WO2020197367A1 - Composition for preventing or treating diabetes by using st8sia1, and method for screening for antidiabetic agents - Google Patents

Composition for preventing or treating diabetes by using st8sia1, and method for screening for antidiabetic agents Download PDF

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WO2020197367A1
WO2020197367A1 PCT/KR2020/095049 KR2020095049W WO2020197367A1 WO 2020197367 A1 WO2020197367 A1 WO 2020197367A1 KR 2020095049 W KR2020095049 W KR 2020095049W WO 2020197367 A1 WO2020197367 A1 WO 2020197367A1
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st8sia1
cells
protein
diabetes
composition
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Korean (ko)
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황인후
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황정후
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a composition for preventing or treating diabetes using ST8SIA1 and a method for screening a diabetes therapeutic agent, and more particularly, to a diabetes mellitus comprising as an active ingredient an inhibitor of the expression or activity of the ST8SIA1 protein or insulin-secreting cells in which the ST8SIA1 gene is deleted.
  • Glucose toxicity in diabetes causes apoptosis of ⁇ -cells and affects various organ systems, including the pancreas.
  • the basic mechanism is not fully known. Impairment of ⁇ -cells and impaired insulin production are typical features of diabetes (Xu, G et al., Nat. Med. 19, 1141-1146. 2013), but apoptosis of ⁇ -cells is prevented despite the rapid spread of diabetes. The exact molecular mechanism of the resulting glucose toxicity is still unknown (Chen, J et al., Diabetes 57, 938-944. 2008).
  • Apoptosis is a process that naturally occurs in the body by activating specialized intracellular signal transduction to destroy cells. It is also a homeostatic mechanism for maintaining cell populations in tissues. Inadequate apoptosis is fundamentally common in a variety of pathologies, including metabolic stress such as ischemic injury, autoimmune diseases and many types of cancer, neurodegenerative diseases, and glucose toxicity (Elmore, S et al., Toxicol. Pathol. 35, 495-516. 2007).
  • Such apoptosis is caused by intrinsic pathways that activate caspase-9 and -3 by cytochrome c released from mitochondria, and caspase-8 and -10 by death receptors such as TNF- ⁇ (tumor nerosis factor- ⁇ ) and Fas ligand. This activation is divided into two pathways of the extrinsic pathway. In addition, it is known that activation of various proteins independent of caspase is involved in apoptosis signaling.
  • the ST8SIA1 gene is a gene that encodes an enzyme called alpha-N-acetyl neuraminide alpha-2,8-sialyl transferase, and the enzyme transfers sialic acid from CMP-sialic acid to GM3 to ganglioside ( Ganglioside) is a type II membrane protein that produces GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells.
  • the ST8SIA1 protein can often be found in the Golgi apparatus and is a member of the glycosyltransferase family.
  • ST8SIA1 can act as a target of diabetes treatment as in the present invention.
  • ST8SIA1 has a function of inhibiting apoptosis of pancreatic beta cells, particularly in a high glucose state caused by diabetes, and inhibiting this.
  • the present invention was completed by confirming that the amount of insulin secreted in the body is increased by the insulin secreting cells and that blood sugar is normally regulated.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • compositions for preventing or treating diabetes comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • compositions for preventing or treating diabetes consisting essentially of an inhibitor of the expression or activity of the ST8SIA1 protein as an active ingredient.
  • Another object of the present invention is to provide a third object of the present invention.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
  • compositions for preventing or treating diabetes comprising the insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
  • compositions for preventing or treating diabetes consisting essentially of insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
  • Another object of the present invention is to provide the use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
  • Another object of the present invention is to provide a method for treating diabetes, comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes consisting essentially of an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes consisting essentially of insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
  • the present invention provides a use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
  • the present invention provides a method for treating diabetes comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • treatment refers to suppression of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction of disease progression rate, improvement of disease state, improvement, alleviation or improved prognosis do.
  • prevention' as used in the present invention means any action that suppresses the onset or delays the progression of a disease.
  • 'Protein' is used interchangeably with'polypeptide' or'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in nature.
  • a fragment of the FGF12 protein refers to a peptide of a portion of the FGF12 protein.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is composed of uracil (U) instead of thymine.
  • A forms a hydrogen bond with T or U
  • C forms a hydrogen bond with the G base, and the relationship between these bases is called'complementary'.
  • mRNA messenger RNA or messenger RNA
  • RNA messenger RNA or messenger RNA
  • diabetes which is an object of treatment or prevention, is a metabolic disorder syndrome characterized by hyperglycemia caused by a lack of insulin hormone or an abnormality in insulin resistance produced by beta cells of the pancreas and further defects of both.
  • IDDM insulin-dependent diabetes
  • NIDDM insulin-independent diabetes
  • diabetes can be divided into insulin-dependent diabetes (IDDM, Type 1) and insulin-independent diabetes (NIDDM, Type 2) caused by impaired insulin resistance and insulin secretion.
  • IDDM insulin-dependent diabetes
  • NIDDM insulin-independent diabetes
  • Type 2 diabetes various complications such as heart disease, bowel disease, eye disease, neurological disease, stroke, etc. occur, which result in chronic neurological disease and cardiovascular disease due to elevated blood sugar and insulin levels for a long time.
  • the short-term hypoglycemic and hyperglycemic reactions cause acute complications.
  • Diabetes as hyperglycemia persists chronically, causes disorders in lipid and protein metabolism as well as sugar metabolism.
  • the conditions are diverse and are directly caused by hyperglycemia, and include diabetic peripheral nerve disorders, diabetic retinopathy, diabetic nephropathy, diabetic cataract, cornea, diabetic arteriosclerosis, etc. in the retina, kidney, nerve, and cardiovascular system.
  • ST8SIA1 modulates the p38MAPK/ERK signaling system in human pancreatic ⁇ cells and prevents apoptosis by inhibiting ST8SIA1 It has been identified that it has a role to play. Therefore, it can be understood that by inhibiting the expression or activity of the ST8SIA1 protein, it is possible to inhibit the occurrence or progression of diabetes by preventing the death of the pancreas, particularly ⁇ cells, due to glucose toxicity.
  • the diabetes in the present invention may be a target of diabetes caused by glucose toxicity or deep or advanced diabetes, and more specifically, selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes. Can be.
  • the term'ST8SIA1' used in the present invention refers to a gene or protein thereof encoding alpha-N-acetylneuraminide alpha-2,8-sialyltransferase. It is a type II membrane protein that moves sialic acid from CMP-sialic acid to GM3 to produce ganglioside GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells.
  • the ST8SIA1 protein (alpha-N-acetylneuraminide alpha-2,8-sialyltransferase) in the present invention may be derived from mammals, preferably derived from humans. Most preferably, the ST8SIA1 protein in the present invention comprises the amino acid sequence of human ST8SIA1 isoform 1 (NP_003025.1) represented by SEQ ID NO: 1 or human ST8SIA1 isoform 2 (NP_001291379.1) represented by SEQ ID NO: 2 Characterized in that (NCBI Genbank accession number in parentheses).
  • the ST8SIA1 protein expression inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, or protein nucleic acid (PNA) that complementarily binds to ST8SIA1 mRNA.
  • the ST8SIA1 mRNA most preferably contains a nucleotide sequence of human ST8SIA1 mRNA transcript variant 1 (NM_003034.3) represented by SEQ ID NO: 3 or human ST8SIA1 mRNA transcript variant 2 (NM_001304450.1) represented by SEQ ID NO: 4 (NCBI Genbank accession number in parentheses).
  • NM_003034.3 represented by SEQ ID NO: 3
  • human ST8SIA1 mRNA transcript variant 2 NM_001304450.1
  • SEQ ID NO: 4 NCBI Genbank accession number in parentheses
  • The'siRNA small interfering RNA or short interfering RNA or silencing RNA
  • Interference is a short double-stranded RNA that is composed of 20 to 25 nucleotides complementary to a specific site of the target mRNA.
  • an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex and binds to the target mRNA, and the argonaute protein in the RISC complex cleaves and degrades the target mRNA. It suppresses the expression of specific genes through mechanisms such as suppressing the binding of proteins and ribosomes important for protein translation with mRNA.
  • RISC RNA-induced silencing complex
  • the shRNA according to the present invention is a substance that induces RNA interference, and is a molecule having a double-stranded structure in the molecule and a hairpin-like structure by partially containing a palindromic nucleotide sequence in a single-stranded RNA.
  • a shRNA expression plasmid is introduced into a cell and expressed, a 21-23 base pair siRNA is generated by RNase III (ribonuclease III) enzyme called dicer in the cell to induce RNAi.
  • the siRNA or shRNA according to the present invention may be obtained by applying various modifications to improve the stability of oligonucleotides in vivo, impart resistance to nucleases, and reduce non-specific immune responses.
  • the modification of the oligonucleotide is the OH group at the 2′ carbon position of the sugar structure in one or more nucleotides -CH 3 (methyl), -OCH 3 (methoxy), -NH 2 , -F, -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylami Modification by substitution with dooxyethyl; Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from modifications of nucleotide bonds to phosphorothi
  • the ST8SIA1 protein activity inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody, natural extract or synthetic compound that specifically binds to the ST8SIA1 protein.
  • the pharmaceutical composition according to the present invention may be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier for the treatment of diabetes.
  • the carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes.
  • it is formulated using a transfection reagent, and can be delivered to cells using subcutaneous, blood, bone marrow, or abdominal cavity.
  • the pharmaceutical composition according to the present invention may be administered to a patient in a pharmaceutically effective amount, that is, in an amount sufficient to prevent diabetes or alleviate and treat symptoms.
  • a typical daily dosage may be administered in the range of about 0.01 to 1000 mg/kg, and preferably, it may be administered in the range of about 1 to 100 mg/kg.
  • the pharmaceutical composition of the present invention can be administered in one or several divided doses within a preferred dosage range.
  • the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, age, sex, weight, individual difference and disease state.
  • the route of administration may be administered orally or parenterally.
  • Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or pancreatic It may be intravenous administration, but is not limited thereto, and most preferably it is administered directly to the pancreas.
  • powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier for oral administration. It can be formulated in the form of such as.
  • suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.
  • the pharmaceutical composition of the present invention when administered parenterally, can be formulated according to a method known in the art in the form of injections, transdermal administrations and nasal inhalants together with a suitable parenteral carrier.
  • the pharmaceutical composition can be administered by any device capable of moving the active substance to the target cell.
  • Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections or instillations.
  • injectables include aqueous solvents such as physiological saline or Ringel's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin).
  • aqueous solvent etc.
  • stabilizers for preventing deterioration e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.
  • emulsifiers e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.
  • emulsifiers e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.
  • emulsifiers e.g., buffers for pH adjustment
  • Pharmaceutical carriers such as preservatives for inhibiting the growth of microorganisms (eg, phenyl mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.
  • suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil.
  • suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used.
  • the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
  • transdermal administration ointments, creams, lotions, gels, external solutions, pasta, liniment, air rolls, and the like are included.
  • transdermal administration means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin.
  • the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle or applying it directly to the skin. These formulations are described in prescriptions generally known in pharmaceutical chemistry.
  • the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray.
  • the dosage unit can be determined by providing a valve that delivers a metered amount.
  • gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.
  • compositions according to the present invention include one or more buffers (e.g., saline or PBS), carbohythrate (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.
  • buffers e.g., saline or PBS
  • carbohythrate e.g., glucose, mannose, sucrose or dextran
  • antioxidants e.g., bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.
  • bacteriostatic agents e.g., EDTA or glutathione
  • adjuvants
  • compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
  • composition of the present invention may be administered alone or may be administered in combination with a known compound that protects pancreatic cells and has an effect of treating diabetes.
  • (c) It provides a diabetes treatment screening method comprising the step of selecting a test substance that reduces the ST8SIA1 gene expression level compared to the control cells as a diabetes treatment candidate substance.
  • the step (a) is a step of contacting a cell expressing the ST8SIA1 gene in order to confirm whether the test substance to be analyzed has an activity to inhibit the expression of the ST8SIA1 gene.
  • 'contacting' is in a general sense, and combining two or more agents (eg, two polypeptides), or combining an agent and cells (eg, proteins and cells).
  • agents eg, two polypeptides
  • cells eg, proteins and cells.
  • Contact can occur in vitro. For example, combining two or more agents in a test tube or other container, or combining a test agent with cells or cell lysates and a test agent. Contact can also occur in cells or in situ.
  • two polypeptides are brought into contact in a cell or cell lysate by coexpressing a recombinant polynucleotide encoding two polypeptides in a cell.
  • a protein chip or a protein array in which the protein to be tested is arranged on the surface of a stationary bed may be used.
  • the'test substance' can be used interchangeably with a test agent or agent, and any substance, molecule, element, compound ( Includes a compound, entity, or combination thereof. Examples include proteins, polypeptides, small organic molecules, polysaccharides, and polynucleotides. It may also be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances.
  • Test formulations can be synthetic or natural. The test formulations can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Combinatorial libraries can be produced with several types of compounds that can be synthesized in a step-by-step manner.
  • Compounds of multiple combinatorial libraries can be prepared by the encoded synthetic libraries (ESL) method (WO 95/12608, WO93/06121, WO 94/08051, WO 95/395503 and WO 95/30642).
  • Peptide libraries can be prepared by the phage display method (WO91/18980).
  • Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field.
  • Known pharmacological agents can be directed or applied to a random chemical modification, such as acylation, alkylation, esterification, amidification, to prepare structural analogues.
  • the test agent may be a naturally occurring protein or fragment thereof. Such test formulations can be obtained from natural sources, such as cell or tissue lysates. Libraries of polypeptide preparations can be produced, for example, by conventional methods or obtained from commercially available cDNA libraries.
  • the test agent may be a peptide, such as a peptide having about 5 to 30 amino acids, preferably about 5 to 20, more preferably about 7 to 15 amino acids.
  • the peptide may be a naturally occurring protein, a random peptide, or a cut product of a'biased' random peptide.
  • the test agent may be a nucleic acid.
  • the nucleic acid test agent can be a naturally occurring nucleic acid, a random nucleic acid, or a'biased' random nucleic acid.
  • cuts of prokaryotic or eukaryotic genomes can be used analogously to those described above.
  • test agent may be a small molecule (eg, a molecule having a molecular weight of about 1,000 Da or less).
  • a high throughput assay may be preferably applied to a method for screening a small molecule modulating agent. Many assays are useful for this screening.
  • expression means that a protein or nucleic acid is produced in a cell.
  • the cell expressing ST8SIA1 may be a cell that internally expresses ST8SIA1, or may be a cell transformed with a recombinant expression vector containing a polynucleotide encoding ST8SIA1 to overexpress ST8SIA1.
  • the cells expressing the ST8SIA1 gene may be pancreatic beta cells or cells derived from pancreatic beta cells.
  • the present inventors have used the IPCs cell line derived from human pancreatic beta cells as cells that internally express ST8SIA1.
  • the step (b) is a step of measuring the gene expression level of ST8SIA1 in cells expressing ST8SIA1 contacted with the test substance and cells expressing ST8SIA1 without contact with the test substance.
  • the method of measuring the expression level of the ST8SIA1 gene may be performed by measuring the mRNA or protein level of ST8SIA1.
