6 cytopathic method.
  • the method is as follows: Human peripheral blood mononuclear cells with a density of 5xl0 5 / hole Inoculation into U-bottom 96-well plates, human peripheral blood mononuclear cells in the presence of the same concentration (16 g/ml) of three oligonucleotides mt01, mt01a, mtOlb or control oligonucleotide msl9 The cells were co-cultured with CpG 2216 (2 ⁇ ⁇ / ⁇ 1 ) for 48 hours, and the supernatant was collected.
  • Vero ⁇ 6 cells (2 ⁇ 10 4 /well) were inoculated into a flat-bottom 96-well plate, cultured for 12 hours, and the supernatant collected above was added for 18 hours, and then 10XTCID50 (TCID50, half tissue infection dose) of VSV virus was added to continue culture. hour. After the end of the culture, stain with 0.5% crystal violet for 15 minutes at 37 ° C, discard the crystal violet staining solution, gently rinse the remaining crystal violet staining solution, pat dry the plate, add crystal violet decoloring solution 100 ⁇ 1 per well, decolorize the shaker for 1 h. The A value at 578 nm was measured with a microplate reader (Thermo Labsystems Multiskan Ascent 345). Each group was given 3 duplicate wells and the results were statistically detected.
  • oligonucleotides mt01, mtOlas mtOlb inhibited the production of interferon by human peripheral blood mononuclear cells stimulated by CpG 2216, thereby reducing CpG 2216-stimulated human peripheral blood alone.
  • the protective effect of nuclear cell culture supernatant on Vero E6 cells challenged with VSV virus (p ⁇ 0.01).
  • Oligonucleotides mt01, mt01a, mtO lb can inhibit the secretion of immunologically active substances (including antiviral substances) from human immune cells (human peripheral blood mononuclear cells) stimulated by Toll-like receptor 9 agonist (CpG 2216). Such as IFN, etc.).
  • CpG 2216 human peripheral blood mononuclear cells
  • IFN Toll-like receptor 9 agonist
  • Human peripheral blood mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Human peripheral blood mononuclear cells are seeded at a density of 5xl0 5 /well into a U-bottom 96-well plate, in the presence of the same concentration (16 g/ml) of three oligonucleotides mt01, mt01a, mtOlb or In the case of the control oligonucleotide msl9, human peripheral blood mononuclear cells were incubated with CpG 684 (1 g/ml) for 48 hours, followed by addition of 3 H-thymidine (0.5 ⁇ /?) for 16 hours. In fiberglass The cells were harvested on the filter and the results were detected with a scintillation counter. Each group was given 3 duplicate wells and the results were statistically detected.
  • oligonucleotides mt01, mt01a, and mtOlb inhibited proliferation of human peripheral blood mononuclear cells stimulated by CpG 2006 compared with the control group, thereby reducing CpG 2006-stimulated human peripheral blood mononuclear cells.
  • Cell proliferation ( ⁇ 0.01).
  • Oligonucleotides mt01, mt01a, and mtOlb can inhibit the proliferation of human immune cells (human peripheral blood mononuclear cells) stimulated by the Toll-like receptor 9 agonist (CpG 2006).
  • human immune cells human peripheral blood mononuclear cells
  • Toll-like receptor 9 agonist CpG 2006
  • Example 3 Different concentrations of oligonucleotides mtOl inhibits human peripheral blood mononuclear cells to produce interferon under CpG 2216 stimuli
  • Mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Vero E6 cells were incubated with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) in DM medium at 37 ° C, 5% CO 2 cells. Cultivate in the box. The antiviral activity of the culture supernatant of treated human peripheral blood mononuclear cells was examined by Vero E6 cytopathic method.
  • the method is as follows: Human peripheral blood mononuclear cells are co-cultured with CpG2216 (2 ⁇ ⁇ / ⁇ 1) in the presence of different concentrations of mtOl (16, 8, 4, 2, 1, 0.5, C ⁇ g/ml). Hours, collect the supernatant. Vero E6 cells (2> ⁇ 10 4 /well;) were inoculated into a flat-bottomed 96-well plate, cultured for 12 hours, and the supernatant collected above was added for further 18 hours, followed by lOx TCID50 (TCID50, half tissue infection dose). The VSV virus was cultured for 48 hours.
  • Oligonucleotide mtO1 inhibits the secretion of immunologically active substances (including antiviral substances) from human immune cells (human peripheral blood mononuclear cells) stimulated by an agonist of Toll-like receptor 9 (CpG 2216) For example, IFN, etc., the inhibitory ability is enhanced as the concentration of the oligonucleotide mtOl is increased.
  • Example 4 mtOl inhibits human peripheral blood mononuclear cells to produce interferon under inactivation of virus HSV-1, inactivated virus PR8-F1U, and inactivated virus FM1.
