WO2019017680A2 - Micro-arn utilisé en tant que biomarqueur de la maladie de parkinson et kit diagnostique le mettant en oeuvre - Google Patents

Micro-arn utilisé en tant que biomarqueur de la maladie de parkinson et kit diagnostique le mettant en oeuvre Download PDF

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WO2019017680A2
WO2019017680A2 PCT/KR2018/008085 KR2018008085W WO2019017680A2 WO 2019017680 A2 WO2019017680 A2 WO 2019017680A2 KR 2018008085 W KR2018008085 W KR 2018008085W WO 2019017680 A2 WO2019017680 A2 WO 2019017680A2
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mir
parkinson
disease
mirna
present
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PCT/KR2018/008085
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Korean (ko)
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WO2019017680A3 (fr
WO2019017680A9 (fr
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류현정
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국민대학교 산학협력단
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Priority claimed from KR1020170116583A external-priority patent/KR101956315B1/ko
Application filed by 국민대학교 산학협력단 filed Critical 국민대학교 산학협력단
Priority to EP18834700.9A priority Critical patent/EP3656876A4/fr
Priority to CN201880059124.3A priority patent/CN111344417A/zh
Priority to JP2020502648A priority patent/JP7030358B2/ja
Priority to US16/631,954 priority patent/US11447772B2/en
Publication of WO2019017680A2 publication Critical patent/WO2019017680A2/fr
Publication of WO2019017680A3 publication Critical patent/WO2019017680A3/fr
Publication of WO2019017680A9 publication Critical patent/WO2019017680A9/fr
Priority to US17/888,084 priority patent/US20220389426A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • 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

Definitions

  • the present invention relates to a method for diagnosing Parkinson's disease using miRNA whose expression is decreased or increased in Parkinson's disease, and a composition for preventing or treating Parkinson's disease using the miRNA.
  • Parkinson's disease is a degenerative disease of the central nervous system. It is caused by the deformation of the substantia nigra pars compacta of the middle cerebral cortex and the reduction of brain volume and the aggregation of ⁇ -synuclein ( ⁇ Syn) The disease has an incomplete gait, hand gestation, and stiff behavior.
  • Drug products for dopamine agonists which accounted for 47% of total sales in 2011, are expected to decline to 42% in 2021, while new pipeline drugs are expected to account for about 20% of sales by 2021 (R & D Trends: Parkinson's Disease, Datamonitor, 2012).
  • Positron emission tomography is a test to detect the presence of dopaminergic neurons in the brain.
  • a brain dopamine transporter positron emission tomography is performed, Symptoms can be confirmed. Parkinsonism, accompanied by Alzheimer's disease, Parkinson's disease, and Parkinson's disease, are similar to Parkinson's symptoms, but dopaminergic neurons are normal .
  • the brain MRI magnetic resonance imaging
  • MRI magnetic resonance imaging
  • Parkinson's disease blood test, urine test, electrocardiogram, and chest X-ray
  • medical problems may be misdiagnosed as Parkinson's disease, It is a test to confirm that there is no disease.
  • the present inventors made efforts to develop a technique capable of quickly and economically diagnosing Parkinson's disease and distinguishing it from the similar disease, and at the same time developing a technique capable of treating the disease. As a result, it has been confirmed that the expression level of specific miRNAs is specifically increased or decreased in the Parkinson's disease model, and that the diagnosis and treatment of Parkinson's disease can be efficiently performed, thereby completing the present invention.
  • Another object of the present invention is to provide a kit for the diagnosis or prognosis of Parkinson's disease.
  • the present invention provides a method for providing information for diagnosis of Parkinson's disease comprising the steps of:
  • the present invention further comprises the following steps:
  • the present invention provides a method for providing information for diagnosis of Parkinson's disease comprising the steps of:
  • miRNA &quot is transcribed as an RNA precursor of a hairpin structure, is cleaved by a dsRNA cleaving enzyme having RNase III cleavage activity, introduced into a protein complex called RISC, 15 to 25 bases of non-coding RNA are used.