  • RT-PCR reverse transcription polymerase chain reaction
  • competitive RT-PCR competitive RT-PCR
  • RPA RNase protection assay
  • northern blotting DNA microarray chip
  • RNA sequencing RNA sequencing
  • methods for measuring the expression level of proteins can be used without limitation, methods known in the art, such as western blotting, dot blotting, enzyme-linked immunosorbent assay. , Radioactive Immunoassay (RIA), Radioimmune Diffusion Method, Ouchteroni Immunity Diffusion Method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS) or protein chip method, etc.
  • RIA Radioactive Immunoassay
  • FACS flow cytometry
  • FACS flow cytometry
  • the step (c) is a step of selecting a test substance that reduces the expression level of the ST8SIA1 gene compared to the control cells as a candidate substance for treating diabetes.
  • the test substance that reduces the expression level of the ST8SIA1 gene reduces the protein level and the activity level of the protein of ST8SIA1 to prevent apoptosis caused by glucose toxicity in pancreatic ⁇ cells, and thus delay or symptomatic diabetes caused by this. It can be seen that it can alleviate.
  • compositions for preventing or treating diabetes comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
  • IPCs insulin producing cells
  • the method of producing the'ST8SIA1 gene-deleted insulin secreting cell' of the present invention is not limited thereto, but may include the following steps:
  • step (b) transfecting the exosomes isolated in step (a) with an antibody that specifically binds to ST8SIA1;
  • step (c) transfecting the exosomes prepared in step (c) into insulin secreting cells.
  • the exosome can serve as a carrier for transporting the protein of interest to the target cells or tissues of the target protein, including the protein of interest (antibody in the present invention) inside.
  • the protein of interest can be used to delete a specific gene (suppress the expression or activity of a gene) by acting on a target cell or tissue.
  • the exosomes are isolated from insulin-secreting cells, the ST8SIA1 antibody is transfected into the exosomes, and then the prepared ST8SIA1 antibody-transfected exosomes are transfected into the insulin-secreting cells. Expression or activity of the protein was knocked out. Deleting the ST8SIA1 gene of insulin-secreting cells using exosomes was the first discovery by the present inventors.
  • the insulin-secreting cells from which the ST8SIA1 gene is knocked-out when administered or transplanted to an individual, insulin secretion is increased, so that the blood sugar in the individual decreases and the ability to control blood glucose level is very excellent. there was.
  • The'insulin secreting cells' may be differentiated from stem cells, but are not limited thereto.
  • the method of differentiating insulin-secreting cells from stem cells is not limited as long as it is known, for example, Soria et al. [Soria B, Roche E, Berna G and Leon-Quinto T (2000) Insulin-secreting cells derived from embryonic stem. cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157-162], Assady et al. [Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, and Tzukerman M. (2001) Insulin production by human embryonic stem cells.
  • the stem cells are undifferentiated cells having the ability to differentiate into various body tissues, which are classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells. Can be.
  • Stem cells in the present invention may be adult stem cells, embryonic stem cells, mesenchymal stem cells, tumor stem cells, or induced pluripotent stem cells, depending on their origin or type.
  • the term'mesenchymal stem cell (MSC)' as used herein has the ability to differentiate into various mesenchymal cells including bone, cartilage, fat, muscle cells, or ectodermal cells such as neurons. It is a multipotent stem cell.
  • the mesenchymal stem cells may preferably be derived from those selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic membrane, deciduous membrane, and placenta.
  • the mesenchymal stem cells may be derived from humans, fetuses, or mammals other than humans.
  • the mammals other than humans are more preferably canine, feline, monkey, cow, sheep, pig, horse, rat, mouse, guinea pig, and the like, and the origin is not limited.
  • the active ingredient is a concept including a cell culture medium containing the cells, a concentrate of a cell culture, and the like.
  • the present invention provides a use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
  • the present invention provides a method for treating diabetes, comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  • The'effective amount' of the present invention refers to an amount that improves, treats, prevents, detects, diagnoses, or inhibits or reduces diabetes when administered to an individual, and the'individual' refers to an animal, preferably a mammal , In particular, it may be an animal, including humans, and may be cells, tissues, organs, etc. derived from animals. The individual may be a patient in need of the effect.
  • The'treatment' of the present invention refers generically to improving the symptoms of diabetes or diabetes, which may include curing, substantially preventing, or improving the condition, as long as it originates from the disease. It includes, but is not limited to, alleviating, curing or preventing the symptoms or most of the symptoms.
  • the term'comprising' is used in the same manner as'containing' or'as a feature', and does not exclude additional component elements or method steps that are not mentioned in the composition or method. .
  • the term'consisting of' is used in the same way as'consisting of', and means excluding additional elements, steps, or ingredients that are not separately described.
  • the term'essentially consisting of' or'essentially consisting of' means, in the scope of a composition or method, a component element or step that does not substantially affect its basic properties in addition to the described component elements or steps. Means to include.
  • the present invention can prevent apoptosis by controlling the expression of related factors upon inhibition of ST8SIA1 in hyperglycemic conditions, decrease insulin resistance and increase sensitivity through increased tyrosine phosphorylation, and increase insulin secretion due to ST8SIA1 inhibition. It can be usefully used to develop a therapeutic agent for diabetes with a completely new mechanism of action that inhibits the expression or activity of the ST8SIA1 protein.
  • ST8SIA1 plays a role in promoting apoptosis in pancreatic beta cells under hyperglycemic conditions, lowering the activity of ST8SIA1 inhibits apoptosis caused by hyperglycemia and has the effect of protecting pancreatic beta cells.
  • antidiabetic candidate substances can be selected by screening for agents that inhibit activity.
  • insulin secretion is increased, so that blood sugar in the individual decreases and the ability to regulate blood glucose level is very excellent.
  • Figure 3 shows the change in the amount of insulin secretion when ST8SIA1 is inhibited in IPCs cells. PBS was used as a control.
  • FIG. 4 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
  • 5 shows the results of measuring insulin sensitivity by dividing 9-week-old db/db mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
  • Figure 6 is a 9-week-old db/db mice divided by 8 per group, and ST8SIA1KO-IPCs were transplanted at 1 week of breeding, and glucose tolerance was measured.
  • Figure 7 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group, using anti-ST8SIA1-Ab as an adjuvant with 0.5% saponin and 100 ul of saponin at 1 week.
  • IPCs cells Insulin secreting cell line IPCs cells were differentiated from mesenchymal stem cells.
  • Glucose toxicity was induced by treatment with 33 mM glucose for 48 hours. Then, 10 ⁇ M ST8SIA1 antibody (ST pharm) was treated for 48 hours.
  • Cells were lysed using RIPA buffer (50mMTris-HCl, 150mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche Diagnostics).
  • the protein composition of the cell lysate was quantified with a BCA protein assay kit (Roche Diagnostics), and then the cell lysate containing the same amount of protein was separated by SDS-PAGE. Then, the protein was transferred to Immobilon NC membranes. Blocking was performed for 1 hour with TBS and Tween 20 solution (0.05% Tween 20) containing 5% skim milk or 5% bovine albumin serum (BSV).
  • BD-MSC bone marrow derived mesenchyma stem cells
  • IPCs insulin producing cells
  • mice 9-week-old db/db mice were reared by dividing 8 mice per group. At 1 week of breeding, 3x10 6 ST8SIA1KO-IPCs were transplanted into the mouse pancreas.
  • Exosomes from 10 ml of IPCs culture media using ExoQuick kt (SBI bio). Exosomes in 500ul of sterile PBS are approximately 50-300ug.
  • 10ul Exo-Fect solution, 10ul PBS solution, 50ul purified exosomes, and ST8SIA1 antibody (20ul) were put together and mixed well by repeating 3 times.
  • the mixed exosome transfection solution was incubated with a shaker at 37° C. for 1 hour and immediately placed the tube on ice. Transfect 30ul of ExoQuickTC reagent into the exosome sample suspension, mix well and mix 6 times. Again, the transfection sample is stabilized on ice or at 4° C.
  • the sample was centrifuged for 3 minutes at 13,000-14,000 rpm in a microfuge (maximum speed), the supernatant was removed, and the transfected exosome pellet was released in 300ul 1x PBS.
  • Example 1 Effect of the expression level of ST8SIA1 on apoptosis
  • IPCs ST8SIA1KO-IPCs cells transfected with exosomes were cultured for 2 days under conditions of normal glucose (NG) or high glucose (HG: 33 mM), respectively.
  • NG normal glucose
  • HG high glucose
  • ⁇ -actin was used as a protein loading control.
  • the expression of ST8SIA1 was inhibited with the ST8SIA1 antibody under high glucose conditions and the expression level of cleaved caspase 3 was measured.
  • Diabetes is known to be mainly caused by an abnormality in sub-receptor signaling, especially inhibition of IRS-1 (insulin receptor substrate-1)-associated PI3K activity, rather than a defect in the insulin receptor (IR) itself.
  • IRS-1 insulin receptor substrate-1
  • This IRS-1-associated PI3K activity is regulated by the tyrosine phosphorylation of IRS-1 or the amount of IRS-1 protein itself, and a decrease in the amount of tyrosine phosphorylation protein of IRS-1 is observed in various diabetic animal models or diabetic environments.
  • ST8SIA1KO-IPCs cells were not treated with or treated with 100 nM insulin for 10 minutes to measure tyrosine phosphorylation by Western blot.
  • the ST8SIA1KO-IPCs group showed that blood sugar was rapidly decreased to and maintained at a normal level, whereas in the control group, it was confirmed that the blood sugar was continuously increased or increased.
  • Example 4 Effect on insulin sensitivity and glucose tolerance
  • IPGTT oral glucose tolerance test
  • glucose (dosage: 2g/kg body weight) was administered intraperitoneally (IPGTT) at 10 weeks of age.
  • Glucose levels were monitored at 60 and 120 minutes after testing by the tail bleed method.
  • insulin resistance test insulin (0.5U/kg body weight) was administered by intraperitoneal injection at 14 weeks of age. Glucose levels were monitored at 10, 20, 40 and 60 minutes after testing by tail bleed method.
  • the present invention can screen an agent that inhibits the expression or activity of ST8SIA1 to screen for antidiabetic candidate substances, and when the insulin-secreting cells from which the ST8SIA1 gene is knocked-out is administered or transplanted to an individual, insulin secretion is increased. As a result, the blood sugar in the individual decreases and the ability to control blood sugar levels is very good, so it has industrial applicability.

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Abstract

The present invention relates to a composition for preventing or treating diabetes by using ST8SIA1, and a method for screening for antidiabetic agents, and, more specifically, to: a composition for preventing or treating diabetes, containing, as an active ingredient, a ST8SIA1 protein expression or activity inhibitor or an insulin-secreting cell in which a ST8SIA1 gene is deleted; and a method for screening for antidiabetic agents.

Description

ST8SIA1를 이용한 당뇨병의 예방 또는 치료용 조성물과 당뇨병 치료제 스크리닝 방법Composition for preventing or treating diabetes using ST8SIA1 and screening method for diabetes treatment
본 출원은 2019년 3월 25일에 출원된 대한민국 특허출원 제10-2019-0033947호를 우선권으로 주장하고, 상기 명세서 전체는 본 출원의 참고문헌이다.This application claims priority to Korean Patent Application No. 10-2019-0033947 filed on March 25, 2019, and the entire specification is a reference to this application.
본 발명은 ST8SIA1를 이용한 당뇨병의 예방 또는 치료용 조성물과 당뇨병 치료제 스크리닝 방법에 관한 것으로, 보다 상세하게는 ST8SIA1 단백질의 발현 또는 활성 억제제 또는 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물과 (a) 시험 물질을 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계; (b) 상기 세포와 시험 물질을 접촉하지 않은 대조군 세포에서 ST8SIA1 유전자 발현 수준을 측정하는 단계; 및 (c) 대조군 세포와 비교하여 ST8SIA1 유전자 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계를 포함하는 당뇨병 치료제 스크리닝 방법에 대한 것이다. The present invention relates to a composition for preventing or treating diabetes using ST8SIA1 and a method for screening a diabetes therapeutic agent, and more particularly, to a diabetes mellitus comprising as an active ingredient an inhibitor of the expression or activity of the ST8SIA1 protein or insulin-secreting cells in which the ST8SIA1 gene is deleted. Contacting the prophylactic or therapeutic pharmaceutical composition and (a) a test substance to cells expressing the ST8SIA1 gene; (b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And (c) selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.
당뇨에서 글루코스 독성은 β-세포의 세포사멸(apoptosis)을 야기하고, 췌장을 포함한 다양한 장기 시스템에 영향을 미친다. 그러나 기본적인 메커니즘은 완전하게 알려져 있지는 않다. β-세포의 장애 및 손상된 인슐린 생산은 당뇨의 전형적인 특징이나(Xu, G et al., Nat. Med. 19, 1141-1146. 2013), 당뇨병의 급속한 확산에도 불구하고 β-세포의 세포사멸을 야기하는 글루코스 독성의 정확한 분자적 메커니즘은 여전히 알려지지 않았다(Chen, J et al., Diabetes 57, 938-944. 2008). Glucose toxicity in diabetes causes apoptosis of β-cells and affects various organ systems, including the pancreas. However, the basic mechanism is not fully known. Impairment of β-cells and impaired insulin production are typical features of diabetes (Xu, G et al., Nat. Med. 19, 1141-1146. 2013), but apoptosis of β-cells is prevented despite the rapid spread of diabetes. The exact molecular mechanism of the resulting glucose toxicity is still unknown (Chen, J et al., Diabetes 57, 938-944. 2008).
세포사멸(apoptosis)은 특화된 세포 내 신호 전달이 활성화되어 세포를 소멸시키는 것으로, 체내에서 자연적으로 일어나는 과정이다. 이는 조직에서 세포 개체수를 유지하기 위한 항상성 메커니즘이기도 하다. 부적절한 세포사멸은 근본적으로 허혈성 손상, 자가면역질환 및 많은 종류의 암과 같은 대사성 스트레스, 신경퇴행성 질환, 글루코스 독성을 포함하는 다양한 병리 현상에서 공통적으로 나타난다(Elmore, S et al., Toxicol. Pathol. 35, 495-516. 2007). 이러한 세포사멸은 mitochondria에서 배출된 cytochrome c에 의해 caspase-9, -3을 활성화시키는 intrinsic pathway와, TNF-α(tumor nerosis factor-α)와 Fas ligand 등의 death receptor에 의하여 caspase-8과 -10이 활성화되는 extrinsic pathway의 두 가지 경로로 나뉜다. 이외에도 caspase와는 무관한 다양한 단백의 활성화가 세포사멸 신호전달에 관여하는 것으로 알려져 있다.Apoptosis is a process that naturally occurs in the body by activating specialized intracellular signal transduction to destroy cells. It is also a homeostatic mechanism for maintaining cell populations in tissues. Inadequate apoptosis is fundamentally common in a variety of pathologies, including metabolic stress such as ischemic injury, autoimmune diseases and many types of cancer, neurodegenerative diseases, and glucose toxicity (Elmore, S et al., Toxicol. Pathol. 35, 495-516. 2007). Such apoptosis is caused by intrinsic pathways that activate caspase-9 and -3 by cytochrome c released from mitochondria, and caspase-8 and -10 by death receptors such as TNF-α (tumor nerosis factor-α) and Fas ligand. This activation is divided into two pathways of the extrinsic pathway. In addition, it is known that activation of various proteins independent of caspase is involved in apoptosis signaling.