  • HSV virus herpes simplex virus, from the Department of Microbiology, School of Medicine, Jilin University
  • PR8-F1U influenza virus PR8 strain, from the Department of Microbiology, School of Medicine, Jilin University
  • FM1 influenza virus sub-type mouse lung-adapted strain
  • Mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Vero cell culture amplifies HSV-1, chicken embryo amplifies PR8-Flu, and chicken embryo amplifies FM1.
  • 50% tissue cell infection (TCID50) was determined separately. 70 ° C water bath lO min inactivated HSV-1, 56V water bath 30 min inactivated PR8-Flu, 56 ° C water bath 30 min inactivated FMl. The treated HSV-1, PR8-Flu and FMl were separately added to the Vero cell culture system, and the cells were stably passaged three times. No cells were infected by the virus, and the virus was completely inactivated.
  • Vero E6 cells were incubated with IMDM medium containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C, 5% CO 2 cells. Cultivate in the box. The antiviral activity of human peripheral blood mononuclear cell culture supernatants treated with the aforementioned three inactivated viruses was examined by Vem E6 cytopathic method.
  • the method is as follows: In the case of mtOl (8 ⁇ ⁇ / ⁇ 1) or control oligo. acid msl9, 'human peripheral blood mononuclear cells and inactivated virus HSV-1, inactivated virus PR8-Flu, inactivated virus FM1 was cultured for 48 hours and the upper jaw was collected. Vero E6 cells (2 ⁇ 10 4 /well) were inoculated into a flat-bottom 96-well plate, cultured for 12 hours, and the supernatant collected above was added for further 18 hours, and 10 ⁇ TCID50 (TCID50, half tissue infection dose) of VSV virus was added. 48 hours.
  • TCID50 TCID50, half tissue infection dose
  • mtOl can inhibit the production of human peripheral blood mononuclear cells stimulated by inactivated virus HSV-1, inactivated virus PR8-Flu, and inactivated virus FM1.
  • Antiviral Substance thereby reducing the protective effect of human peripheral blood mononuclear cell culture supernatant stimulated by inactivated virus HSV-1, inactivated virus PR8-Flu, inactivated virus FM1 on Vero E6 cells challenged by VSV virus ( ⁇ 0 ⁇ 01).
  • Oligonucleotide mtOl can inhibit the secretion of immune activity from human immune cells (human peripheral blood mononuclear cells) stimulated by viral components (inactivated virus HSV-1, inactivated virus PR8-Flu, inactivated virus FM1). Substance (including antiviral substances such as IFN, etc.), and since the inactivated virus HSV-1 is a Toll-like receptor 9 agonist, the inactivated virus PR8-Flu, the inactivated virus FM1 is a Toll-like receptor 7 agonist, The oligonucleotide mtOl can inhibit immune activation mediated by Toll-like receptors 7, 9.
  • Oligonucleotide mtOl inhibited the production of interferon by mouse spleen cells under CpG 2216 stimuli.
  • L929 cells mouse fibroblast strain, American Type Culture Collection
  • Mouse spleen cells were routinely isolated. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • L929 cells were cultured in IMDM with 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C, 5% CO 2 in a cell incubator Internal culture.
  • antibiotics 100 IU/ml penicillin and 100 IU/ml streptomycin
  • mice spleen cells were co-cultured with CpG2216 (2 ⁇ ⁇ / ⁇ 1) for 48 hours, and the supernatant was collected.
  • L929 cells (2x10 4 /? L) were inoculated into a flat-bottom 96-well plate and cultured for 12 hours. The supernatant collected above was added for 18 hours, and 10 ⁇ TCID50 (TCID50, half tissue infection dose) of VSV virus was added to continue the culture. 48 hours.
  • mtO1 inhibited the production of antiviral substances by mouse spleen cells stimulated by CpG 2216, thereby reducing the protective effect of CpG 2216-stimulated mouse spleen cell culture supernatant on Vero E6 cells challenged by VSV virus. ( ⁇ 0.01 ).
  • Oligonucleotide mtOl can inhibit the secretion of immunologically active substances (including antiviral substances) from mouse immune cells (spleen cells) stimulated by Toll-like receptor 9 agonist, which contains the bacterial genome immunostimulatory sequence CpG 2216. IFN, etc.).
  • Example 6 Different concentrations of oligonucleotide mtOl inhibited proliferation of human peripheral blood mononuclear cells stimulated by CpG 2006, CpG C274 or CpG 684.
