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WO2024029755A1 - Modèle animal de maladie de charcot-marie-tooth - Google Patents

Modèle animal de maladie de charcot-marie-tooth Download PDF

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WO2024029755A1
WO2024029755A1 PCT/KR2023/009373 KR2023009373W WO2024029755A1 WO 2024029755 A1 WO2024029755 A1 WO 2024029755A1 KR 2023009373 W KR2023009373 W KR 2023009373W WO 2024029755 A1 WO2024029755 A1 WO 2024029755A1
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hspb8
marie
charcot
tooth disease
protein
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Korean (ko)
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고형종
강경화
한지은
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Research Foundation for Industry Academy Cooperation of Dong A University
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/60—New or modified breeds of invertebrates
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    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/056—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to mutation of coding region of the transgene (dominant negative)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates to an animal model of Charcot-Marie-Tooth disease caused by K141N or K141T mutation of HSPB8.
  • Hereditary peripheral neuropathy is largely divided into three types: hereditary motor and sensory neuropathy (HMSN), hereditary motor neuropathy (HMN), and hereditary sensory neuropathy (HSN).
  • HMSN hereditary motor and sensory neuropathy
  • HSN hereditary sensory neuropathy
  • CMT Charcot-MarieTooth disease
  • Charcot-Marie-Tooth disease was first known in 1886 by the Frenchmen Charcot and Marie, and the Englishman Tooth, and is commonly called CMT disease, named after the first letters of their names.
  • Charcot-Marie-Tooth (CMT) disease, or hereditary motor and sensory neuropathy (HMSN) is also known as distal hereditary motor neuropathy (dHMN), hereditary sensory neuropathy (HMSN).
  • HSN Hereditary Neuropathy with a liability to pressure palsy
  • HNPP Hereditary Neuropathy with a liability to pressure palsy
  • Charcot-Marie-Tooth disease is a general term for all genetic diseases in which motor and sensory nerves are abnormal on nerve conduction tests.
  • the incidence of CMT is one in 2,500 people, which is the highest among rare inherited diseases.
  • CMT patients the muscles in the feet and hands gradually atrophy, resulting in weakened strength, deformed foot and hand shapes, facial paralysis, and hearing impairment. Onset usually occurs around the age of 10, but rarely occurs after the age of 30.
  • patients' symptoms range from mild, almost normal, to very severe, requiring assistance with walking or relying on a wheelchair.
  • CMT myelin defect type
  • CMT2 axonal type
  • CMTX CMTX type
  • CMT genetic diagnosis of CMT relies on direct sequencing of the primary causative gene, but despite being a difficult process, it has low isolation efficiency.
  • WES Whole Exome Sequencing
  • NGS Next-generation Sequencing
  • CMT1A genetic research on various diseases, especially heterogeneous diseases such as CMT, is expanding and accelerating.
  • onapriston, ascorbic acid, and NT-3 neurotrophin-3 have been reported as reasonable and potential drug treatments for CMT1A, the most common type of hereditary motor sensory neuropathy. .
  • the object of the present invention is to provide a composition for diagnosing Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a diagnostic kit for Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide an animal model for Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a method for providing information necessary to diagnose the possibility of developing Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a screening method for a candidate drug for the treatment of Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a method for diagnosing Charcot-Marie-Tooth disease.
  • an object of the present invention is to provide a method of treating Charcot-Marie-Tooth.
  • an object of the present invention is to provide a diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.
  • the object of the present invention is to provide a use for the prevention or treatment of Charcot-Marie-Tooth disease of a vector containing PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same.
  • the present invention provides a composition for diagnosing Charcot-Marie-Tooth disease, which includes an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.
  • the present invention provides a kit for diagnosing Charcot-Marie-Tooth disease comprising the composition.
  • the present invention provides a pharmaceutical composition for preventing or treating Charcot-Marie-Tooth disease, comprising PINK1 or Parkin protein, a nucleic acid molecule encoding it, or an activator thereof.