한편, ST8SIA1 유전자는 알파-N-아세틸 뉴라민니드 알파-2,8-시알릴 트랜스퍼라제라는 효소를 암호화하는 유전자로, 상기 효소는 시알산을 CMP-sialic acid에서 GM3로 이동시켜 갱글리오사이드 (Ganglioside) GD3과 GT3을 생산하는 II형 막 단백질이다. 갱글리오사이드 GD3는 배양된 악성 세포의 세포 부착 및 성장에 중요하다고 알려져 있다. ST8SIA1 단백질은 흔히 골지체에서 발견될 수 있으며 glycosyltransferase family의 구성원이다. On the other hand, the ST8SIA1 gene is a gene that encodes an enzyme called alpha-N-acetyl neuraminide alpha-2,8-sialyl transferase, and the enzyme transfers sialic acid from CMP-sialic acid to GM3 to ganglioside ( Ganglioside) is a type II membrane protein that produces GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells. The ST8SIA1 protein can often be found in the Golgi apparatus and is a member of the glycosyltransferase family.
하지만, 현재까지, 본 발명과 같이 ST8SIA1가 당뇨 치료의 타겟으로 작용할 수 있는 점은 보고된 바가 없다.However, until now, it has not been reported that ST8SIA1 can act as a target of diabetes treatment as in the present invention.
이에 본 발명자들은 새로운 당뇨병 치료법을 개발하기 위하여 연구한 결과, ST8SIA1가 췌장 베타 세포의 세포사멸, 특히 당뇨병 등에 의해서 초래되는 고글루코스 상태에서 췌장 베타 세포의 세포사멸을 억제하는 기능이 있고, 이를 저해한 인슐린 분비 세포에 의해서 체내 인슐린 분비량이 증가되고, 혈당이 정상적으로 조절되는 것을 확인하여 본 발명을 완성하였다. Accordingly, the inventors of the present invention have studied to develop a new diabetes treatment, and as a result, ST8SIA1 has a function of inhibiting apoptosis of pancreatic beta cells, particularly in a high glucose state caused by diabetes, and inhibiting this. The present invention was completed by confirming that the amount of insulin secreted in the body is increased by the insulin secreting cells and that blood sugar is normally regulated.
따라서 본 발명의 목적은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
또한, ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
또한 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 필수적으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for preventing or treating diabetes consisting essentially of an inhibitor of the expression or activity of the ST8SIA1 protein as an active ingredient.
본 발명의 다른 목적은Another object of the present invention
(a) 시험 물질을 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계;(a) contacting the test substance with cells expressing the ST8SIA1 gene;
(b) 상기 세포와 시험 물질을 접촉하지 않은 대조군 세포에서 ST8SIA1 유전자 발현 수준을 측정하는 단계; 및(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And
(c) 대조군 세포와 비교하여 ST8SIA1 유전자 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계를 포함하는 당뇨병 치료제 스크리닝 방법을 제공하는 것이다. (c) To provide a diabetes treatment screening method comprising the step of selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.
본 발명의 또 다른 목적은 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
또한 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for preventing or treating diabetes comprising the insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
또한 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 필수적으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.In addition, it is to provide a pharmaceutical composition for preventing or treating diabetes consisting essentially of insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
본 발명의 또 다른 목적은 당뇨병의 예방 또는 치료용 제제를 제조하기 위한 ST8SIA1 단백질의 발현 또는 활성 억제제의 용도를 제공하는 것이다.Another object of the present invention is to provide the use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
본 발명의 또 다른 목적은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 당뇨병의 치료 방법을 제공하는 것이다.Another object of the present invention is to provide a method for treating diabetes, comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
상기와 같은 목적을 달성하기 위하여, 본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
또한, 본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
또한, 본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 필수적으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다. In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes consisting essentially of an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
본 발명의 다른 목적을 달성하기 위하여, 본 발명은In order to achieve another object of the present invention, the present invention
(a) 시험 물질을 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계;(a) contacting the test substance with cells expressing the ST8SIA1 gene;
(b) 상기 세포와 시험 물질을 접촉하지 않은 대조군 세포에서 ST8SIA1 유전자 발현 수준을 측정하는 단계; 및(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And
(c) 대조군 세포와 비교하여 ST8SIA1 유전자 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계를 포함하는 당뇨병 치료제 스크리닝 방법을 제공하는 것이다. (c) To provide a diabetes treatment screening method comprising the step of selecting a test substance that decreases the ST8SIA1 gene expression level compared to the control cells as a candidate substance for the treatment of diabetes.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다.In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
또한, 본 발명은 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
또한, 본 발명은 ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 필수적으로 이루어지는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating diabetes consisting essentially of insulin-secreting cells in which the ST8SIA1 gene is deleted as an active ingredient.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 당뇨병의 예방 또는 치료용 제제를 제조하기 위한 ST8SIA1 단백질의 발현 또는 활성 억제제의 용도를 제공한다.In order to achieve another object of the present invention, the present invention provides a use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
본 발명의 또 다른 목적을 달성하기 위하여, 본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 당뇨병의 치료 방법을 제공한다.In order to achieve another object of the present invention, the present invention provides a method for treating diabetes comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
이하 본 발명을 상세히 설명한다. Hereinafter, the present invention will be described in detail.
본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
본 명세서에서 '치료'는 질환의 발생 또는 재발 억제, 증상의 완화, 질병의 직접 또는 간접적인 병리학적 결과의 감소, 질병 진행 속도의 감소, 질병 상태의 개선, 호전, 완화 또는 개선된 예후를 의미한다. 본 발명에서 사용되는 용어 '예방'은 질환의 발병을 억제시키거나 진행을 지연시키는 모든 행위를 의미한다.In the present specification,'treatment' refers to suppression of occurrence or recurrence of a disease, alleviation of symptoms, reduction of direct or indirect pathological consequences of a disease, reduction of disease progression rate, improvement of disease state, improvement, alleviation or improved prognosis do. The term'prevention' as used in the present invention means any action that suppresses the onset or delays the progression of a disease.
'단백질'은 '폴리펩타이드(polypeptide)' 또는 '펩타이드(peptide)'와 호환성 있게 사용되며, 예컨대, 자연 상태의 단백질에서 일반적으로 발견되는 바와 같이 아미노산 잔기의 중합체를 말한다. FGF12 단백질의 단편(fragment)'은 FGF12 단백질의 일부분의 펩타이드를 말한다. 'Protein' is used interchangeably with'polypeptide' or'peptide', for example, refers to a polymer of amino acid residues as commonly found in proteins in nature. A fragment of the FGF12 protein refers to a peptide of a portion of the FGF12 protein.
본 발명에서 '폴리뉴클레오티드(polynucleotide)' 또는 '핵산'은 단일 또는 이중 가닥의 형태로 된 데옥시리보핵산(deoxyribonucleic acid, DNA) 또는 리보핵산(ribonucleic acid, RNA)를 말한다. 다른 제한이 없는 한, 자연적으로 생성되는 뉴클레오티드와 비슷한 방법으로 핵산에 혼성화되는 자연적 뉴클레오티드의 공지된 아날로그도 포함된다. 일반적으로 DNA는 아데닌(adenine, A), 구아닌(guanine, G), 시토신(cytosine, C), 티민(thymine, T) 등 네 가지 염기로 구성되어 있으며, RNA는 티민 대신 우라실(Uracil, U)을 가지고 있다. 핵산 이중 가닥에서 A는 T 또는 U, C는 G 염기와 수소결합을 이루는데, 이러한 염기의 관계를 '상보적(complementary)'이라고 한다.In the present invention,'polynucleotide' or'nucleic acid' refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in the form of single or double strands. Unless otherwise limited, also include known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to those of naturally occurring nucleotides. In general, DNA is composed of four bases: adenine (A), guanine (G), cytosine (C), and thymine (T), and RNA is composed of uracil (U) instead of thymine. Have. In the double-stranded nucleic acid, A forms a hydrogen bond with T or U, and C forms a hydrogen bond with the G base, and the relationship between these bases is called'complementary'.
한편, 'mRNA(messenger RNA 또는 전령 RNA)'는 단백질 합성 과정에서 특정 유전자의 염기서열의 유전 정보를 리보솜(ribosome)으로 전달하여 폴리펩티드 합성(단백질 번역, translation)의 청사진 역할을 하는 RNA이다. 유전자를 주형(template)으로 하여 단일 가닥의 mRNA가 전사(transcription) 과정을 통하여 합성된다.On the other hand,'mRNA (messenger RNA or messenger RNA)' is an RNA that serves as a blueprint for polypeptide synthesis (protein translation, translation) by transferring the genetic information of the nucleotide sequence of a specific gene to the ribosome during protein synthesis. Using the gene as a template, single-stranded mRNA is synthesized through a transcription process.
본 발명에서 치료 또는 예방의 대상인 '당뇨병'은 췌장의 베타 세포에서 생성되는 인슐린 호르몬 부족 또는 인슐린 저항성의 이상과 나아가 이러한 두 가지 모두의 결함으로 발생하는 고혈당을 특징으로 하는 대사장애증후군이다. 이러한 당뇨병은 인슐린 의존형 당뇨병(IDDM, Type 1)과 인슐린 저항 및 인슐린 분비 손상에 의해 발생하는 인슐린 비의존형 당뇨병(NIDDM, Type 2)으로 나눌 수 있다. 제1형과 제2형 당뇨병 모두에서 심장 질환, 장 질환, 안과 질환, 신경 질환, 뇌졸중 등과 같은 다양한 합병증이 발생하게 되는데, 이는 장시간 동안 혈당과 인슐린 수준이 상승하여 만성신경질환과 심혈관질환이 발생하게 되고 단시간의 저혈당과 고혈당 반응으로 급성 합병증을 야기시키게 되는 것이다. 당뇨병은 고혈당이 만성으로 지속되면서 당질 대사뿐만 아니라 지질 및 단백질 대사 장애도 함께 일으킨다. 그 병태는 다양하며 직접 고혈당에 기인하는 것으로 망막, 신장, 신경, 심혈관계 등에서 당뇨병성 말초신경장해, 당뇨병성 망막증, 당뇨병성 신증, 당뇨병성 백내장, 각막증, 당뇨병성 동맥경화증 등이 있다. In the present invention,'diabetes', which is an object of treatment or prevention, is a metabolic disorder syndrome characterized by hyperglycemia caused by a lack of insulin hormone or an abnormality in insulin resistance produced by beta cells of the pancreas and further defects of both. These diabetes can be divided into insulin-dependent diabetes (IDDM, Type 1) and insulin-independent diabetes (NIDDM, Type 2) caused by impaired insulin resistance and insulin secretion. In both type 1 and type 2 diabetes, various complications such as heart disease, bowel disease, eye disease, neurological disease, stroke, etc. occur, which result in chronic neurological disease and cardiovascular disease due to elevated blood sugar and insulin levels for a long time. The short-term hypoglycemic and hyperglycemic reactions cause acute complications. Diabetes, as hyperglycemia persists chronically, causes disorders in lipid and protein metabolism as well as sugar metabolism. The conditions are diverse and are directly caused by hyperglycemia, and include diabetic peripheral nerve disorders, diabetic retinopathy, diabetic nephropathy, diabetic cataract, cornea, diabetic arteriosclerosis, etc. in the retina, kidney, nerve, and cardiovascular system.
당뇨병을 일으키고 심화시키는 중요한 병리 현상의 하나는 고혈당에 의한 췌장 β세포의 사멸이다. 본 발명자들은 혈당이 높아진 상태에서 췌장을 비롯한 다양한 장기에 영향을 미치는 글루코스 독성(glucose toxicity 또는 glucotoxicity)에서 ST8SIA1이 인간의 췌장 β세포에서 p38MAPK/ERK 신호체계를 조절하고 ST8SIA1을 억제함으로써 세포사멸을 방어하는 역할을 하는 것을 규명하였다. 따라서 ST8SIA1 단백질의 발현 또는 활성을 억제함으로써 글루코스 독성에 의한 췌장, 특히 β세포의 사멸을 방지하여 당뇨병의 발생 또는 진행을 억제할 수 있음을 이해할 수 있다. One of the important pathologies that cause and deepen diabetes is the death of pancreatic β-cells due to hyperglycemia. In the present inventors, in the glucose toxicity (glucose toxicity or glucotoxicity) that affects various organs including the pancreas in a state of elevated blood sugar, ST8SIA1 modulates the p38MAPK/ERK signaling system in human pancreatic β cells and prevents apoptosis by inhibiting ST8SIA1 It has been identified that it has a role to play. Therefore, it can be understood that by inhibiting the expression or activity of the ST8SIA1 protein, it is possible to inhibit the occurrence or progression of diabetes by preventing the death of the pancreas, particularly β cells, due to glucose toxicity.
이에 따라, 본 발명에서의 당뇨병은 글루코스 독성에 의하여 유발되는 당뇨병 또는 심화 또는 진행된 당뇨병이라면 그 대상이 될 수 있으며, 보다 구체적으로는 제1형 당뇨병, 제2형 당뇨병 및 임신성 당뇨병으로 이루어진 군에서 선택된 것일 수 있다. Accordingly, the diabetes in the present invention may be a target of diabetes caused by glucose toxicity or deep or advanced diabetes, and more specifically, selected from the group consisting of type 1 diabetes, type 2 diabetes, and gestational diabetes. Can be.
본 발명에서 사용된 용어 'ST8SIA1'는 알파-N-아세틸뉴라미니드 알파-2,8-시아릴 전달효소 (alpha-N-acetylneuraminide alpha-2,8-sialyltransferase)를 암호화하는 유전자 또는 이의 단백질로 시알산을 CMP-sialic acid에서 GM3로 이동시켜 갱글리오사이드 (Ganglioside) GD3과 GT3을 생산하는 II형 막 단백질이다. 갱글리오사이드 GD3는 배양된 악성 세포의 세포 부착 및 성장에 중요하다고 알려져 있다.The term'ST8SIA1' used in the present invention refers to a gene or protein thereof encoding alpha-N-acetylneuraminide alpha-2,8-sialyltransferase. It is a type II membrane protein that moves sialic acid from CMP-sialic acid to GM3 to produce ganglioside GD3 and GT3. Ganglioside GD3 is known to be important for cell adhesion and growth of cultured malignant cells.
본 발명에서의 ST8SIA1 단백질 (alpha-N-acetylneuraminide alpha-2,8-sialyltransferase)은 포유류에서 유래한 것일 수 있으며, 바람직하게는 인간에서 유래한 것이다. 가장 바람직하게, 본 발명에서의 ST8SIA1 단백질은 서열번호 1로 표시되는 인간의 ST8SIA1 isoform 1(NP_003025.1) 또는 서열번호 2로 표시되는 인간의 ST8SIA1 isoform 2(NP_001291379.1)의 아미노산 서열을 포함하는 것을 특징으로 한다(괄호 안은 NCBI Genbank accession number). The ST8SIA1 protein (alpha-N-acetylneuraminide alpha-2,8-sialyltransferase) in the present invention may be derived from mammals, preferably derived from humans. Most preferably, the ST8SIA1 protein in the present invention comprises the amino acid sequence of human ST8SIA1 isoform 1 (NP_003025.1) represented by SEQ ID NO: 1 or human ST8SIA1 isoform 2 (NP_001291379.1) represented by SEQ ID NO: 2 Characterized in that (NCBI Genbank accession number in parentheses).