  • Human peripheral blood mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Human peripheral blood mononuclear cells are seeded at a density of 5 ⁇ 10 5 /well into a U-bottom 96-well plate in the presence of mt01 (8 g/ml) or control oligonucleotide msl9, with CpG 2006 (1 g/ml), CpG C274 (1 g/ml) or CpG 684 (1 g/ml) were incubated for 48 hours, followed by addition of 3H-thymidine (0.5 ⁇ /well) for 16 hours. The cells were harvested on a glass fiber filter and the results were detected using a scintillation counter. Each group was given 3 duplicate wells and the results were statistically detected.
  • the oligonucleotide mtO1 inhibited the proliferation of human peripheral blood mononuclear cells stimulated by CpG 2006, CpG C274 or CpG 684 (p ⁇ 0.01).
  • Oligonucleotide mtOl can inhibit the proliferation of human immune cells (human peripheral blood mononuclear cells) stimulated by Toll-like receptor 9 agonists containing the bacterial genomic immunostimulatory sequence CpG 2006, CpG C274 or CpG 684.
  • Example 7 Oligonucleotide mtOl inhibited the proliferation of mouse spleen cells under CpG 2006, CpG C274, CpG 684 stimulation.
  • L929 cells mouse fibroblast cell line, American Type Culture Collection
  • BALB/c mouse Animal Room of Jilin University School of Basic Medicine
  • Mouse spleen cells were routinely isolated. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • mice spleen cells were inoculated into a U-bottom 96-well plate at a density of 5 ⁇ 10 5 /well in the presence of mtOl Or co-incubation with CpG 2006 (1 g/ml), CpG C274 (lg/ml), CpG 684 (1 g/ml) or CpG 2395 (1 g/ml) for 48 hours with the control oligonucleotide msl9 Then, 3H-thymidine (0.5 ⁇ /well;) was added and cultured for 16 hours. The cells were harvested on a glass fiber filter and the results were detected using a scintillation counter. Each group was given 3 duplicate wells and the results were statistically detected.
  • the oligonucleotide mtO1 inhibited the proliferation of mouse spleen cells stimulated by CpG 2006, CpG C274 or CpG 684 (/? ⁇ 0.01).
  • Oligonucleotide mtOl can inhibit the proliferation of mouse immune cells (spleen cells) stimulated by Toll-like receptor 9 agonists (CpG 2006, 0 0 0274 or .0 684).
  • Example 8 Oligonucleotides mt01, mt01c, mt01d, mt01f3 ⁇ 43 Human peripheral blood mononuclear cells produce interferon under CpG 2216 stimuli.
  • Oligonucleotides mt01c, mt01d, mtOlf sequences are shown in Table 1, wherein two bases in the mtOla sequence were changed and tandemly repeated three times to obtain mtOlc; three bases in the mtOla sequence were changed and tandemly repeated Three times, mtOl mountain was obtained, and one base in the mtOla sequence was changed and then repeated in series three times to obtain mt01f. The remaining materials are the same as in the first embodiment.
  • Mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Vero E6 cells were incubated with IMDM medium containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C, 5% CO 2 cells. Cultivate in the box. The antiviral activity of the culture supernatant of treated human peripheral blood mononuclear cells was detected by Vem E6 cytopathic method.
  • the method is as follows: human peripheral blood mononuclear cells were co-cultured with CpG2216 (2 g/ml) in the presence of the same concentration (8 g/ml) of mt01, mt01c, mt01d, mtOlf or control oligonucleotide msl9. For 48 hours, collect the supernatant. Vero E6 cells (2x l0 4 / well) were inoculated into a flat-bottom 96-well plate, cultured for 12 hours, and the supernatant collected above was added for further 18 hours, and then VSV of TCID50 (TCID50, half tissue infection dose) was added. The virus was cultured for 48 hours.
  • TCID50 TCID50, half tissue infection dose
  • oligonucleotides mt01, mt01c, Both mt01d and mtOlf can inhibit the production of antiviral substances from human peripheral blood mononuclear cells stimulated by CpG 2216, thereby reducing the protection of CpG 2216-stimulated human peripheral blood mononuclear cell culture supernatant against Vero E6 cells challenged by VSV virus. Function (;? ⁇ 0.01).
  • oligonucleotide obtained by changing the 1-3 base sequence of mtOla still inhibits the production of interferon by human peripheral blood mononuclear cells stimulated by CpG 2216.
  • Oligonucleotides mt01, mt01c, mt01d, mtOlf inhibited human peripheral blood mononuclear cells. Interferon was produced under the stimulation of inactivated virus FM1.
  • Mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • TCID50 50% tissue cell infection
  • Vero E6 cells were incubated with IMDM medium containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C, 5% CO 2 cells. Cultivate in the box. The antiviral activity of human peripheral blood mononuclear cell culture supernatants treated with the aforementioned three inactivated viruses was examined by Vero E6 cytopathic method.