  • the present invention provides a Charcot-Marie-Tooth disease animal model expressing HSPB8 protein containing a K141N or K141T mutation.
  • the present invention provides a method for providing information necessary for diagnosing the possibility of developing Charcot-Marie-Tooth disease, which includes detecting the K141N or K141T mutation of the HSPB8 protein in a sample isolated from a subject.
  • the present invention provides a screening method for a candidate drug for the treatment of Charcot-Marie-Tooth disease.
  • the present invention provides a method for diagnosing Charcot-Marie-Tooth disease, comprising detecting a K141N or K141T mutation in the HSPB8 protein.
  • the present invention provides a method for treating Charcot-Marie-Tooth, comprising administering a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same to an individual suffering from Charcot-Marie-Tooth disease. provides.
  • the present invention provides diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting K141N or K141T mutations in the HSPB8 protein.
  • the present invention provides the use of a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same for the prevention or treatment of Charcot-Marie-Tooth disease.
  • Figure 1 shows the results of HSPB8 expression and lifespan analysis of transgenic Drosophila:
  • Figure 1A Results of immunoblot analysis of control Drosophila elav (elav-GAL4), wild-type HSPB8-expressing Drosophila ( elav HSPB8 WT ), HSPB8 K141T -expressing Drosophila ( elav HSPB8 K141T ) and HSPB8 K141E -expressing Drosophila ( elav HSPB8 K141E );
  • Figure 1B Photomicrograph of a 5-day-old fruit fly
  • Figure 1D Lifespan curve of female Drosophila.
  • Figure 2 is a diagram confirming the occurrence of sensory defects due to HSPB8 mutation:
  • FIG. 2A Heat nociception assay results of L3 larvae ( ppk ) transgenic with the ppk-GAL4 driver;
  • Figure 2b Result of response delay analysis for thermal nociception in ppk- expressing larvae, HSPB8 WT -expressing larvae ( ppk HSPB8 WT ), HSPB8 K141T -expressing larvae ( ppk HSPB8 K141T ) and HSPB8 K141E- expressing larvae ( ppk HSPB8 K141E ) at 40°C.
  • Figure 3 is a diagram confirming changes in exercise capacity due to HSPB8 mutation:
  • elav HSPB8 WT Drosophila expressing wild type HSPB8;
  • elav HSPB8 K141E Drosophila expressing HSPB8 K141E ;
  • Figure 3a and b Average walking speed of 5-day-old fruit flies (A) and 15-day-old fruit flies (B); and
  • Figure 3c and d Movement trajectories of 5-day-old fruit flies (C) and 15-day-old fruit flies (D).
  • Figure 4 shows the occurrence of mitochondrial dysfunction caused by mutations and the expression of PINK1 and parkin . This is a diagram confirming the recovery of mitochondrial dysfunction by introducing:
  • elav HSPB8 WT Drosophila expressing wild type HSPB8
  • elav HSPB8 WT Drosophila expressing wild type HSPB8;
  • elav HSPB8 K141E Drosophila expressing HSPB8 K141E ;
  • elav HSPB8 K141T PINK1 Drosophila expressing HSPB8 K141T and PINK1 ;
  • elav HSPB8 K141E PINK1 Drosophila expressing HSPB8 K141E and PINK1 ;
  • FIG. 4A Mitochondrial membrane potential ( ⁇ ) measured in Drosophila larva VNC
  • Figure 4b mt-Keima fluorescence image of Drosophila larva VNC
  • FIG. 4C Quantitative analysis of mitophagy in Drosophila larvae VNC
  • Figure 4d Climbing ability analysis of 15-day-old fruit flies
  • Figure 4e Average walking speed of 15-day-old fruit flies.
  • Figure 4f Movement trajectory of female fruit fly.