상기 ST8SIA1 단백질의 발현 억제제는 ST8SIA1 mRNA에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide), siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme 또는 PNA(protein nucleic acid)일 수 있다. 또한 상기 ST8SIA1 mRNA는 가장 바람직하게는 서열번호 3으로 표시되는 인간 ST8SIA1 mRNA transcript variant 1(NM_003034.3) 또는 서열번호 4로 표시되는 인간 ST8SIA1 mRNA transcript variant 2(NM_001304450.1)의 염기서열을 포함하는 것이다(괄호 안은 NCBI Genbank accession number). 상기한 인간의 ST8SIA1 mRNA transcript variant들의 coding region(exon) 등의 특징 사항은 괄호 안에 기재된 Genbank accession number로 NCBI 데이터베이스를 검색하여 나오는 서열정보에서 확인된다.The ST8SIA1 protein expression inhibitor may be an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, or protein nucleic acid (PNA) that complementarily binds to ST8SIA1 mRNA. In addition, the ST8SIA1 mRNA most preferably contains a nucleotide sequence of human ST8SIA1 mRNA transcript variant 1 (NM_003034.3) represented by SEQ ID NO: 3 or human ST8SIA1 mRNA transcript variant 2 (NM_001304450.1) represented by SEQ ID NO: 4 (NCBI Genbank accession number in parentheses). Features such as the coding region (exon) of the above-described human ST8SIA1 mRNA transcript variants are identified in the sequence information displayed by searching the NCBI database with the Genbank accession number in parentheses.
상기 'siRNA(small interfering RNA 또는 short interfering RNA 또는 silencing RNA)'는 세포 안에 인위적으로 도입되어 특정 유전자의 mRNA의 분해를 유도하여 단백질 번역(translation)이 나지 않도록 하여 유전자 발현을 억제하는 RNA 간섭(RNA interference) 현상을 일으키는 짧은 이중가닥의 RNA로, 표적 mRNA의 특정 부위에 상보적인 20 내지 25개의 뉴클레오티드로 구성되어 있다. 세포 내에서 siRNA의 이중 가닥 중 표적 mRNA에 상보적인 가닥(antisense strand)이 RISC(RNA-induced silencing complex) 단백질 복합체와 결합하여 표적 mRNA에 결합하고, RISC 복합체 안의 argonaute 단백질이 표적 mRNA를 절단하여 분해시키거나, 단백질 번역에 중요한 단백질과 리보솜이 mRNA와 결합하는 것을 억제하는 등의 기작으로 특정 유전자의 발현을 억제한다. The'siRNA (small interfering RNA or short interfering RNA or silencing RNA)' is artificially introduced into the cell to induce the degradation of the mRNA of a specific gene, thereby preventing protein translation, thereby inhibiting gene expression. Interference) is a short double-stranded RNA that is composed of 20 to 25 nucleotides complementary to a specific site of the target mRNA. Among the double strands of siRNA in cells, an antisense strand complementary to the target mRNA binds to the RNA-induced silencing complex (RISC) protein complex and binds to the target mRNA, and the argonaute protein in the RISC complex cleaves and degrades the target mRNA. It suppresses the expression of specific genes through mechanisms such as suppressing the binding of proteins and ribosomes important for protein translation with mRNA.
본 발명에 따른 shRNA는 RNA 간섭을 유도하는 물질로서, 1본쇄 RNA에서 부분적으로 회문상의 염기서열을 포함함으로써, 분자 내에서 2본쇄 구조를 가지고 헤어핀과 같은 구조가 되는 약 20염기 이상의 분자이다. 또한, shRNA 발현용 플라스미드(plasmid)를 세포 내로 도입시켜 발현하게 되면, 세포 내의 다이서(dicer)라는 RNaseⅢ(ribonucleaseⅢ) 효소에 의해 21~23개 염기쌍의 siRNA가 생성되어 RNAi를 유도한다.The shRNA according to the present invention is a substance that induces RNA interference, and is a molecule having a double-stranded structure in the molecule and a hairpin-like structure by partially containing a palindromic nucleotide sequence in a single-stranded RNA. In addition, when a shRNA expression plasmid is introduced into a cell and expressed, a 21-23 base pair siRNA is generated by RNase III (ribonuclease III) enzyme called dicer in the cell to induce RNAi.
본 발명에 따른 siRNA 또는 shRNA는 올리고뉴클레오티드의 생체 내 안정성 향상, 핵산 분해효소 저항성 부여 및 비특이적 면역반응 감소를 위한 다양한 변형(modification)을 가한 것일 수 있다. 상기 올리고뉴클레오티드의 변형은 하나 이상의 뉴클레오티드 내 당 구조의 2´ 탄소 위치에서 OH기가 -CH 3(메틸), -OCH 3(methoxy), -NH 2, -F, -O-2-메톡시에틸, -O-프로필(propyl), -O-2-메틸티오에틸(methylthioethyl), -O-3-아미노프로필, -O-3-디메틸아미노프로필, -O-N-메틸아세트아미도 또는 -O-디메틸아미도옥시에틸로의 치환에 의한 변형; 뉴클레오티드 내 당(sugar) 구조 내의 산소가 황으로 치환된 변형; 또는 뉴클레오티드결합의 포스포로티오에이트(phosphorothioate) 또는 보라노포스페이트(boranophosphate), 메틸포스포네이트(methyl phosphonate) 결합으로의 변형에서 선택된 하나 이상의 변형이 조합되어 사용될 수 있으며, PNA(peptide nucleic acid), LNA(locked nucleic acid) 또는 UNA(unlocked nucleic acid) 형태로의 변형도 사용이 가능하다.The siRNA or shRNA according to the present invention may be obtained by applying various modifications to improve the stability of oligonucleotides in vivo, impart resistance to nucleases, and reduce non-specific immune responses. The modification of the oligonucleotide is the OH group at the 2′ carbon position of the sugar structure in one or more nucleotides -CH 3 (methyl), -OCH 3 (methoxy), -NH 2 , -F, -O-2-methoxyethyl, -O-propyl, -O-2-methylthioethyl, -O-3-aminopropyl, -O-3-dimethylaminopropyl, -ON-methylacetamido or -O-dimethylami Modification by substitution with dooxyethyl; Modification in which oxygen in the sugar structure in the nucleotide is substituted with sulfur; Alternatively, one or more modifications selected from modifications of nucleotide bonds to phosphorothioate, boranophosphate, and methyl phosphonate bonds may be used in combination, and PNA (peptide nucleic acid), Modification into the form of LNA (locked nucleic acid) or UNA (unlocked nucleic acid) can also be used.
또한 상기 ST8SIA1 단백질 활성 억제제는 ST8SIA1 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 항체, 천연추출물 또는 합성화합물일 수도 있다. In addition, the ST8SIA1 protein activity inhibitor may be a compound, peptide, peptide mimetics, aptamer, antibody, natural extract or synthetic compound that specifically binds to the ST8SIA1 protein.
본 발명에 따른 약학적 조성물은 당뇨병 치료를 위해 약학적으로 허용되는 담체와 함께 당업계에 공지된 방법으로 투여 경로에 따라 다양하게 제형화될 수 있다. 상기 담체로는 모든 종류의 용매, 분산매질, 수중유 또는 유중수 에멀젼, 수성 조성물, 리포좀, 마이크로비드 및 마이크로좀이 포함된다. 바람직하게는 transfection reagent를 이용하여 제형화하고, 피하, 혈액, 골수, 복강 등을 이용 세포에 전달할 수 있다.The pharmaceutical composition according to the present invention may be variously formulated according to the route of administration by a method known in the art together with a pharmaceutically acceptable carrier for the treatment of diabetes. The carrier includes all kinds of solvents, dispersion media, oil-in-water or water-in-oil emulsions, aqueous compositions, liposomes, microbeads and microsomes. Preferably, it is formulated using a transfection reagent, and can be delivered to cells using subcutaneous, blood, bone marrow, or abdominal cavity.
상기 본 발명에 따른 약학적 조성물은 약학적으로 유효한 양, 즉 당뇨병을 예방하거나 증상을 완화하고 치료하기 충분한 양으로 환자에게 투여될 수 있다. 예를 들어 일반적인 1일 투여량으로는 약 0.01 내지 1000㎎/㎏의 범위로 투여될 수 있으며, 바람직하게는, 약 1 내지 100mg/kg의 범위로 투여될 수 있다. 본 발명의 약학적 조성물은 바람직한 투여량 범위 내에서 1회 또는 수회로 분할 투여할 수 있다. 또한 본 발명에 따른 약학적 조성물의 투여량은 투여 경로, 투여 대상, 연령, 성별 체중, 개인차 및 질병 상태에 따라 통상의 기술자가 적절하게 선택할 수 있다. The pharmaceutical composition according to the present invention may be administered to a patient in a pharmaceutically effective amount, that is, in an amount sufficient to prevent diabetes or alleviate and treat symptoms. For example, a typical daily dosage may be administered in the range of about 0.01 to 1000 mg/kg, and preferably, it may be administered in the range of about 1 to 100 mg/kg. The pharmaceutical composition of the present invention can be administered in one or several divided doses within a preferred dosage range. In addition, the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by a person skilled in the art according to the route of administration, the subject of administration, age, sex, weight, individual difference and disease state.
투여 경로로는 경구적 또는 비경구적으로 투여될 수 있다. 비경구적인 투여방법으로는 이에 한정되지는 않으나 정맥 내, 근육 내, 동맥 내, 골수 내, 경막 내, 심장 내, 경피, 피하, 복강 내, 비강 내, 장관, 국소, 설하, 직장, 또는 췌장 내 투여일 수 있으나, 이에 제한되지 않으며, 가장 바람직하게는 췌장으로 직접 투여된다.The route of administration may be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual, rectal, or pancreatic It may be intravenous administration, but is not limited thereto, and most preferably it is administered directly to the pancreas.
본 발명의 약학적 조성물을 경구 투여하는 경우, 적합한 경구 투여용 담체와 함께 당업계에 공지된 방법에 따라 분말, 과립, 정제, 환제, 당의정제, 캡슐제, 액제, 겔제, 시럽제, 현탁액, 웨이퍼 등의 형태로 제형화할 수 있다. 적합한 담체의 예로는 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨 및 말티톨 등을 포함하는 당류와 옥수수 전분, 밀 전분, 쌀 전분 및 감자 전분 등을 포함하는 전분류, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸셀룰로즈 등을 포함하는 셀룰로즈류, 젤라틴, 폴리비닐피롤리돈 등과 같은 충전제가 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있다. 나아가, 상기 약학적 조성물은 항응집제, 윤활제, 습윤제, 향료, 유화제 및 방부제 등을 추가로 포함할 수 있다.In the case of oral administration of the pharmaceutical composition of the present invention, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier for oral administration. It can be formulated in the form of such as. Examples of suitable carriers include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, and starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as celluloses including methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethylcellulose, gelatin, and polyvinylpyrrolidone may be included. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anti-aggregating agent, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent and a preservative.
또한, 비경구적으로 투여하는 경우, 본 발명의 약학적 조성물은 적합한 비경구용 담체와 함께 주사제, 경피투여제 및 비강 흡입제의 형태로 당 업계에 공지된 방법에 따라 제형화될 수 있다. In addition, when administered parenterally, the pharmaceutical composition of the present invention can be formulated according to a method known in the art in the form of injections, transdermal administrations and nasal inhalants together with a suitable parenteral carrier.
또한, 약학적 조성물은 활성 물질이 표적 세포로 이동할 수 있는 임의의 장치에 의해 투여될 수 있다. 바람직한 투여방식 및 제제는 정맥 주사제, 피하 주사제, 피내 주사제, 근육 주사제 또는 점적 주사제 등이다. 주사제는 생리식염액 또는 링겔액 등의 수성 용제, 식물유, 고급 지방산 에스테르 (예를 들어, 올레인산에칠 등), 알코올류(예를 들어, 에탄올, 벤질알코올, 프로필렌글리콜 또는 글리세린 등) 등의 비수성 용제 등을 이용하여 제조할 수 있고, 변질 방지를 위한 안정화제 (예를 들어, 아스코르빈산, 아황산수소나트륨, 피로아황산나트륨, BHA, 토코페롤, EDTA 등), 유화제, pH 조절을 위한 완충제, 미생물 발육을 저지하기 위한 보존제 (예를 들어, 질산페닐수은, 치메로살, 염화벤잘코늄, 페놀, 크레솔, 벤질알코올 등) 등의 약제학적 담체를 포함할 수 있다. In addition, the pharmaceutical composition can be administered by any device capable of moving the active substance to the target cell. Preferred modes of administration and formulations are intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections or instillations. Injectables include aqueous solvents such as physiological saline or Ringel's solution, vegetable oils, higher fatty acid esters (e.g., ethyl oleic acid, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, or glycerin). It can be prepared using an aqueous solvent, etc., and stabilizers for preventing deterioration (e.g., ascorbic acid, sodium hydrogen sulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, Pharmaceutical carriers such as preservatives for inhibiting the growth of microorganisms (eg, phenyl mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.) may be included.
상기 주사제의 경우에는 반드시 멸균되어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올, 폴리올(예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등), 이들의 혼합물 및/또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는, 적합한 담체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS(phosphate buffered saline) 또는 주사용 멸균수, 10% 에탄올, 40% 프로필렌 글리콜 및 5% 덱스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤, 클로로부탄올, 페놀, 소르빈산, 티메로살 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있다. In the case of such injections, they must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for injections include, but are not limited to, water, ethanol, polyol (eg, glycerol, propylene glycol and liquid polyethylene glycol, etc.), a mixture thereof and/or a solvent or dispersion medium containing vegetable oil. I can. More preferably, suitable carriers include isotonic solutions such as Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine or sterile water for injection, 10% ethanol, 40% propylene glycol and 5% dextrose. Etc. can be used. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, and the like may be additionally included. In addition, the injection may further include an isotonic agent such as sugar or sodium chloride in most cases.
경피투여제의 경우, 연고제, 크림제, 로션제, 겔제, 외용액제, 파스타제, 리니멘트제, 에어롤제 등의 형태가 포함된다. 상기에서 '경피투여'는 약학적 조성물을 국소적으로 피부에 투여하여 약학적 조성물에 함유된 유효한 양의 활성성분이 피부 내로 전달되는 것을 의미한다. 예컨대, 본 발명의 약학적 조성물을 주사형 제형으로 제조하여 이를 30 게이지의 가는 주사 바늘로 피부를 가볍게 단자(prick)하거나 피부에 직접적으로 도포하는 방법으로 투여될 수 있다. 이들 제형은 제약 화학에 일반적으로 공지된 처방서에 기술되어 있다. In the case of transdermal administration, ointments, creams, lotions, gels, external solutions, pasta, liniment, air rolls, and the like are included. In the above,'transdermal administration' means that the active ingredient in an effective amount contained in the pharmaceutical composition is delivered into the skin by topically administering the pharmaceutical composition to the skin. For example, the pharmaceutical composition of the present invention may be prepared in an injectable formulation and administered by lightly pricking the skin with a 30 gauge thin injection needle or applying it directly to the skin. These formulations are described in prescriptions generally known in pharmaceutical chemistry.