  • the miRNA used in the present invention may be a miRNA represented by a specific nucleotide sequence (or a sequence number), as well as a miRNA precursor (pre-miRNA, pri-miRNA), miRNA having biological functions equivalent thereto, (Homologs or orthologs), polymorphisms such as polymorphisms, and derivatives thereof.
  • Such precursors, homologues, variants or derivatives may be specifically identified by miRBase release 20 (http://www.mirbase.org/) and include, under stringent conditions, the complement of the miRNA of Sequence Listing Nos. 1 to 9 And miRNA having a nucleotide sequence hybridizing with the sequence.
  • the miRNA used in the present invention may be a gene product of a miR gene, and the gene product may be a mature miRNA (for example, a 15-25 base or 19-25 base noncoding RNA) or miRNA precursors (e. G., Pre-miRNA or pri-miRNA as described above).
  • the term " nucleic acid " is used for nucleic acids including both RNA, DNA, and RNA / DNA (chimeras).
  • the DNA includes both cDNA, genomic DNA, and synthetic DNA.
  • the RNA includes both total RNA, mRNA, rRNA, miRNA, siRNA, snoRNA, snRNA, non-coding RNA and synthetic RNA.
  • the terms " synthetic DNA " and " synthetic RNA " refer to nucleic acid sequences that are synthetically produced using an automatic nucleic acid synthesizer, for example, based on a predetermined base sequence (either natural or non- DNA and RNA.
  • &quot unnatural sequence " is meant to be used broadly and includes sequences that differ from the native sequence, including, for example, substitution, deletion, insertion and / or addition of one or more nucleotides , A mutation sequence), a sequence containing one or more modified nucleotides (i.e., a modified sequence), and the like.
  • polynucleotides are used herein interchangeably with nucleic acids.
  • &quot includes mammals such as humans, primates including chimpanzees, pets such as dogs and cats, livestock such as cows, horses, sheep, goats, It is interpreted as meaning.
  • " normal group " is also interpreted in this meaning and means an object not affected by Parkinson's disease to be detected.
  • the sample included in the present invention is not limited as long as it is natural or artificially separated from the subject and includes genetic information related to Parkinson's disease of the subject, preferably feces, cells, blood, plasma, serum, hair or urine Etc., and more preferably, blood, plasma, serum or the like separated out of the body can be used.
  • the method for providing information for diagnosing Parkinson's disease of the present invention can be performed using various known methods such as a hybridization method, an immunoassay method, or a gene amplification method, but is not limited thereto .
  • the hybridization method uses a probe to confirm the presence of miRNA.
  • the term " probe &quot refers to a linear oligomer of natural or modified monomer or linkages and includes deoxyribonucleotides and ribonucleotides and can specifically hybridize to a target nucleotide sequence, Present or artificially synthesized.
  • the probe of the present invention is preferably a single strand, and is an oligodioxyribonucleotide.
  • the probe is used as a hybridizable array element and immobilized on a substrate.
  • substrates include rigid or semi-rigid supports such as membranes, filters, chips, slides, wafers, fibers, magnetic beads or non-magnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the hybridization array elements are arranged and immobilized on the substrate. Such immobilization is carried out by a chemical bonding method or a covalent bonding method such as UV.
  • the hybridization array element may be bonded to a glass surface modified to include an epoxy compound or an aldehyde group, and may also be bound by UV on a polylysine coating surface.
  • the hybridization array element can be coupled to the substrate via a linker (e.g., ethylene glycol oligomer and diamine).
  • sample DNA to be applied to the microarray of the present invention can be labeled and hybridized with the array elements on the microarray.
  • the hybridization conditions can be varied. Further, the detection and analysis of hybridization degree can be variously performed depending on the labeling substance.
  • the label of the probe may provide a signal to detect hybridization, which may be linked to an oligonucleotide.