  • the oligonucleotides mt01, mt01c, mt01d, mtOlf can inhibit the production of antiviral substances from human peripheral blood mononuclear cells stimulated by inactivated virus FM1, as compared with the control oligonucleotide msl9. Peripheral blood mononuclear cells stimulated by inactivated virus FM1 were reduced. Protective effect of culture supernatant on Vero E6 cells challenged with VSV virus ( ⁇ 0.01). Conclusion: The mtOla 1-3 base sequence changes can still inhibit the production of interferon by human peripheral blood mononuclear cells under the stimulation of inactivated virus FM1.
  • Oligonucleotide mtO1 inhibits SLE patient serum to stimulate interferon production by human peripheral blood mononuclear cells.
  • the serum of SLE patients was from venous blood of patients with renal disease and rheumatology in the Sino-Japanese Friendship Hospital of Jilin University. All patients met the diagnostic criteria for SLE revised by the American College of Rheumatology (ARA) in 1997. All sera were stored at -70 ° C, diluted 2 times with IMDM medium containing 10% FBS, and sterilized by 0.2 ⁇ disposable filter (Acrodisc® Syringe Filter Pall Life Sciences).
  • Human peripheral blood mononuclear cells were isolated from human peripheral blood-enriched leukocytes by Ficoll-Hypaque density gradient centrifugation. The survival rate of human peripheral blood mononuclear cells was determined by phenol blue staining method to be 95%-99%.
  • Vero E6 cells were cultured with IMDM medium containing 10% (v/v) heat-inactivated fetal bovine serum and antibiotics (100 IU/ml penicillin and 100 IU/ml streptomycin) at 37 ° C: 5% CO 2 cells Cultivate in the incubator. The antiviral activity of the treated human peripheral blood mononuclear cell culture supernatant was examined by Vero E6 cytopathic method.
  • the method is as follows: Human peripheral blood mononuclear cells are seeded into U-bottom 96-well plates at a density of 5 ⁇ 10 5 /well, in the presence of the same concentration (8 g/ml) of mtOl or control oligonucleotide msl9, Human peripheral blood mononuclear cells were co-cultured with 2-fold diluted SLE serum for 48 hours, and the supernatant was collected. Vero E6 cells (2 ⁇ 10 4 /well) were inoculated into a flat-bottom 96-well plate, cultured for 12 hours, and then the supernatant collected above was added for 18 hours, and 10 ⁇ TCID50 (TCID50, half tissue infection dose) of VSV virus was added for further 48 hours.
  • TCID50 TCID50, half tissue infection dose
  • the cells were stained with 0.5% crystal violet for 15 minutes at 37 ° C, the crystal violet staining solution was discarded, and the remaining crystal violet staining solution was gently washed with running water, and the plate was patted dry, and 100 ⁇ l of crystal violet decolorizing solution was added to each well.
  • the plate was decolorized for 1 h, and the A value of 578 nm was measured with a microplate reader. Three replicate wells were set in each group, and the results were statistically analyzed.
  • Stimulation supernatants from SLE patients alone can protect Vero cells from VSV cleavage, whereas normal human serum-stimulated supernatants do not have this protective effect, and mtOl alone does not have the ability to stimulate PBMC to secrete interferon. . This suggests that serum from SLE patients can stimulate interferon production in human peripheral blood mononuclear cells.
  • mtOl was used in combination with serum from patients with SLE, mtO1 showed an inhibitory effect on serum-stimulated human peripheral blood mononuclear cells from SLE patients (P ⁇ 0.05) (Fig. 14).
  • An immune complex formed by autoantibodies and DNA is present in the serum of SLE patients.
  • This immune complex is a causative agent of SLE that stimulates the production of interferon by activating TLR9 (Means TK, et al. Human lupus autoantibody-DNA complexes activate DCs through cooperation of CD32 and TLR9. J Clin Invest 2005; 115: 407-17).
  • TLR9 Means TK, et al. Human lupus autoantibody-DNA complexes activate DCs through cooperation of CD32 and TLR9. J Clin Invest 2005; 115: 407-17.
  • the results of mtO1 inhibition of interferon production in this example and the above examples demonstrate that mtO1 can treat SLE by inhibiting the production of interferon by immune complexes, viruses, and Toll-like receptor 9 activators which inhibit the formation of autoantibodies and DNA.

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Abstract

La présente invention concerne des oligodésoxynucléotides ayant une fonction d’immunosuppression et leur utilisation pharmaceutique dans le traitement et la prévention de maladies associées à l’activation de récepteurs du type Toll, de maladies induites par l’hyperstimulation de microorganismes envers le système immunitaire et de maladies auto-immunes.
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