흡입투여제의 경우, 본 발명에 따라 사용되는 화합물은 적합한 추진제, 예를 들면, 디클로로플루오로메탄, 트리클로로플루오로메탄, 디클로로테트라플루오로에탄, 이산화탄소 또는 다른 적합한 기체를 사용하여, 가압 팩 또는 연무기로부터 에어로졸 스프레이 형태로 편리하게 전달할 수 있다. 가압 에어로졸의 경우, 투약 단위는 계량된 양을 전달하는 밸브를 제공하여 결정할 수 있다. 예를 들면, 흡입기 또는 취입기에 사용되는 젤라틴 캡슐 및 카트리지는 화합물 및 락토오즈 또는 전분과 같은 적합한 분말 기제의 분말 혼합물을 함유하도록 제형화할 수 있다.In the case of inhalation administration, the compounds used according to the invention can be used in pressurized packs or with suitable propellants, for example dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. It can be conveniently delivered from a nebulizer in the form of an aerosol spray. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve that delivers a metered amount. For example, gelatin capsules and cartridges for use in an inhaler or insufflator can be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.
그 밖의 약학적으로 허용되는 담체로는 당업계에 공지되어 있는 것을 참고로 할 수 있다.Other pharmaceutically acceptable carriers may refer to those known in the art.
본 발명에 따른 약학적 조성물은 하나 이상의 완충제(예를 들어, 식염수 또는 PBS), 카보하이트레이트(예를 들어, 글루코스, 만노즈, 슈크로즈 또는 덱스트란), 항산화제, 정균제, 킬레이트화제(예를 들어, EDTA 또는 글루타치온), 아쥬반트(예를 들어, 알루미늄 하이드록사이드), 현탁제, 농후제 및/또는 보존제를 추가로 포함할 수 있다.Pharmaceutical compositions according to the present invention include one or more buffers (e.g., saline or PBS), carbohythrate (e.g., glucose, mannose, sucrose or dextran), antioxidants, bacteriostatic agents, chelating agents (e.g. For example, EDTA or glutathione), adjuvants (eg, aluminum hydroxide), suspending agents, thickening agents and/or preservatives may further be included.
또한, 본 발명의 약학적 조성물은 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 사용하여 제형화될 수 있다. In addition, the pharmaceutical compositions of the present invention may be formulated using methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal.
또한, 본 발명의 약학적 조성물은 단독으로 투여하거나, 췌장 세포를 보호하고 당뇨병 치료의 효과가 있는 공지의 화합물과 병용하여 투여할 수 있다. In addition, the pharmaceutical composition of the present invention may be administered alone or may be administered in combination with a known compound that protects pancreatic cells and has an effect of treating diabetes.
또한 본 발명은 In addition, the present invention
(a) 시험 물질을 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계;(a) contacting the test substance with cells expressing the ST8SIA1 gene;
(b) 상기 세포와 시험 물질을 접촉하지 않은 대조군 세포에서 ST8SIA1 유전자 발현 수준을 측정하는 단계; 및(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And
(c) 대조군 세포와 비교하여 ST8SIA1 유전자 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계를 포함하는 당뇨병 치료제 스크리닝 방법을 제공한다. (c) It provides a diabetes treatment screening method comprising the step of selecting a test substance that reduces the ST8SIA1 gene expression level compared to the control cells as a diabetes treatment candidate substance.
상기 (a) 단계는 분석의 대상인 시험 물질이 ST8SIA1 유전자의 발현을 억제하는 활성이 있는지 확인하기 위하여 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계이다. The step (a) is a step of contacting a cell expressing the ST8SIA1 gene in order to confirm whether the test substance to be analyzed has an activity to inhibit the expression of the ST8SIA1 gene.
본 발명의 방법에서 '접촉(contacting)'은 일반적인 의미이며, 2개 이상의 제제(예를 들어, 2개의 폴리펩티드)를 결합시키거나, 제제와 세포(예를 들어, 단백질과 세포)를 결합시키는 것을 말한다. 접촉은 시험관 내(in vitro)에서 일어날 수 있다. 예컨대, 시험관(test tube) 또는 다른 컨테이너(container)에서 2개 이상의 제제를 결합시키거나 시험 제제와 세포 또는 세포 용해물과 시험 제제를 결합시키는 것이다. 또한 접촉은 세포 또는 인 시투(in situ)에서 일어날 수도 있다. 예컨대, 2개의 폴리펩티드를 암호화하는 재조합 폴리뉴클레오티드를 세포 내에서 공동발현(coexpression)시킴으로써 세포 또는 세포 용해물에서 2개의 폴리펩티드를 접촉시키는 것이다. 또한 테스트하고자 하는 단백질이 고정상의 표면에 배열된 단백질 칩(protein chip)이나 단백질 어레이(protein array)를 이용할 수도 있다.In the method of the present invention,'contacting' is in a general sense, and combining two or more agents (eg, two polypeptides), or combining an agent and cells (eg, proteins and cells). Say. Contact can occur in vitro. For example, combining two or more agents in a test tube or other container, or combining a test agent with cells or cell lysates and a test agent. Contact can also occur in cells or in situ. For example, two polypeptides are brought into contact in a cell or cell lysate by coexpressing a recombinant polynucleotide encoding two polypeptides in a cell. In addition, a protein chip or a protein array in which the protein to be tested is arranged on the surface of a stationary bed may be used.
또한 본 발명의 방법에서 '시험 물질'은 시험 제제(test agent) 또는 제제(agent)와 호환가능하게 사용할 수 있는 것으로, 임의의 물질(substance), 분자(molecule), 원소(element), 화합물(compound), 실재물(entity) 또는 이들의 조합을 포함한다. 예를 들어 단백질, 폴리펩티드, 저분자 유기화합물(small organic molecule), 다당류(polysaccharide), 폴리뉴클레오티드 등을 포함한다. 또한 자연 산물(natural product), 합성 화합물 또는 화학 화합물 또는 2개 이상의 물질의 조합일 수도 있다. In addition, in the method of the present invention, the'test substance' can be used interchangeably with a test agent or agent, and any substance, molecule, element, compound ( Includes a compound, entity, or combination thereof. Examples include proteins, polypeptides, small organic molecules, polysaccharides, and polynucleotides. It may also be a natural product, a synthetic compound or a chemical compound, or a combination of two or more substances.
보다 구체적으로 본 발명의 방법으로 스크리닝할 수 있는 시험 제제는, 폴리펩티드, 베타-턴 유도체(beta-turn mimetics), 다당류, 인지질, 호르몬, 프로스타글란딘, 스테로이드, 방향족 화합물, 헤테로사이클릭 화합물, 벤조디아제핀(benzodiazepines), 올리고머릭 N-치환 글리신(oligomeric N-substituted glycines), 올리고카르바메이트(oligocarbamates), 당류(saccharides), 지방산, 퓨린, 피리미딘 또는 이들의 유도체, 구조적 아날로그 또는 이들의 조합을 포함한다. 시험 제제는 합성 물질 또는 천연물질일 수 있다. 상기 시험 제제는 합성 또는 자연 화합물의 라이브러리를 포함하는 광범위하고 다양한 출처로부터 얻어질 수 있다. 조합(combinatorial) 라이브러리는 스텝-바이-스텝 방식으로 합성될 수 있는 여러 종류의 화합물로 생산될 수 있다. 다수의 조합 라이브러리의 화합물들은 ESL(encoded synthetic libraries) 방법(WO 95/12608, WO93/06121, WO 94/08051, WO 95/395503 및 WO 95/30642)에 의해 제조될 수 있다. 펩티드 라이브러리는 파지 디스플레이 방법(WO91/18980)에 의해 제조될 수 있다. 박테리아, 곰팡이, 식물 및 동물 추출물 형태의 자연 화합물의 라이브러리는 상업적인 출처로부터 얻거나 또는 필드(field)에서 수집될 수 있다. 공지된 약리학적(pharmacological) 제제가 구조적 아날로그를 제조하기 위하여 아실화, 알킬화, 에스테르화 반응(esterification), 아미드화 반응(amidification)과 같이 지시되거나(direct) 무작위한 화학적 수식에 적용될 수 있다.More specifically, test agents that can be screened by the method of the present invention include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins, steroids, aromatic compounds, heterocyclic compounds, benzodiazepines. ), oligomeric N-substituted glycines, oligocarbamates, saccharides, fatty acids, purines, pyrimidines or derivatives thereof, structural analogues, or combinations thereof. Test formulations can be synthetic or natural. The test formulations can be obtained from a wide variety of sources, including libraries of synthetic or natural compounds. Combinatorial libraries can be produced with several types of compounds that can be synthesized in a step-by-step manner. Compounds of multiple combinatorial libraries can be prepared by the encoded synthetic libraries (ESL) method (WO 95/12608, WO93/06121, WO 94/08051, WO 95/395503 and WO 95/30642). Peptide libraries can be prepared by the phage display method (WO91/18980). Libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts can be obtained from commercial sources or collected in the field. Known pharmacological agents can be directed or applied to a random chemical modification, such as acylation, alkylation, esterification, amidification, to prepare structural analogues.
상기 시험 제제는 자연적으로 생성되는 단백질 또는 이의 단편일 수 있다. 이런 시험 제제는 자연 출처(natural source), 예컨대, 세포 또는 조직 용해물로부터 수득될 수 있다. 폴리펩티드 제제의 라이브러리는 예컨대, 통상적인 방법에 의해 생성되거나 상업적으로 입수할 수 있는 cDNA 라이브러리로부터 수득될 수 있다. 상기 시험 제제는 펩티드, 예컨대, 약 5~30개, 바람직하게는 약 5~20개, 보다 바람직하게는 약 7~15개의 아미노산을 가지는 펩티드일 수 있다. 상기 펩티드는 자연적으로 생성되는 단백질, 랜덤 펩티드 또는 '바이어스(biased)' 랜덤 펩티드의 절단물일 수 있다. 또한 상기 시험 제제는 핵산일 수 있다. 핵산 시험 제제는 자연적으로 생성되는 핵산, 랜덤 핵산, 또는 '바이어스(biased)' 랜덤 핵산일 수 있다. 예컨대, 원핵 또는 진핵 게놈의 절단물을 위에서 기재한 바와 유사하게 사용할 수 있다.The test agent may be a naturally occurring protein or fragment thereof. Such test formulations can be obtained from natural sources, such as cell or tissue lysates. Libraries of polypeptide preparations can be produced, for example, by conventional methods or obtained from commercially available cDNA libraries. The test agent may be a peptide, such as a peptide having about 5 to 30 amino acids, preferably about 5 to 20, more preferably about 7 to 15 amino acids. The peptide may be a naturally occurring protein, a random peptide, or a cut product of a'biased' random peptide. In addition, the test agent may be a nucleic acid. The nucleic acid test agent can be a naturally occurring nucleic acid, a random nucleic acid, or a'biased' random nucleic acid. For example, cuts of prokaryotic or eukaryotic genomes can be used analogously to those described above.
또한 상기 시험 제제는 소분자(small molecule; 예를 들어, 약 1,000Da이하의 분자량을 갖는 분자)일 수 있다. 소분자의 조절 제제를 스크리닝하기 위한 방법에는 바람직하게는 고속 분석 어세이(high throughput assay)가 적용될 수 있다. 많은 어세이가 상기 스크리닝에 유용하다.Further, the test agent may be a small molecule (eg, a molecule having a molecular weight of about 1,000 Da or less). A high throughput assay may be preferably applied to a method for screening a small molecule modulating agent. Many assays are useful for this screening.
본 명세서에서 '발현(expression)'이라 함은 세포에서 단백질 또는 핵산이 생성되는 것을 의미한다. 상기 ST8SIA1을 발현하는 세포는 ST8SIA1을 내재적으로 발현하는 세포일 수도 있고, ST8SIA1을 암호화하는 폴리뉴클레오티드를 포함하는 재조합 발현 벡터로 형질전환되어 ST8SIA1을 과발현하는 세포일 수도 있다. 바람직하게는 상기 ST8SIA1 유전자를 발현하는 세포는 췌장 베타 세포(pancreatic β cell) 또는 췌장 베타 세포에서 유래한 세포일 수 있다. 본 발명자들은 ST8SIA1을 내재적으로 발현하는 세포로서 인간의 췌장 베타 세포에서 유래한 IPCs 세포주를 사용한 바 있다. In the present specification, "expression" means that a protein or nucleic acid is produced in a cell. The cell expressing ST8SIA1 may be a cell that internally expresses ST8SIA1, or may be a cell transformed with a recombinant expression vector containing a polynucleotide encoding ST8SIA1 to overexpress ST8SIA1. Preferably, the cells expressing the ST8SIA1 gene may be pancreatic beta cells or cells derived from pancreatic beta cells. The present inventors have used the IPCs cell line derived from human pancreatic beta cells as cells that internally express ST8SIA1.
상기 (b) 단계는 시험 물질을 접촉시킨 ST8SIA1을 발현하는 세포와 시험 물질을 접촉시키지 않은 ST8SIA1을 발현하는 세포에서 ST8SIA1의 유전자 발현 수준을 측정하는 단계이다.The step (b) is a step of measuring the gene expression level of ST8SIA1 in cells expressing ST8SIA1 contacted with the test substance and cells expressing ST8SIA1 without contact with the test substance.
상기 ST8SIA1 유전자의 발현 수준을 측정하는 방법은 ST8SIA1의 mRNA 또는 단백질 수준을 측정하여 실시할 수 있다.The method of measuring the expression level of the ST8SIA1 gene may be performed by measuring the mRNA or protein level of ST8SIA1.
mRNA 발현의 측정은 당업계에서 통상적인 발현 수준 확인 방법을 제한 없이 사용할 수 있으며, 분석 방법의 예로 역전사중합체연쇄반응(reverse transcription polymerase chain reaction, RT-PCR), 경쟁적 RT-PCR(competitive RT-PCR), 실시간 RT-PCR(real-time RT-PCR), RNase 보호 분석법(RPA:RNase protection assay), 노던 블랏팅(northern blotting), DNA 마이크로어레이 칩(microarray chip), RNA 염기서열분석(RNA sequencing) 등이 있으나, 이들에 한정되는 것은 아니다.For the measurement of mRNA expression, conventional methods for determining expression levels in the art can be used without limitation, and examples of analysis methods include reverse transcription polymerase chain reaction (RT-PCR), competitive RT-PCR (competitive RT-PCR). ), real-time RT-PCR, RNase protection assay (RPA), northern blotting, DNA microarray chip, RNA sequencing ), but is not limited to these.
또한 단백질의 발현 수준 측정 방법은 당업계에서 공지되어 있는 방법은 제한 없이 사용할 수 있으며, 그 예로 웨스턴 블랏팅(western blotting), 닷 블랏팅(dot blotting), 효소면역분석법(enzyme-linked immunosorbent assay), 방사능 면역분석법(RIA), 방사면역확산법, 오우크테로니 면역 확산법, 로케트 면역 전기영동, 면역조직화학염색, 면역침전법(immunoprecipitation), 보체 고정 분석법, 유세포 분석법(FACS) 또는 단백질 칩 방법 등이 있으나, 이들에 한정되는 것은 아니다. In addition, methods for measuring the expression level of proteins can be used without limitation, methods known in the art, such as western blotting, dot blotting, enzyme-linked immunosorbent assay. , Radioactive Immunoassay (RIA), Radioimmune Diffusion Method, Ouchteroni Immunity Diffusion Method, rocket immunoelectrophoresis, immunohistochemical staining, immunoprecipitation, complement fixation assay, flow cytometry (FACS) or protein chip method, etc. However, it is not limited to these.