  • Suitable labels include, but are not limited to, fluorescent moieties such as fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), terminal deoxynucleotidyl transferase (TdT)
  • fluorescent moieties such as fluorescein, phycoerythrin, rhodamine, lissamine, and Cy3 and Cy5 (Pharmacia), terminal deoxynucleotidyl transferase (TdT)
  • TdT terminal deoxynucleotidyl transferase
  • magnetic particles radioactive isotopes
  • mass labels electron dense particles
  • enzymes alkaline phosphatase or horseradish peroxidase
  • joins substrates for enzymes, heavy metals
  • Markers can be obtained using a variety of methods routinely practiced in the art, such as nick translation, the Multiprime DNA labeling systems booklet (Amersham, 1989) and the kaination method (Maxam & Gilbert, Methods in Enzymology, 65: 499 (1986)).
  • the label provides signals that can be detected by fluorescence, radioactivity, color measurement, weighing, X-ray diffraction or absorption, magnetism, enzymatic activity, mass analysis, binding affinity, hybridization high frequency, and nanocrystals.
  • suitable hybridization conditions can be determined by a series of procedures by an optimization procedure. This procedure is performed by a person skilled in the art in a series of procedures to establish a protocol for use in the laboratory. Conditions such as, for example, temperature, concentration of components, hybridization and washing time, buffer components and their pH and ionic strength depend on various factors such as probe length and GC amount and target nucleotide sequence. Detailed conditions for hybridization are described in Joseph Sambrook, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001); And M.L.M. Anderson, Nucleic Acid Hybridization, Springer-Verlag New York Inc .; N.Y. (1999).
  • hybridization signal generated through the hybridization reaction is detected.
  • the hybridization signal can be carried out in various ways depending on, for example, the type of label attached to the probe. For example, when a probe is labeled with an enzyme, the substrate of the enzyme can be reacted with the result of hybridization reaction to confirm hybridization.
  • the combination of enzymes / substrates that may be used is a combination of a peroxidase (e.g., horseradish peroxidase) with a chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis- Acetyl-3,7-dihydroxyphenox), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2, 2'-Azine-di [3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol / pyronine; (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate, and ECF substrate; alkaline phosphatase and bromochloroindoleyl phosphate; Glucose oxidas
  • the hybridization signal to the sequence of the miRNA in the biological sample is up-regulated or down-regulated compared to the normal sample, it is diagnosed as Parkinson's disease.
  • the method for providing information for diagnosing Parkinson's disease of the present invention can be carried out by an immunoassay method.
  • the immunoanalytical methods can be used for immunoassays such as radioimmunoassay, radioactive immunoprecipitation, immunoprecipitation, enzyme-linked immunosorbent assay, capture-ELISA, inhibition or hardwood analysis, sandwich analysis, flow cytometry, But are not limited to, chemical purification. Methods for such immunoassays are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), inMethodsin Molecular Biology, Vol. 1, Walker, J.M. ed., Humana Press, NJ, 1984; And Ed Harlow and David Lane, UsingAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999, which is incorporated herein by reference.
  • the method for providing information for diagnosis of Parkinson's disease of the present invention can be carried out by a gene amplification method. Detection of miRNA using the gene amplification method can be carried out using various methods known in the art. In this case, a primer or a probe of miRNA can be used.
  • the gene amplification reaction is performed to examine the expression level of the miRNA gene. Since the present invention analyzes the expression level of the gene, the amount of the mRNA of the marker is examined in a sample (for example, a cell) to be analyzed to determine the expression level of the marker gene. Therefore, in principle, the present invention carries out a gene amplification reaction using mRNA in a biological sample as a template and a primer binding to mRNA or cDNA.
  • RNA is isolated from the sample to obtain mRNA. Separation of total RNA can be carried out according to conventional methods known in the art (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001); Tesniere, C. et al., Plant Mol Biol. Rep., 9: 242 (1991); Ausubel, FM et al., Current Protocols in Molecular Biology, John Willey & Sons (1987) and Chomczynski, P. et al., Anal. Biochem. 162: 156 (1987)).
  • Trizol can be used to easily isolate total RNA in a cell.