  • Figure 5 is a diagram confirming motor defects caused by motor neuron-specific HSPB8 mutant expression:
  • D42 Drosophila expressing motor neuron-specific D42 -GAL4;
  • D42 HSPB8 WT Drosophila expressing motor neuron-specific HSPB8 WT ;
  • D42 HSPB8 K141T Drosophila expressing motor neuron-specific HSPB8 K141T ;
  • D42 HSPB8 K141E Drosophila expressing motor neuron-specific HSPB8 K141E ;
  • Figure 5A Average walking speed of 5-day-old fruit flies
  • Figure 5b Average walking speed of 15-day-old fruit flies
  • Figure 5d Movement trajectory of 15-day-old fruit flies.
  • Figure 6 shows 15-day-old male transgenic Drosophila ( elav HSPB8 WT , elav HSPB8 K141T ) following PINK1 activation by KR administration (1 and 5 mM). and elav HSPB8 K141E ), which confirmed the recovery of motor skills:
  • Figure 6A Average walking speed of fruit flies
  • step of or “step of” do not mean “step for.”
  • the term "combination thereof" included in the Markushi format expression means a mixture or combination of one or more components selected from the group consisting of the components described in the Markushi format expression, It means including one or more selected from the group consisting of.
  • the present invention provides a composition for diagnosing Charcot-Marie-Tooth disease (CMT) disease, comprising an agent capable of detecting the K141N or K141T mutation of the small heat shock protein B8 (HSPB8) protein. It's about.
  • CMT Charcot-Marie-Tooth disease
  • HSPB8 small heat shock protein B8
  • detecting the K141N or K141T mutation of the HSPB8 protein may be detecting a nucleic acid molecule encoding the HSPB8 protein comprising the K141N or K141T mutation or the HSPB8 protein comprising the K141N or K141T mutation.
  • the agent capable of detecting a mutation is an antibody, antibody fragment, aptamer, Affilin, Affibody, Affimer, Affitin, Alphabody, Anticlin, DARpin, Fynomoer, Kunitz domain peptide, avidity multimer, peptidomimetic It may include peptidomimetics, receptors, ligands, or cofactors, and detection of protein mutations can be performed using Western blot, ELISA (enzyme linked immunosorbent assay), RIA (Radioimmunoassay), radioimmunodiffusion, or immunosorbent assay. It may be performed by one or more methods selected from the group consisting of electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, mass spectrometry, or protein microarray.
  • the antibody is a polyclonal antibody, monoclonal antibody, minibody, domain antibody, bispecific antibody, antibody mimetic, chimeric antibody, antibody conjugate, human antibody, or humanized antibody.
  • Fragments with immunological activity include Fab, Fd, Fab', dAb, F(ab'), F(ab') 2 , scFv (single chain fragment variable), Fv, single chain antibody, and Fv of the antibody. It may be a dimer, a complementarity determining region fragment, or a diabody.
  • an agent capable of detecting a mutation may include a primer pair, or probe, or a primer pair and probe that specifically recognizes a nucleic acid molecule encoding the HSPB8 protein, Detection of nucleic acid molecules can be performed using polymerase chain reaction, real-time RT-PCR, reverse transcription polymerase chain reaction, competitive RT-PCR, and nuclease protection assay (RNase, S1 nuclease assay). ), in situ hybridization, nucleic acid microarray, Northern blot, DNA chip, multiplex PCR, or ddPCR.
  • the term “detection” or “measurement” means confirming the presence or absence of a detected or measured object.
  • the terms “gene,” “nucleic acid,” “polynucleotide,” or “oligonucleotide” refer to DNA molecules, RNA molecules, or analogs thereof.
  • the terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” include, but are not limited to, DNA molecules, such as cDNA, genomic DNA, or synthetic DNA and RNA molecules, such as guide RNA, messenger RNA, or Contains synthetic RNA.
  • the terms “nucleic acid” and “polynucleotide” include single-stranded and double-stranded forms.
  • the term "primer” is a nucleic acid sequence with a short free 3-terminal hydroxyl group that can form a base pair with a complementary template and serves as a starting point for copying the template strand. It refers to a short nucleic acid sequence that functions as a point. Primers can initiate DNA synthesis in the presence of four different nucleoside triphosphates and reagents for polymerization (i.e., DNA polymerase or reverse transcriptase) in an appropriate buffer solution and temperature.