상기 (c) 단계는 대조군 세포와 비교하여 ST8SIA1 유전자의 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계이다.The step (c) is a step of selecting a test substance that reduces the expression level of the ST8SIA1 gene compared to the control cells as a candidate substance for treating diabetes.
본 발명자들이 규명한 바와 같이, ST8SIA1 억제시 인슐린 분비가 증가되고, 세포 사멸을 방어하는 역할을 한다. 따라서 ST8SIA1의 유전자의 발현 수준을 감소시키는 시험 물질은 ST8SIA1의 단백질 수준 및 단백질의 활성 수준을 떨어뜨려 췌장 β 세포에서 글루코스 독성으로 유발되는 세포사멸을 방지하고 따라서 이로 인하여 유발되는 당뇨병을 지연시키거나 증상을 완화할 수 있음을 알 수 있다. As identified by the present inventors, upon inhibition of ST8SIA1, insulin secretion is increased, and it serves to prevent cell death. Therefore, the test substance that reduces the expression level of the ST8SIA1 gene reduces the protein level and the activity level of the protein of ST8SIA1 to prevent apoptosis caused by glucose toxicity in pancreatic β cells, and thus delay or symptomatic diabetes caused by this. It can be seen that it can alleviate.
또한, 본 발명은In addition, the present invention
ST8SIA1 유전자가 결실된 인슐린 분비 세포를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물을 제공한다.It provides a pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which the ST8SIA1 gene is deleted.
본 발명의 '인슐린 분비 세포(insulin producing cells; IPCs)'란 인슐린을 분비할 수 있는 세포로 인슐린 분비능이 있으면 그 종류가 제한되지 않으나, 일반적으로는 췌장 베타 세포를 의미한다.The term'insulin producing cells (IPCs)' of the present invention is a cell capable of secreting insulin, and its type is not limited as long as it has insulin secretion ability, but generally refers to pancreatic beta cells.
본 발명의 'ST8SIA1 유전자가 결실된 인슐린 분비 세포'를 제조하는 방법은 이에 제한되지 않으나, 다음의 단계를 포함할 수 있다:The method of producing the'ST8SIA1 gene-deleted insulin secreting cell' of the present invention is not limited thereto, but may include the following steps:
(a) 인슐린 분비 세포에서 엑소좀을 분리하는 단계;(a) separating exosomes from insulin-secreting cells;
(b) 상기 단계(a)에서 분리된 엑소좀에 ST8SIA1에 특이적으로 결합하는 항체를 형질주입(transfection)하는 단계; 및(b) transfecting the exosomes isolated in step (a) with an antibody that specifically binds to ST8SIA1; And
(c) 상기 (c)단계에서 제조된 엑소좀을 인슐린 분비 세포에 형질주입하는 단계.(c) transfecting the exosomes prepared in step (c) into insulin secreting cells.
본 발명에 있어서, 상기 엑소좀은 목적 단백질(본 발명에서는 항체)을 내부에 포함하여 목적 단백질의 표적 세포 또는 조직으로 목적단백질을 운반하는 운반체로서 역할을 수행할 수 있는데, 상기 엑소좀에 의하여 운반된 목적 단백질은 표적 세포 또는 조직에 작용하여 특정 유전자를 결실(유전자의 발현 또는 활성을 억제)시키는데 사용될 수 있다.In the present invention, the exosome can serve as a carrier for transporting the protein of interest to the target cells or tissues of the target protein, including the protein of interest (antibody in the present invention) inside. The protein of interest can be used to delete a specific gene (suppress the expression or activity of a gene) by acting on a target cell or tissue.
상기 제조방법에 따라 항체가 형질주입된 엑소좀을 이용하여 인슐린 분비 세포 내 ST8SIA1을 결실(ST8SIA1의 발현 또는 활성을 억제)시키는 경우, 불안정한 상태인 항체를 엑소좀 안에 내재함으로써, 대식세포에 의한 식균작용이나 분해를 피할 수 있으며 신체 내에서 장시간 순환할 수 있다. 또한, 항체가 형질주입된 엑소좀은 잠재적으로 엔도솜 경로 및 리소좀 분해를 피할 수 있어, 항체를 인슐린 분비세포 내 표적에 직접적으로 전달할 수 있다는 장점이 있다(WO2013084000A2 참조).In the case of deletion of ST8SIA1 (inhibiting the expression or activity of ST8SIA1) in insulin secreting cells using exosomes transfected with antibodies according to the above manufacturing method, phagocytosis by macrophages by internalizing the antibody in an unstable state into exosomes It can avoid action or decomposition and can circulate in the body for a long time. In addition, the antibody-transfected exosomes have the advantage that they can potentially avoid endosome pathways and lysosome degradation, and thus can directly deliver the antibody to targets in insulin-secreting cells (see WO2013084000A2).
본 발명의 일실시예에서는 인슐린 분비세포에서 엑소좀을 분리하고, ST8SIA1 항체를 엑소좀에 형질주입한 뒤, 제조된 ST8SIA1 항체 형질주입된 엑소좀을 인슐린 분비세포에 형질주입하여 인슐린 분비세포의 ST8SIA1 단백질의 발현 또는 활성을 제거(knock-out)하였다. 엑소좀을 이용하여 인슐린 분비세포의 ST8SIA1 유전자를 결실시키는 것은 본 발명자들이 최초로 발견한 것이다. In one embodiment of the present invention, the exosomes are isolated from insulin-secreting cells, the ST8SIA1 antibody is transfected into the exosomes, and then the prepared ST8SIA1 antibody-transfected exosomes are transfected into the insulin-secreting cells. Expression or activity of the protein was knocked out. Deleting the ST8SIA1 gene of insulin-secreting cells using exosomes was the first discovery by the present inventors.
또한, 본 발명의 일실시예에서는 ST8SIA1 유전자가 제거(knock-out)된 인슐린 분비 세포를 개체에 투여 또는 이식하는 경우 인슐린 분비가 증가되어, 개체내 혈당이 감소하고 혈당량 조절능이 매우 우수함을 확인할 수 있었다.In addition, in one embodiment of the present invention, when the insulin-secreting cells from which the ST8SIA1 gene is knocked-out is administered or transplanted to an individual, insulin secretion is increased, so that the blood sugar in the individual decreases and the ability to control blood glucose level is very excellent. there was.
상기 '인슐린 분비 세포'는 줄기세포에서 분화된 것일 수 있으나 이에 제한되지는 않는다. 줄기세포에서 인슐린 분비 세포를 분화하는 방법은 공지된 것이라면 그 방법이 제한되지 않으나, 그 예로 Soria 등[Soria B, Roche E, Berna G and Leon-Quinto T (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157-162], Assady 등[Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, and Tzukerman M. (2001) Insulin production by human embryonic stem cells. Diabetes 50, 1691-1707], Fujikawa 등[Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, and Petersen BE (2005) Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin-producing cells. Am J Pathol 166, 1781-1791] 등의 문헌을 참조할 수 있다.The'insulin secreting cells' may be differentiated from stem cells, but are not limited thereto. The method of differentiating insulin-secreting cells from stem cells is not limited as long as it is known, for example, Soria et al. [Soria B, Roche E, Berna G and Leon-Quinto T (2000) Insulin-secreting cells derived from embryonic stem. cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157-162], Assady et al. [Assady S, Maor G, Amit M, Itskovitz-Eldor J, Skorecki KL, and Tzukerman M. (2001) Insulin production by human embryonic stem cells. Diabetes 50, 1691-1707], Fujikawa et al. [Fujikawa T, Oh SH, Pi L, Hatch HM, Shupe T, and Petersen BE (2005) Teratoma formation leads to failure of treatment for type I diabetes using embryonic stem cell-derived insulin -producing cells. Am J Pathol 166, 1781-1791, et al.
상기 줄기세포는 다양한 신체 조직으로 분화할 수 있는 능력을 갖는 미분화 세포로서, 이는 만능 줄기 세포(totipotent stem cell), 전분화능 줄기세포 (pluripotent stem cell), 다분화능 줄기 세포(multipotent stem cell)로 분류될 수 있다.The stem cells are undifferentiated cells having the ability to differentiate into various body tissues, which are classified into totipotent stem cells, pluripotent stem cells, and multipotent stem cells. Can be.
본 발명에서 줄기세포는 그 유래 또는 유형에 따라, 성체줄기세포, 배아줄기세포, 중간엽 줄기세포, 종양줄기세포 또는 유도만능줄기세포일 수 있다.Stem cells in the present invention may be adult stem cells, embryonic stem cells, mesenchymal stem cells, tumor stem cells, or induced pluripotent stem cells, depending on their origin or type.
본 명세서에서 사용된 용어 '중간엽 줄기세포(mesenchymal stem cell, MSC)'는 뼈, 연골, 지방, 근육세포를 포함한 여러 가지 중배엽성 세포 또는 신경세포와 같은 외배엽성 세포로도 분화하는 능력을 가진 다분화능 줄기세포(multipotent stem cell)이다. 상기 중간엽 줄기세포는 바람직하게는 제대, 제대혈, 골수, 지방, 근육, 신경, 피부, 양막, 융모막, 탈락막, 및 태반으로 구성된 군에서 선택되는 것으로부터 유래될 수 있다. 또한, 상기 중간엽 줄기세포는 인간, 태아, 또는 인간을 제외한 포유동물로부터 유래될 수 있다. 상기 인간을 제외한 포유동물은 보다 바람직하게는 개과 동물, 고양이과 동물, 원숭이과 동물, 소, 양, 돼지, 말, 랫트, 마우스 또는 기니피그 등일 수 있으며, 그 유래를 제한하지 않는다.The term'mesenchymal stem cell (MSC)' as used herein has the ability to differentiate into various mesenchymal cells including bone, cartilage, fat, muscle cells, or ectodermal cells such as neurons. It is a multipotent stem cell. The mesenchymal stem cells may preferably be derived from those selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, chorionic membrane, deciduous membrane, and placenta. In addition, the mesenchymal stem cells may be derived from humans, fetuses, or mammals other than humans. The mammals other than humans are more preferably canine, feline, monkey, cow, sheep, pig, horse, rat, mouse, guinea pig, and the like, and the origin is not limited.
상기 유효성분은 상기 세포를 포함하는 세포 배양액, 세포 배양물의 농축물 등을 포함하는 개념이다.The active ingredient is a concept including a cell culture medium containing the cells, a concentrate of a cell culture, and the like.
본 발명은 당뇨병의 예방 또는 치료용 제제를 제조하기 위한 ST8SIA1 단백질의 발현 또는 활성 억제제의 용도를 제공한다.The present invention provides a use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
본 발명은 ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 당뇨병의 치료 방법을 제공한다.The present invention provides a method for treating diabetes, comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
본 발명의 상기 ‘유효량’이란 개체에게 투여하였을 때, 당뇨병의 개선, 치료, 예방, 검출, 진단 또는 당뇨병의 억제 또는 감소 효과를 나타내는 양을 말하며, 상기 ‘개체’란 동물, 바람직하게는 포유동물, 특히 인간을 포함하는 동물일 수 있으며, 동물에서 유래한 세포, 조직, 기관 등일 수도 있다. 상기 개체는 상기 효과가 필요한 환자(patient) 일 수 있다.The'effective amount' of the present invention refers to an amount that improves, treats, prevents, detects, diagnoses, or inhibits or reduces diabetes when administered to an individual, and the'individual' refers to an animal, preferably a mammal , In particular, it may be an animal, including humans, and may be cells, tissues, organs, etc. derived from animals. The individual may be a patient in need of the effect.
본 발명의 상기 ‘치료’는 당뇨병 또는 당뇨병의 증상을 개선시키는 것을 포괄적으로 지칭하고, 이는 상기 질환을 치유하거나, 실질적으로 예방하거나, 또는 상태를 개선시키는 것을 포함할 수 있으며, 상기 질환으로부터 비롯된 한 가지 증상 또는 대부분의 증상을 완화시키거나, 치유하거나 예방하는 것을 포함하나, 이에 제한되는 것은 아니다.The'treatment' of the present invention refers generically to improving the symptoms of diabetes or diabetes, which may include curing, substantially preventing, or improving the condition, as long as it originates from the disease. It includes, but is not limited to, alleviating, curing or preventing the symptoms or most of the symptoms.
본 발명에서 용어 '~을 포함하는(comprising)'이란 '함유하는' 또는 '특징으로 하는'과 동일하게 사용되며, 조성물 또는 방법에 있어서, 언급되지 않은 추가적인 성분 요소 또는 방법 단계 등을 배제하지 않는다. 용어 '~로 이루어지는(consisting of)'이란 '~로 구성되는'과 동일하게 사용되며, 별도로 기재되지 않은 추가적인 요소, 단계 또는 성분 등을 제외하는 것을 의미한다. 용어 '필수적으로 이루어지는(essentially consisting of)' 또는 '필수적으로 구성되는'이란 조성물 또는 방법의 범위에 있어서, 기재된 성분 요소 또는 단계와 더불어 이의 기본적인 특성에 실질적으로 영향을 미치지 않는 성분 요소 또는 단계 등을 포함하는 것을 의미한다.In the present invention, the term'comprising' is used in the same manner as'containing' or'as a feature', and does not exclude additional component elements or method steps that are not mentioned in the composition or method. . The term'consisting of' is used in the same way as'consisting of', and means excluding additional elements, steps, or ingredients that are not separately described. The term'essentially consisting of' or'essentially consisting of' means, in the scope of a composition or method, a component element or step that does not substantially affect its basic properties in addition to the described component elements or steps. Means to include.
본 발명은 고혈당 조건에서 ST8SIA1 억제시 관련 인자의 발현을 조절하여 세포 사멸을 방어할 수 있고, 티로신 인산화 증가를 통해 인슐린 저항성을 감소시키고 감수성을 증가시킬 뿐만 아니라, ST8SIA1 억제로 인슐린 분비량이 증가된 점을 통하여 ST8SIA1 단백질의 발현 또는 활성을 억제하는 전혀 새로운 작용기작의 당뇨병의 치료제를 개발하는 데 유용하게 이용될 수 있다. 또한, ST8SIA1는 췌장 베타 세포에서 고혈당 조건에서 세포사멸을 촉진시키는 역할을 하므로, ST8SIA1의 활성을 낮추면, 고혈당에 의한 세포사멸을 억제하고 췌장 베타 세포를 보호하는 효과가 있으며, 이를 이용하여 ST8SIA1의 발현 또는 활성을 억제하는 제제를 스크리닝하여 항당뇨병 후보 물질을 선별할 수 있다. 또한, ST8SIA1 유전자가 제거(knock-out)된 인슐린 분비 세포를 개체에 투여, 이식하는 경우, 또는 피하등에 직접투여의 경우 인슐린 분비가 증가되어, 개체내 혈당이 감소하고 혈당량 조절능이 매우 우수하다.The present invention can prevent apoptosis by controlling the expression of related factors upon inhibition of ST8SIA1 in hyperglycemic conditions, decrease insulin resistance and increase sensitivity through increased tyrosine phosphorylation, and increase insulin secretion due to ST8SIA1 inhibition. It can be usefully used to develop a therapeutic agent for diabetes with a completely new mechanism of action that inhibits the expression or activity of the ST8SIA1 protein. In addition, since ST8SIA1 plays a role in promoting apoptosis in pancreatic beta cells under hyperglycemic conditions, lowering the activity of ST8SIA1 inhibits apoptosis caused by hyperglycemia and has the effect of protecting pancreatic beta cells. Using this, ST8SIA1 expression Alternatively, antidiabetic candidate substances can be selected by screening for agents that inhibit activity. In addition, in the case of administering or transplanting insulin-secreting cells from which the ST8SIA1 gene has been knocked-out to an individual, or when directly administered subcutaneously, insulin secretion is increased, so that blood sugar in the individual decreases and the ability to regulate blood glucose level is very excellent.