  • cDNA is synthesized from the separated mRNA, and this cDNA is amplified. Since the total RNA of the present invention is isolated from a human sample, it has a poly-A tail at the end of the mRNA, and the cDNA can be easily synthesized using the oligo dT primer and the reverse transcriptase using such a sequence characteristic ( (1989); and Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed., Cold Spring Harbor Press (2001)). Next, the synthesized cDNA is amplified through gene amplification reaction. The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure.
  • Suitable conditions for nucleic acid hybridization to form such a double-stranded structure include Nucleic Acid Hybridization, A (Hayes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, PracticalApproach, IRL Press, Washington, DC (1985).
  • a variety of DNA polymerases can be used in the amplification of the present invention, including the " Clenow " fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase.
  • Polymerases are thermostable DNA polymerases that can be obtained from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermusfiliformis, Thermisflavus, Thermococcus literalis, and Pyrococcus furiosus (Pfu).
  • Taq Thermus aquaticus
  • Tth Thermus thermophilus
  • Thermusfiliformis Thermisflavus
  • Thermococcus literalis Thermococcus literalis
  • Pyrococcus furiosus Pyrococcus furiosus
  • the excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with a joinder such as Mg2 +, dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Therefore, the amplification process of the present invention can be carried out in a single reaction without changing the conditions such as the addition of reactants.
  • annealing or hybridization is carried out under stringent conditions which allow specific binding between the target nucleotide sequence and the primer.
  • the stringent conditions for annealing are sequence-dependent and vary with environmental variables.
  • &quot refers to a reaction to amplify a nucleic acid molecule.
  • a variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- , The method of Miller, HI (WO 89/06700) and Davey, C. et al (EP 329,822), the method of ligase chain reaction (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed.
  • the gene amplification method of the present invention is carried out according to the polymerase chain reaction (PCR) disclosed in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159.
  • PCR polymerase chain reaction
  • PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR.
  • real-time PCR differential display PCR (DD-PCR), rapid amplification of cDNA ends (RACE), multiplex PCR, inverse polymerase chain reaction chain reaction (IPCR), vectorette PCR, thermal asymmetric interlaced PCR (TAIL-PCR) and multiplex PCR have been developed for specific applications.
  • DD-PCR differential display PCR
  • RACE rapid amplification of cDNA ends
  • IPCR inverse polymerase chain reaction chain reaction
  • TAIL-PCR thermal asymmetric interlaced PCR
  • multiplex PCR have been developed for specific applications.
  • the primer used in the gene amplification method is an oligonucleotide having a sequence complementary to the cDNA sequence of the miRNA.
  • " primer " refers to a single-stranded DNA strand that can act as a starting point for template-directed DNA synthesis under suitable conditions (i. E., Four other nucleoside triphosphates and polymerase) Means an oligonucleotide.
  • the suitable length of the primer is typically 15-30 nucleotides, although it varies depending on various factors such as temperature and use of the primer. Short primer molecules generally require lower temperatures to form sufficiently stable hybrid complexes with the template.
  • the sequence of the primer does not need to have a sequence completely complementary to a partial sequence of the template, and it is sufficient if the primer has sufficient complementarity within a range capable of hybridizing with the template and acting as a primer. Therefore, the primer set in the present invention does not need to have a perfectly complementary sequence to the cDNA sequence of the marker, which is a template, and it is sufficient if it has sufficient complementarity within the range capable of hybridizing to the sequence and acting as a primer.
  • the primer used in the present invention has a sequence completely complementary to the cDNA sequence of the marker.
  • a primer design can be easily carried out by those skilled in the art with reference to the cDNA sequence of the miRNA, for example, by using a program for primer design (e.g., PRIMER 3 program).
  • a program for primer design e.g., PRIMER 3 program.
  • the amplified cDNA of the marker is analyzed by a suitable method to examine the expression level of the marker gene.
  • a suitable method to examine the expression level of the marker gene For example, the result of amplification reaction described above is subjected to gel electrophoresis, and the resultant band is observed and analyzed to examine the expression level of the marker gene.
  • miRNA is a biomolecule whose expression is decreased or increased in Parkinson's disease.
  • " high expression (or overexpression) " or " up-regulation &quot means that the degree of expression of a subject nucleotide sequence or protein in a biological sample to be investigated is higher than that of a normal sample .
  • expression analysis methods conventionally used in the art, such as RT-PCR or ELISA methods (see Sambrook, J. et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001) In the case of expression analysis, it means that the expression is analyzed to be high.
  • the miRNA of the present invention when the miRNA of the present invention is highly expressed or underexpressed by 10% or more as compared with the normal sample, it is determined as " high expression " or " low expression " It is judged as a bottle.
  • the miRNA of the present invention is up-regulated or down-regulated in patients with Parkinson's disease as compared with normal individuals, Preferably upwards or downwards by more than 50%.
  • a method of inhibiting miR-494-3p, miR-501-5p, miR-1244, miR-6768-5p, miR- miR-4767 and miR-3064-5p <
  • RTI ID 0.0 > 1. < / RTI >
  • the kit of the present invention is a kit comprising hsa-miR-494-3p, hsa-miR-1244, hsa-miR-6768-5p, hsa- further comprising a nucleic acid capable of specifically binding to one or more miRNAs selected from the group consisting of hsa-miR-501-5p, hsa-miR-1226-5p, hsa-miR-4767 and hsa-miR- do.
  • kits of the present invention may include known nucleic acids or nucleic acids that can be detected in the future that enable the detection of Parkinson's disease in addition to the nucleic acids and may also include antibodies for measuring known markers for Parkinson's disease have.
  • the nucleic acids contained in the kits of the present invention may be packaged separately or in any combination in other containers.
  • the kit of the present invention may contain a kit for extracting nucleic acid (for example, total RNA) from body fluids, cells or tissues, a labeling fluorescent substance, an enzyme for nucleic acid amplification and a medium, instructions for use and the like.
  • nucleic acid for example, total RNA
  • the kit of the present invention is a device for measuring the Parkinson's disease marker in which the nucleic acid is bound or attached to, for example, a solid phase.
  • a solid phase examples include plastic, paper, glass, and silicone.
  • the preferred solid phase material is plastic because of ease of processing.
  • the shape of the solid phase is arbitrary, for example, a square, a circle, a rectangle, a film, or the like.
  • the kit of the present invention may include a nucleic acid capable of specifically binding to at least one, preferably at least two, more preferably at least three of each of the miRNAs.
  • the present invention provides a method for screening a Parkinson's disease-inducing substance comprising the steps of:
  • the screening method of the present invention further comprises the following steps:
  • the present invention provides a method for screening a Parkinson's disease-inducing substance comprising the steps of:
  • the present invention provides a method of screening for a therapeutic agent for Parkinson's disease comprising the steps of:
  • the screening method of the present invention further comprises the following steps:
  • the present invention provides a method of screening for a therapeutic agent for Parkinson's disease comprising the steps of:
  • candidate substance used in referring to the screening method of the present invention refers to an unknown substance (for example, various natural substances used in screening) for examining whether or not there is activity to induce, reduce, , Compound libraries, gene or protein libraries, etc.).
  • the term "therapeut agent for Parkinson's disease " means a drug (pharmaceutical composition), a health functional food or a diuretic known to exhibit or exhibit pharmacological activity against Parkinson's disease.
  • drugs and functional foods known in the art having pharmacological activity against Parkinson's disease can be used to diagnose the sensitivity of a subject diagnosed with Parkinson's disease or to predict the prognosis.
  • the present invention can be applied to screening a substance having pharmacological activity against Parkinson's disease among candidate substances whose pharmacological activity against Parkinson's disease is not known.
  • the present invention provides a pharmaceutical composition comprising 1 (1) selected from the group consisting of miR-494-3p, miR-501-5p, miR-1244, miR-6768-5p, miR- Or more miRNA as an active ingredient.
  • the present invention also provides a composition for preventing, ameliorating or treating Parkinson's disease.
  • the present invention provides a pharmaceutical composition comprising 1 (1) selected from the group consisting of miR-494-3p, miR-501-5p, miR-1244, miR-6768-5p, miR- Or more miRNA to a patient in need thereof.