도 1은 중간엽줄기세포로부터 분화시킨 인슐린 분비 세포(insulin producing cells; IPCs)에서 ST8SIA1 억제된 경우(ST8SIA1KO-IPCs) 세포사멸 관련 단백질인 cleaved caspase 3의 단백질 수준을 웨스턴 블랏팅으로 조사한 결과이다. β-actin를 단백질의 로딩컨트롤로서 사용하였다.1 is a result of investigation of the protein level of cleaved caspase 3, an apoptosis-related protein, by Western blotting when ST8SIA1 is inhibited (ST8SIA1KO-IPCs) in insulin producing cells (IPCs) differentiated from mesenchymal stem cells. β-actin was used as a protein loading control.
도 2는 IPCs 세포에서 ST8SIA1 억제된 경우 ERK의 인산화 결과를 웨스턴 블랏팅으로 조사한 결과이다. β-actin를 단백질의 로딩컨트롤로서 사용하였다.2 is a result of investigation of the phosphorylation result of ERK by Western blotting when ST8SIA1 is inhibited in IPCs cells. β-actin was used as a protein loading control.
도 3은 IPCs 세포에 ST8SIA1 억제된 경우 인슐린 분비량 변화를 나타낸 것이다. PBS를 대조군으로 사용하였다.Figure 3 shows the change in the amount of insulin secretion when ST8SIA1 is inhibited in IPCs cells. PBS was used as a control.
도 4은 9주령 마우스를 군당 8마리씩 나누어 사육 1주에 ST8SIA1KO-IPCs는 이식하고, db/db 마우스의 혈당 농도를 측정한 결과이다.FIG. 4 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
도 5는 9주령 db/db 마우스를 군당 8마리씩 나누어 사육 1주에 ST8SIA1KO-IPCs를 이식하고, 인슐린 감수성을 측정한 결과이다.5 shows the results of measuring insulin sensitivity by dividing 9-week-old db/db mice into 8 mice per group and transplanting ST8SIA1KO-IPCs at 1 week of breeding.
도 6은 9주령 db/db 마우스를 군당 8마리씩 나누어 사육 1주에 ST8SIA1KO-IPCs를 이식하고, 내당능을 측정한 결과이다.Figure 6 is a 9-week-old db/db mice divided by 8 per group, and ST8SIA1KO-IPCs were transplanted at 1 week of breeding, and glucose tolerance was measured.
도 7은 9주령 마우스를 군당 8마리씩 나누어 사육 1주에 anti-ST8SIA1-Ab를 사포닌 0.5% 100ul를 어드주번트로 사용하고, db/db 마우스의 혈당 농도를 측정한 결과이다.Figure 7 shows the results of measuring the blood glucose concentration of db/db mice by dividing 9-week-old mice into 8 mice per group, using anti-ST8SIA1-Ab as an adjuvant with 0.5% saponin and 100 ul of saponin at 1 week.
이하 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.However, the following examples are only illustrative of the present invention, and the contents of the present invention are not limited to the following examples.
실험 방법Experimental method
1. 세포 배양1. Cell culture
중간엽줄기세포로부터 인슐린분비 세포주인 IPCs 세포를 분화시켰다. 10% FBS, 1% 페니실린-스트렙토마이신, 10uM 트로글리타존(troglitazone) 및 16.7μM 황산아연(zinc sulfate)을 함유하는 저포도당(5mM glucose) DMEM을 이용하고, 37℃, 5% CO 2 조건에서 배양하였다. 48시간 동안 33mM 글루코스를 처리하여 글루코스 독성(glucose toxicity)을 유도하였다. 그 후 10μM ST8SIA1 항체(ST pharm)를 48시간 동안 처리하였다.Insulin secreting cell line IPCs cells were differentiated from mesenchymal stem cells. Low glucose (5 mM glucose) DMEM containing 10% FBS, 1% penicillin-streptomycin, 10 uM troglitazone and 16.7 μM zinc sulfate was used, and cultured at 37° C. and 5% CO 2 . Glucose toxicity was induced by treatment with 33 mM glucose for 48 hours. Then, 10 μM ST8SIA1 antibody (ST pharm) was treated for 48 hours.
2. 웨스턴 블롯 분석2. Western Blot Analysis
세포를 protease inhibitor cocktail(Roche Diagnostics)을 포함하는 RIPA buffer(50mMTris-HCl, 150mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS)를 이용하여 용해하였다. 세포 용해물의 단백질 구성물은 BCA protein assay kit(Roche Diagnostics)로 정량한 뒤 같은 양의 단백질을 함유하는 세포 용해물을 SDS-PAGE로 분리하였다. 그 다음 Immobilon NC membranes으로 단백질을 이동시켰다. 5% 스킴 밀크 또는 5% BSV(bovine albumin serum)를 포함하는 TBS와 Tween 20 용액(0.05% Tween 20)으로 1시간 동안 블라킹하였다. ST8SIA1(Abnova, Taiwan), GAPDH(Santa Cruz Biotechnology), p38MAPK(pMAPK, Santa Cruz), 그리고 phosphated ERK(pERK, cell Signaling)에 대한 항체와 프로브를 4℃에서 밤새 반응시켰고, 1시간 동안 peroxidase가 결합된 2차 항체를 반응시켰다. 멤브레인을 TBS와 Tween 20으로 5분씩 3번 세척하였고, chemiluminescence system(Thermo Fisher Scientific)을 사용하여 ChemiDoc MP system (Bio-Rad)으로 밴드를 관찰하였다. Image J 프로그램을 이용하여 밴드의 세기를 측정하였다.Cells were lysed using RIPA buffer (50mMTris-HCl, 150mMNaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS) containing protease inhibitor cocktail (Roche Diagnostics). The protein composition of the cell lysate was quantified with a BCA protein assay kit (Roche Diagnostics), and then the cell lysate containing the same amount of protein was separated by SDS-PAGE. Then, the protein was transferred to Immobilon NC membranes. Blocking was performed for 1 hour with TBS and Tween 20 solution (0.05% Tween 20) containing 5% skim milk or 5% bovine albumin serum (BSV). Antibodies and probes against ST8SIA1 (Abnova, Taiwan), GAPDH (Santa Cruz Biotechnology), p38MAPK (pMAPK, Santa Cruz), and phosphated ERK (pERK, cell signaling) were reacted overnight at 4°C, and peroxidase bound for 1 hour The secondary antibody was reacted. The membrane was washed 3 times with TBS and Tween 20 for 5 minutes each, and the band was observed with a ChemiDoc MP system (Bio-Rad) using a chemiluminescence system (Thermo Fisher Scientific). The intensity of the band was measured using the Image J program.
3. ST8SIA1 녹아웃 인슐린 분비 세포 유도3. ST8SIA1 knockout insulin secreting cell induction
골수로부터 중간엽줄기세포(bone marrow derived mesenchyma stem cells; BD-MSC)를 분리하기 위해 암컷 비당뇨병성 C57BL/6 mouse의 bone marrow에서 얻어낸 골수세포를 alpha-MEM(15% fbs) media에서 8~12일간 배양을 한다. 3일마다 media 교체를 하고 이후 5% FBS high DMEM 15일 배양을 진행하여 MSC를 분리하였다. 다음 단계에서 BD-MSC를 5% FBS, 20μmol/L 니코틴 아미드를 함유한 Low DMEM 배지에서 7일간 배양하고 마지막 3단계에서 10 μmol/L의 Exclusive-4를 첨가한 Low DMEM 배지에서 7일간 더 배양하여 인슐린 분비 β 세포(insulin producing cells; IPCs)를 유도하였다. 세포에서 엑소좀에 ST8SIA1 항체를 넣어 다시 엑소좀을 IPCs에 트랜스펙션하여 ST8SIA1 유전자를 제거하여 ST8SIA1KO-IPCs 세포주를 생성하였다. In order to separate bone marrow derived mesenchyma stem cells (BD-MSC) from bone marrow, bone marrow cells obtained from bone marrow of female non-diabetic C57BL/6 mice were used in alpha-MEM (15% fbs) media. Incubate for 12 days. The media was replaced every 3 days, and then, 5% FBS high DMEM was cultured for 15 days to separate MSCs. In the next step, BD-MSC was incubated for 7 days in Low DMEM medium containing 5% FBS and 20 μmol/L nicotinamide, and incubated for 7 more days in Low DMEM medium containing 10 μmol/L Exclusive-4 in the last 3 steps. As a result, insulin producing cells (IPCs) were induced. In the cells, the ST8SIA1 antibody was added to the exosomes, and the exosomes were again transfected into IPCs to remove the ST8SIA1 gene to generate the ST8SIA1KO-IPCs cell line.
9주령 db/db 마우스를 군당 8마리씩 나누어 사육하였다. 사육 1주에 3x10 6개의 ST8SIA1KO-IPCs를 마우스 췌장에 이식하였다.9-week-old db/db mice were reared by dividing 8 mice per group. At 1 week of breeding, 3x10 6 ST8SIA1KO-IPCs were transplanted into the mouse pancreas.
4. 엑소좀 트랜스펙션 (exosome transfection)4. Exosome transfection
IPCs의 배양 media 10ml에서 ExoQuick kt (SBI bio)를 이용하여 엑소좀을 분리한다. 멸균된 PBS 500ul에 엑소좀은 대략 50~300ug 이다. 멸균된 1.5ml 튜브에 10ul Exo-Fect 용액과 10ul PBS용액, 50ul 정제된 엑소좀, ST8SIA1 안티바디(20ul)를 함께 넣어 3번 반복하여 잘 혼합한다. 혼합된 엑소좀 트랜스펙션 솔루션을 37℃에서 1시간 동안 쉐이커로 인큐베이트하고 즉시 얼음에 튜브를 놓는다. ExoQuickTC 시약 30ul를 엑소좀 샘플 현탄액에 트랜스펙션하고 6번 잘 혼합하여 섞는다. 다시 트랜스펙션 샘플을 얼음 또는 4℃에 30분간 안정화시킨다. 트랜스펙션된 엑소좀을 얻기 위해 microfuge (최고 속도)에서 13,000-14,000 rpm으로 3분 동안 시료를 원심 분리한 후 상등액을 제거하고 transfected exosome 펠렛을 300ul 1x PBS에 풀어준다. 6-well에 약 1X10 5개로 성장한 IPCs에 트랜스펙션된 엑소좀(300ul)를 첨가해주고 24시간동안 37 ℃ 인큐베이션에서 배양해준다.Isolate exosomes from 10 ml of IPCs culture media using ExoQuick kt (SBI bio). Exosomes in 500ul of sterile PBS are approximately 50-300ug. In a sterilized 1.5ml tube, 10ul Exo-Fect solution, 10ul PBS solution, 50ul purified exosomes, and ST8SIA1 antibody (20ul) were put together and mixed well by repeating 3 times. The mixed exosome transfection solution was incubated with a shaker at 37° C. for 1 hour and immediately placed the tube on ice. Transfect 30ul of ExoQuickTC reagent into the exosome sample suspension, mix well and mix 6 times. Again, the transfection sample is stabilized on ice or at 4° C. for 30 minutes. To obtain the transfected exosomes, the sample was centrifuged for 3 minutes at 13,000-14,000 rpm in a microfuge (maximum speed), the supernatant was removed, and the transfected exosome pellet was released in 300ul 1x PBS. Add the transfected exosomes (300ul) to the IPCs grown to about 1×10 5 in a 6-well and incubate at 37° C. incubation for 24 hours.
실시예 1 :ST8SIA1의 발현량이 세포사멸에 미치는 영향Example 1: Effect of the expression level of ST8SIA1 on apoptosis
ST8SIA1 억제와 세포사멸의 연관성을 알아보았다. The association between ST8SIA1 inhibition and apoptosis was investigated.
엑소좀으로 트랜스펙션된 IPCs (ST8SIA1KO-IPCs)세포를 각각 정상 글루코스(NG) 또는 고농도 글루코스(HG : 33mM)의 조건으로 2일 동안 배양하였다. β-actin을 단백질의 로딩컨트롤로서 사용하였다. ST8SIA1의 발현량이 글루코스 독성으로 유발되는 세포사멸에 미치는 영향을 확인하기 위하여 고농도 글루코오스 조건에서 ST8SIA1 항체로 ST8SIA1의 발현을 억제하고 cleaved caspase 3의 발현량을 측정하였다.IPCs (ST8SIA1KO-IPCs) cells transfected with exosomes were cultured for 2 days under conditions of normal glucose (NG) or high glucose (HG: 33 mM), respectively. β-actin was used as a protein loading control. In order to confirm the effect of ST8SIA1 expression on apoptosis induced by glucose toxicity, the expression of ST8SIA1 was inhibited with the ST8SIA1 antibody under high glucose conditions and the expression level of cleaved caspase 3 was measured.
그 결과, 도 1에 나타난 바와 같이, 항체로 ST8SIA1 억제시 (항-ST8SIA1 항체 lane), 이를 억제하지 않은 대조군 (control lane)에 대비하여 세포사멸에 중요한 cleaved caspase 3 (c-caspase-3)가 감소하는 것으로 나타났다. 이를 통해, 알려진 바와 같이 고농도 글루코스에 의해 세포사멸이 증가하는 반면, ST8SIA1의 발현량이 감소하면 고농도의 포도당에 기인한 세포자살로부터 세포사멸을 방어할 수 있음을 확인할 수 있었다.As a result, as shown in FIG. 1, when ST8SIA1 is inhibited with an antibody (anti-ST8SIA1 antibody lane), cleaved caspase 3 (c-caspase-3), which is important for apoptosis compared to a control lane that does not inhibit it, is Appeared to decrease. Through this, it was confirmed that apoptosis was increased by high glucose concentration, as known, whereas when the expression level of ST8SIA1 decreased, apoptosis could be prevented from apoptosis caused by high glucose concentration.
실시예 2: 인슐린 수용체의 티로신 인산화의 관계Example 2: Relationship of tyrosine phosphorylation of insulin receptor
당뇨는 인슐린 수용체(insulin receptor; IR) 자체의 결함보다는 수용체 하위 신호전달의 이상, 특히 IRS-1(insulin receptor substrate-1)-associated PI3K 활성의 저해가 주된 원인으로 알려져 있다. 이러한 IRS-1-associated PI3K 활성은 IRS-1의 티로신 인산화 혹은 IRS-1 단백질양 자체에 의해 조절되며 여러 당뇨 동물 모델 혹은 당뇨 환경에서 IRS-1의 티로신 인산화 단백질양의 감소가 관찰된다.Diabetes is known to be mainly caused by an abnormality in sub-receptor signaling, especially inhibition of IRS-1 (insulin receptor substrate-1)-associated PI3K activity, rather than a defect in the insulin receptor (IR) itself. This IRS-1-associated PI3K activity is regulated by the tyrosine phosphorylation of IRS-1 or the amount of IRS-1 protein itself, and a decrease in the amount of tyrosine phosphorylation protein of IRS-1 is observed in various diabetic animal models or diabetic environments.