  • the composition of the present invention is a pharmaceutical composition.
  • the pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not.
  • the pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, kaolin, sorbiol, sorbitol, etc.
  • Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
  • composition of the present invention can be administered orally or parenterally.
  • parenteral administration the composition can be administered by nasal administration, topical administration, intravenous infusion, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.
  • the appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient, Usually, a skilled physician can readily determine and prescribe dosages effective for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.001-100 mg / kg.
  • the pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container.
  • the formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
  • the pharmaceutical composition of the present invention can be prepared in the form of external preparation for skin, aerosol, spray, eye drop, oral preparation and injection.
  • the composition of the present invention is a food composition.
  • composition of the present invention When the composition of the present invention is prepared with a food composition, it includes components that are ordinarily added in the manufacture of food, and includes, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavors.
  • carbohydrates are monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose, oligosaccharides and the like; And polysaccharides such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol.
  • Natural flavorings tau martin, stevia extract [e.g., rebaudioside A, glycyrrhizin, etc.]
  • synthetic flavors sacharin, aspartame, etc.
  • the food composition of the present invention when prepared as a drink, citric acid, liquid fructose, sugar, glucose, acetic acid, malic acid, juice, mulberry extract, jujube extract and licorice extract may be further added.
  • the present invention provides a method for providing information for diagnosis of Parkinson's disease.
  • the present invention also provides a composition for preventing, ameliorating or treating Parkinson's disease.
  • the miRNA of the present invention is specifically reduced or increased in the Parkinson's disease model and can be effectively used for diagnosis and treatment of Parkinson's disease.
  • FIG. 1 shows miRNAs with reduced expression levels in the death phase of Parkinson's disease model cells.
  • Figure 2 shows miRNAs with increased expression levels in the death phase of Parkinson's disease model cells.
  • Figure 3 is a photograph of an internal control with 6-OHDA injected.
  • Substantia nigra (A) in the left hemisphere without any treatment of brain tissue is fully preserved, whereas 6-hydroxy dopamine injected in the right hemisphere is over-constricted by degeneration of tissue as well as nerve cell death of Substantia nigra (B) have.
  • Figure 4 shows the results of confirming that the nerve cells of Substantia nigra are alive due to the effect of 494-3p injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • Figure 5 shows the results of confirming that the nerve cells of Substantia nigra were alive due to the effect of miR1244 injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • FIG. 6 shows the results of confirming that the nerve cells of Substantia nigra were alive due to the effect of miR4324 injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • Figure 7 shows the results of confirming that the nerve cells of Substantia nigra were alive due to the effect of miR4726-5p injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • Figure 8 shows the results of confirming that the nerve cells of Substantia nigra were alive due to the effect of miR6768-5p injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • Figure 9 shows the results of confirming that the nerve cells of Substantia nigra are alive due to the effect of miR501-5p injected with 6-OHDA. Lesions in A (normal) and B (injected) in the same subject were confirmed by H & E staining.
  • C57BL / 6 SH-SY5Y neuroblast cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium, Invitrogen, MD, USA) supplemented with heat-inactivated fetal bovine serum (GIBCO, MD, USA) (Jang, S.-W., Oh, M.-S., Yang, SI & Cho, E.-M., Gene expression profiles of human neuroblastoma cells exposed to CuO nanoparticles and Cu ions. BioChip Journal 10, 140-149 (2016)). 6-hydroxy dopamine (Sigma-Aldrich, St. Louis, Mo., USA) was dissolved in PBS (phosphate buffered saline) and stored at -80 ° C., , Especially light sensitive.
  • PBS phosphate buffered saline
  • a stable tetrazolium salt, WST-1 assay (Sigma-Aldrich, St. Louis, Mo., USA) was used.
  • SH-SY5Y cells were plated in a 96- -Hydroxydopamine (6OHDA, 25 [mu] M, 24h). Only WST-1 solution (10 ⁇ l / well) was added 2 hours before the end of the experiment. After the completion of the experiment, the color changed at 450 nm was measured and confirmed.
  • RNA of SH-SY5Y cell line treated with 6OHDA 25 ⁇ M, 24 h was isolated using Trizol reagent. The total amount and purity of the isolated RNA were measured using a NanoDrop ND-2000 spectrophotometer (Nano Drop, Delaware, USA) at 260/280 nm (ratio 1.8 -2.0) ratio (Kim, GW et al . Integrative analysis of differential gene expression and DNA methylation of ethylbenzene-exposed workers. BioChip Journal 9, 259-267 (2015)).
  • Affymetrix miRNA 4.0 array (Lee, SEE Identification and characterization of microRNAs in acrolein-stimulated endothelial cells: Implications for vascular disease. BioChip Journal 9, 144-155 (2015)) was used for miRNA expression profiling assay. Microarray data Using the Gene Expression Omnibus (GEO) database.
  • GEO Gene Expression Omnibus
  • TargetScan6.2 DB was used and miRanda algorithm (Kim, GW et al., Integrative analyzes of differential gene expression and DNA methylation of ethylbenzene-exposed workers. BioChip Journal 9, 259-267 (2015)).
  • miRNA target that exhibits the most frequent and large expression changes is the Gene Ontology (GO) categories (Cho, H. et al., A relationship between miRNA and gene expression in the mouse Sertoli cell line after exposure to bisphenol A. BioChip Journal 4, 75-81 (2010);..
  • 6-OHDA was injected into the noradrenalin transporter blocker desipramine (12.5 mg / kg; Sigma, St. Louis, USA) so that toxicity by 6-OHDA (Sigma, St. Louis, USA)
  • the animals were injected into the abdominal cavity 30 min before the experiment.
  • the animals were anesthetized by intraperitoneal injection of a mixture of ketamine (40 mg / kg) and xylazine (5 mg / kg), and they were anesthetized with PBS
  • the skin was opened and the head bone was exposed. The parietal point was confirmed, and a small hole was drilled using a dental drill at the point moved 1.1 mm backward and 1.2 mm rightward.
  • a 26-gauge needle was inserted into the medial forebrain bundle (MFB) through the hole at 5.0 mm to the abdomen.
  • MFB medial forebrain bundle
  • a 5 ⁇ L Hamilton syringe was used to inject the miRNA at 0.5 ⁇ L / min. After 5 min, the Hamilton syringe was removed and the skin was sutured. On the left, nothing was injected for use as an internal control.
  • the animals were anesthetized with a mixture of ketamine (70 mg / kg) and xylazine (8 mg / kg) and perfused with 4% paraformaldehyde (0.1 M phosphate buffer, pH 7.4) through the heart.
  • miR which is most prominent in the decrease of expression in the cell death stage of the PD model among the miRNAs which remarkably show the increase or decrease of the expression, is as shown in Fig.
  • miR494-3p has not yet been reported for Parkinson's disease, and a significant reduction has been repeated for each experiment.
  • the miR showing a reduction of more than -2.0 (over 30%) based on the log ratio is listed in FIG.
  • Bioinformatic data analysis of dual miR-1244 showed that it targeted a protein called 'TBC1 domain family member 2B'.
  • FIG. 2 shows the expression of miR which is most prominent in the cell death of the PD model among the miRNAs which remarkably increase or decrease the expression.
  • the miRNAs most prominent in the decrease of expression in the cell death stage of PD model are hsa-miR-494-3p (SEQ ID No. 1: UGAAACAUACACGGGAAACCUC), hsa-miR-1244 (SEQ ID No. 2: AAGUAGUUGGUUUGUAUGAGAUGGUU), hsa- miR-4768-5p (SEQ ID NO: 5 sequence: AGGGCCAGAGGAGCCUGGAGUGG), hsa-miR-4324 (SEQ ID NO: 4 sequence: CCCUGAGACCCUAACCUUAA) -501-5p (SEQ ID NO: 6 sequence: AAUCCUUUGUCCCUGGGUGAGA).