이에, 췌장 베타 세포에서 ST8SIA1의 억제시 인슐린 유도 티로신 인산화가 증가되는지 확인하였다. 이를 위하여 ST8SIA1KO-IPCs 세포에 10분간 100nM의 인슐린을 처리하지 않거나 처리하여 티로신 인산화를 웨스턴 블랏 측정하였다.Accordingly, it was confirmed whether insulin-induced tyrosine phosphorylation was increased when ST8SIA1 was inhibited in pancreatic beta cells. To this end, ST8SIA1KO-IPCs cells were not treated with or treated with 100 nM insulin for 10 minutes to measure tyrosine phosphorylation by Western blot.
그 결과, 도 2에 나타난 바와 같이, 인슐린을 처리한 경우 (항-ST8SIA1 항체 lane) 티로신 인산화가 증가하는 데에 비해서, 항체를 처리하지 않은 대조군 (control lane)에서는 티로신 인산화에 별다른 영향이 없음을 확인하였다. 따라서, 본 발명에서와 같이 ST8SIA1를 억제하는 경우 티로신 인산화의 증가가 일어나고, 인슐린에 대한 인슐린 저항성은 감소시키고, 감수성을 증가시킴을 확인하였다.As a result, as shown in Figure 2, compared to the increase in tyrosine phosphorylation in the case of insulin treatment (anti-ST8SIA1 antibody lane), there is no significant effect on tyrosine phosphorylation in the control lane not treated with the antibody. Confirmed. Therefore, it was confirmed that inhibition of ST8SIA1 as in the present invention results in an increase in tyrosine phosphorylation, decreases insulin resistance to insulin, and increases sensitivity.
실시예 3: 당뇨병에 ST8SIA1KO-IPCs가 미치는 영향Example 3: Effect of ST8SIA1KO-IPCs on diabetes
9주령 db/db 마우스를 군당 8마리씩 나누어 사육하였다. 사육 1주에 엑소좀으로 트랜스펙션된 ST8SIA1KO-IPCs 세포를 이식하였고, 8 주 후에 각 마우스를 희생시켜 db/db 마우스의 혈당 농도 및 인슐린 농도(도 3)을 측정하였다. 혈당 농도는 상용의 키트 (올메디쿠스)를 사용하여 제조사의 지침에 따라서 측정하였으며, 인슐린 농도는 ELISA 방법으로 상용의 키트(peprotec)를 사용하여 제조사의 지침에 따라서 측정하였다. 9-week-old db/db mice were reared by dividing 8 mice per group. ST8SIA1KO-IPCs cells transfected with exosomes were transplanted at 1 week of breeding, and each mouse was sacrificed 8 weeks later to measure the blood glucose concentration and insulin concentration of db/db mice (Fig. 3). Blood glucose concentration was measured according to the manufacturer's instructions using a commercial kit (Allmedicus), and insulin concentration was measured according to the manufacturer's instructions using a commercial kit (peprotec) by ELISA method.
그 결과, 도 3에 나타난 바와 같이, 대조군에 비해 ST8SIA1KO-IPCs 투여군에서 혈중 인슐린 농도가 현격히 증가하는 것으로 나타났다. 이와 같은 결과는 본 발명의 ST8SIA1KO-IPCs의 이식에 의해서 우수한 인슐린 증가를 갖는 것을 확인할 수 있었다. As a result, as shown in FIG. 3, it was found that the blood insulin concentration was significantly increased in the ST8SIA1KO-IPCs administration group compared to the control group. These results confirmed that the ST8SIA1KO-IPCs of the present invention had an excellent insulin increase by implantation.
또한, 도 4에 나타난 바와 같이, 대조군에 비해 ST8SIA1KO-IPCs 군에서는 혈당이 정상 수준으로 빠르게 감소하여 유지되는 것을 나타낸 것에 비해서 대조군에서는 혈당이 지속적으로 증가 또는 증가된 상태가 유지되는 것을 확인할 수 있었다.In addition, as shown in FIG. 4, compared to the control group, the ST8SIA1KO-IPCs group showed that blood sugar was rapidly decreased to and maintained at a normal level, whereas in the control group, it was confirmed that the blood sugar was continuously increased or increased.
실시예 4: 인슐린 감수성 및 내당능에 미치는 영향Example 4: Effect on insulin sensitivity and glucose tolerance
9주령 db/db 마우스를 군당 8마리씩 나누어 사육하였다. 사육 1주에 엑소좀으로 트랜스펙션된 ST8SIA1KO-IPCs는 이식하고, db/db 마우스의 내당능(도 4) 및 인슐린 감수성 (도 5)을 측정하였다.9-week-old db/db mice were reared by dividing 8 mice per group. ST8SIA1KO-IPCs transfected with exosomes at 1 week of breeding were transplanted, and glucose tolerance (FIG. 4) and insulin sensitivity (FIG. 5) of db/db mice were measured.
이를 위하여 경구 포도당 내성 검사 및 인슐린 내성 검사를 수행하였다. To this end, an oral glucose tolerance test and an insulin resistance test were performed.
경구 포도당 내성 검사 (IPGTT)를 위하여 포도당(투여량 : 2g/kg체중)은 10주령에 복강내 투여(IPGTT)하였다. 글루코스 수준은 테일 블리드법에 의해 검사 후 60, 120 분에서 모니터링하였다.For the oral glucose tolerance test (IPGTT), glucose (dosage: 2g/kg body weight) was administered intraperitoneally (IPGTT) at 10 weeks of age. Glucose levels were monitored at 60 and 120 minutes after testing by the tail bleed method.
인슐린 내성 검사 (ITT)를 위하여 인슐린 (체중의 0.5U/kg체중)은 14주령에 복강내주사로 투여하였다. 글루코스 수준은 테일 블리드법에 의해 검사 후 10, 20, 40 및 60분에 모니터링하였다. For the insulin resistance test (ITT), insulin (0.5U/kg body weight) was administered by intraperitoneal injection at 14 weeks of age. Glucose levels were monitored at 10, 20, 40 and 60 minutes after testing by tail bleed method.
그 결과 도 5에서 보듯이, 포도당 투여 후 시간에 따른 혈당량의 변화를 측정한 결과 db/db 마우스 대조군에 비해 ST8SIA1KO-IPCs를 병용하여 투여한 군에서는 혈당이 현격히 조절되는 것을 확인할 수 있었다.As a result, as shown in FIG. 5, as a result of measuring the change in blood glucose level over time after glucose administration, it was confirmed that blood glucose was significantly controlled in the group administered with ST8SIA1KO-IPCs compared to the db/db mouse control group.
또한, 도 6에서 보듯이 인슐린 투여 후 혈당량을 측정한 결과 마우스 대조군(Obese-no injection)에 비하여 ST8SIA1KO-IPCs군의 경우 혈당이 혈액에서 빨리 제거되는 것으로 나타남을 확인할 수 있었다.In addition, as shown in FIG. 6, as a result of measuring the blood glucose level after insulin administration, it was confirmed that the ST8SIA1KO-IPCs group was quickly removed from the blood compared to the mouse control group (Obese-no injection).
또한, 도 7에 나타난 바와 같이, 전달체로 0.5% saponin(시그마) solution 100ul가 혼합된 anti-ST8SIA1-Ab를 피하에 투여한 군에서는 혈당이 정상 수준으로 빠르게 감소하여 유지되는 것을 나타낸 것에 비해서 대조군에서는 혈당이 지속적으로 증가 또는 증가된 상태가 유지되는 것을 확인할 수 있었다.In addition, as shown in FIG. 7, in the group administered subcutaneously with an anti-ST8SIA1-Ab mixed with 100ul of 0.5% saponin (Sigma) solution as a delivery vehicle, blood sugar rapidly decreased to a normal level and maintained, whereas in the control group It was confirmed that the blood sugar was continuously increased or the increased state was maintained.
본 발명은 ST8SIA1의 발현 또는 활성을 억제하는 제제를 스크리닝하여 항당뇨병 후보 물질을 선별할 수 있고, ST8SIA1 유전자가 제거(knock-out)된 인슐린 분비 세포를 개체에 투여 또는 이식하는 경우 인슐린 분비가 증가되어, 개체내 혈당이 감소하고 혈당량 조절능이 매우 우수하므로 산업상 이용가능성이 있다. The present invention can screen an agent that inhibits the expression or activity of ST8SIA1 to screen for antidiabetic candidate substances, and when the insulin-secreting cells from which the ST8SIA1 gene is knocked-out is administered or transplanted to an individual, insulin secretion is increased. As a result, the blood sugar in the individual decreases and the ability to control blood sugar levels is very good, so it has industrial applicability.

Claims (17)

  1. ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating diabetes, comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
  2. 제1항에 있어서, 상기 당뇨병은 제1형 당뇨병, 제2형 당뇨병 및 임신성 당뇨병으로 이루어진 군에서 선택된 것을 특징으로 하는 조성물. The composition of claim 1, wherein the diabetes is selected from the group consisting of type 1 diabetes, type 2 diabetes and gestational diabetes.
  3. 제1항에 있어서, 상기 ST8SIA1 단백질은 서열번호 1 또는 서열번호 2로 표시되는 아미노산 서열을 포함하는 것을 특징으로 하는 조성물.The composition of claim 1, wherein the ST8SIA1 protein comprises an amino acid sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.
  4. 제1항에 있어서, 상기 ST8SIA1 단백질 발현 억제제는 ST8SIA1 mRNA에 상보적으로 결합하는 안티센스 올리고뉴클레오티드(antisense oligonucleotide), siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme 및 PNA(protein nucleic acid)로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 조성물. The method of claim 1, wherein the ST8SIA1 protein expression inhibitor is composed of an antisense oligonucleotide, siRNA, shRNA, miRNA, ribozyme, crispr-cas9, DNAzyme, and protein nucleic acid (PNA) that bind complementarily to ST8SIA1 mRNA. Composition, characterized in that any one selected from the group.
  5. 제4항에 있어서, 상기 ST8SIA1 mRNA는 서열번호 3 또는 서열번호 4로 표시되는 염기 서열을 포함하는 것을 특징으로 하는 조성물. The composition of claim 4, wherein the ST8SIA1 mRNA comprises a nucleotide sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4.
  6. 제1항에 있어서, 상기 ST8SIA1 단백질 활성 억제제는 ST8SIA1 단백질에 특이적으로 결합하는 화합물, 펩티드, 펩티드 유사체(mimetics), 앱타머, 항체, 천연추출물 및 합성화합물로 이루어진 군에서 선택된 어느 하나인 것을 특징으로 하는 조성물. The method of claim 1, wherein the ST8SIA1 protein activity inhibitor is any one selected from the group consisting of compounds, peptides, peptide mimetics, aptamers, antibodies, natural extracts, and synthetic compounds that specifically bind to ST8SIA1 protein. Composition made by.
  7. (a) 시험 물질을 ST8SIA1 유전자를 발현하는 세포에 접촉시키는 단계;(a) contacting the test substance with cells expressing the ST8SIA1 gene;
    (b) 상기 세포와 시험 물질을 접촉하지 않은 대조군 세포에서 ST8SIA1 유전자 발현 수준을 측정하는 단계; 및(b) measuring the ST8SIA1 gene expression level in the control cells not contacting the cells and the test substance; And
    (c) 대조군 세포와 비교하여 ST8SIA1 유전자 발현 수준을 감소시키는 시험 물질을 당뇨병 치료제 후보 물질로 선별하는 단계를 포함하는 당뇨병 치료제 스크리닝 방법. (C) a diabetes treatment screening method comprising the step of selecting a test substance that reduces the ST8SIA1 gene expression level compared to the control cells as a diabetes treatment candidate substance.
  8. 제7항에 있어서, 상기 ST8SIA1 유전자를 발현하는 세포는 췌장 베타 세포(pancreatic β cell) 또는 췌장 베타 세포에서 유래한 세포인 것을 특징으로 하는 방법.The method according to claim 7, wherein the cells expressing the ST8SIA1 gene are pancreatic beta cells or cells derived from pancreatic beta cells.
  9. 제7항에 있어서, 상기 ST8SIA1 유전자 발현 수준은 ST8SIA1 mRNA 또는 단백질 발현 수준을 측정하는 것을 특징으로 하는 방법. The method of claim 7, wherein the ST8SIA1 gene expression level is characterized by measuring the ST8SIA1 mRNA or protein expression level.
  10. ST8SIA1 유전자 발현이 억제된 인슐린 분비 세포를 유효성분으로 포함하는 당뇨병의 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating diabetes, comprising as an active ingredient insulin secreting cells in which ST8SIA1 gene expression is suppressed.
  11. 제10항에 있어서, 상기 ST8SIA1 유전자의 발현 억제는 ST8SIA1에 특이적으로 결합하는 항체를 포함하는 엑소좀으로 형질주입된 것을 특징으로 하는 조성물.11. The composition of claim 10, wherein the ST8SIA1 gene is inhibited by transfection with exosomes containing an antibody that specifically binds to ST8SIA1.
  12. 제10항에 있어서, 상기 조성물은 비경구투여 제제로 제제화된 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 10, wherein the composition is formulated as a parenteral formulation.
  13. 제10항에 있어서, 상기 비경구투여 제제는 주사제 또는 주입제인 것을 특징으로 하는 약학적 조성물.The pharmaceutical composition according to claim 10, wherein the parenteral formulation is an injection or infusion.
  14. 제10항에 있어서, 상기 주사제 또는 주입제는 췌장으로 상기 조성물을 주입하기 위한 것인 약학적 조성물.The pharmaceutical composition of claim 10, wherein the injection or infusion agent is for injecting the composition into the pancreas.
  15. 제10항에 있어서, 상기 인슐린 분비세포는The method of claim 10, wherein the insulin secreting cells
    (a) 인슐린 분비 세포에서 엑소좀을 분리하는 단계;(a) separating exosomes from insulin-secreting cells;
    (b) 상기 단계(a)에서 분리된 엑소좀에 ST8SIA1에 특이적으로 결합하는 항체를 형질주입(transfection)하는 단계; 및(b) transfecting the exosomes isolated in step (a) with an antibody that specifically binds to ST8SIA1; And
    (c) 상기 (c)단계에서 제조된 엑소좀을 인슐린 분비 세포에 형질주입하는 단계를 포함하는 방법에 의해 제조된 것을 특징으로 하는 조성물.(c) A composition, characterized in that it is prepared by a method comprising the step of transfecting the exosome prepared in step (c) into insulin-secreting cells.
  16. 당뇨병의 예방 또는 치료용 제제를 제조하기 위한 ST8SIA1 단백질의 발현 또는 활성 억제제의 용도.Use of an inhibitor of the expression or activity of the ST8SIA1 protein for preparing a preparation for preventing or treating diabetes.
  17. ST8SIA1 단백질의 발현 또는 활성 억제제를 유효성분으로 포함하는 조성물의 유효량을 이를 필요로 하는 개체에 투여하는 것을 포함하는 당뇨병의 치료 방법.A method of treating diabetes, comprising administering to an individual in need thereof an effective amount of a composition comprising an inhibitor of ST8SIA1 protein expression or activity as an active ingredient.
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