  • MiRNA-4767 (SEQ ID No. 8: CGCGGGCGCUCCUGGCCGCCGCC), and hsa-miR-1226-5p (SEQ ID NO: miR-3064-5p (SEQ ID NO: 9 sequence: UCUGGCUGUUGUGGUGUGCAA).
  • the efficacy of the brain was evaluated by comparing H & E staining with cell survival in the substantia nigra pas compacta, a major lesion of Parkinson 's disease.
  • Tissue fixation In order to inactivate the cell's enzymes and convert the tissue components into insoluble state by coagulation or precipitation, they are immersed in a fixative at 4 ° C Or more.
  • FIGS. 3 to 9 in the internal control (FIG. 3) in which only 6-OHDA was injected, the neuronal cell death and the deformation of tissues were excessively contracted, (Fig. 4), miR1244 (Fig. 5), miR4324 (Fig. 6), miR4726-5p It was confirmed to be alive. This proves that the miRNAs of the present invention have an excellent effect in defending the neuronal apoptosis.
  • ⁇ 120> miRNA as a biomarker for parkinson's disease and diagnostic kit

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Abstract

L'invention concerne une méthode destinée à fournir des informations sur le diagnostic de la maladie de Parkinson. L'invention concerne également une composition destinée à prévenir, atténuer ou traiter la maladie de Parkinson. Le micro-ARN selon l'invention peut être utilisé efficacement dans le diagnostic et le traitement de la maladie de Parkinson étant donné qu'il présente une expression régulée spécifiquement à la baisse ou à la hausse dans un modèle de maladie de Parkinson.
PCT/KR2018/008085 2017-07-19 2018-07-17 Micro-arn utilisé en tant que biomarqueur de la maladie de parkinson et kit diagnostique le mettant en oeuvre WO2019017680A2 (fr)

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EP18834700.9A EP3656876A4 (fr) 2017-07-19 2018-07-17 Micro-arn utilisé en tant que biomarqueur de la maladie de parkinson et kit diagnostique le mettant en oeuvre
CN201880059124.3A CN111344417A (zh) 2017-07-19 2018-07-17 作为帕金森病生物标记物的miRNA及利用其的诊断试剂盒
JP2020502648A JP7030358B2 (ja) 2017-07-19 2018-07-17 パーキンソン病バイオマーカーとしてのｍｉＲＮＡ及びこれを用いた診断キット
US16/631,954 US11447772B2 (en) 2017-07-19 2018-07-17 miRNA as biomarker for Parkinson's disease and diagnostic kit using same
US17/888,084 US20220389426A1 (en) 2017-07-19 2022-08-15 miRNA AS BIOMARKER FOR PARKINSON'S DISEASE AND DIAGNOSTIC KIT USING SAME

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KR20170091417 2017-07-19
KR10-2017-0091417 2017-07-19
KR1020170116583A KR101956315B1 (ko) 2017-07-19 2017-09-12 파킨슨병 바이오마커로서의 miR494 및 이를 이용한 진단키트
KR10-2017-0116583 2017-09-12
KR1020170132870A KR102062787B1 (ko) 2017-07-19 2017-10-12 파킨슨병 바이오마커로서의 miR501 및 이를 이용한 진단키트
KR10-2017-0132870 2017-10-12
KR10-2017-0132866 2017-10-12
KR10-2017-0132868 2017-10-12
KR1020170132867A KR101956323B1 (ko) 2017-07-19 2017-10-12 파킨슨병 바이오마커로서의 miR6768 및 이를 이용한 진단키트
KR10-2017-0132869 2017-10-12
KR1020170132866A KR102063244B1 (ko) 2017-07-19 2017-10-12 파킨슨병 바이오마커로서의 miR1244 및 이를 이용한 진단키트
KR10-2017-0132867 2017-10-12
KR1020170132868A KR101956328B1 (ko) 2017-07-19 2017-10-12 파킨슨병 바이오마커로서의 miR4324 및 이를 이용한 진단키트
KR1020170132869A KR101956329B1 (ko) 2017-07-19 2017-10-12 파킨슨병 바이오마커로서의 miR4726 및 이를 이용한 진단키트

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