  • probe refers to a nucleic acid fragment such as RNA or DNA that is as short as a few bases or as long as several hundred bases, capable of forming a specific binding to mRNA, and is labeled to determine the presence or absence of a specific mRNA. You can check. Probes may be manufactured in the form of oligonucleotide probes, single stranded DNA probes, double stranded DNA probes, RNA probes, etc. In the present invention, hybridization is performed using a probe complementary to the AR or AR-V7 gene, and the level of gene expression can be diagnosed based on hybridization. Selection of appropriate probes and hybridization conditions can be modified based on those known in the art, so the present invention is not particularly limited thereto.
  • Primers or probes of the present invention can be chemically synthesized using the phosphoramidite solid support method or other well-known methods. These nucleic acid sequences can also be modified using many means known in the art. Non-limiting examples of such modifications include methylation, capping, substitution of a native nucleotide with one or more homologues, and modifications between nucleotides, such as uncharged linkages (e.g., methyl phosphonate, phosphotriester, phosphoronucleotide). amidate, carbamate, etc.) or charged linkages (e.g. phosphorothioate, phosphorodithioate, etc.).
  • uncharged linkages e.g., methyl phosphonate, phosphotriester, phosphoronucleotide
  • charged linkages e.g. phosphorothioate, phosphorodithioate, etc.
  • an antibody is a term known in the art and refers to a specific protein molecule directed to an antigenic site.
  • an antibody refers to an antibody that specifically binds to the amino acid substitution mutation of HSPB8, which is a marker of the present invention, and the antibody can be produced using a well-known method. This also includes partial peptides that can be made from the above proteins.
  • the form of the antibody of the present invention is not particularly limited, and as long as it is a polyclonal antibody, monoclonal antibody, or has antigen binding properties, a portion thereof is also included in the antibody of the present invention, and all immunoglobulin antibodies are included.
  • the antibodies of the present invention also include special antibodies such as humanized antibodies.
  • the present invention relates to a diagnostic kit for Charcot-Marie-Tooth disease comprising the composition of the present invention.
  • the kit may be an RT-PCR kit, a microarray chip kit, or a protein chip kit.
  • the present invention provides a pharmaceutical for the prevention or treatment of Charcot-Marie-Tooth disease, comprising a vector containing a PINK1 (PTEN-induced putative kinase 1) or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same. It relates to composition.
  • PINK1 PTEN-induced putative kinase 1
  • Parkin protein an activator thereof
  • nucleic acid molecule encoding the same. It relates to composition.
  • the Charcot-Marie-Tooth disease may be Charcot-Marie-Tooth disease caused by a K141N or K141T mutation in the HSPB8 protein.
  • the activator of PINK1 may be kinetin riboside (KR) (N6-furfuryl adenine riboside).
  • KR kinetin riboside
  • the composition can improve symptoms of motor defects, mitochondrial dysfunction, or reduced mitophagy levels caused by the K141N or K141T mutation of the HSPB8 protein.
  • vector refers to any nucleic acid containing a competent nucleotide sequence that is inserted into a host cell and recombines with and integrates into the host cell genome, or replicates spontaneously as an episome.
  • vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors, etc.
  • the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.
  • Pharmaceutically acceptable carriers include, for example, carriers for oral administration, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc., and carriers for parenteral administration, such as water, suitable oils, saline solutions, aqueous glucose and glycols, etc. and may additionally contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid.
  • Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol.
  • benzalkonium chloride methyl- or propyl-paraben and chlorobutanol.
  • other pharmaceutically acceptable carriers those described in the following literature may be referred to (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).
  • the pharmaceutical composition according to the present invention can be formulated in a suitable form according to methods known in the art along with a pharmaceutically acceptable carrier as described above. That is, the pharmaceutical composition of the present invention can be prepared in various forms for parenteral or oral administration according to known methods, and a representative example of the formulation for parenteral administration is an isotonic aqueous solution or suspension as an injectable formulation. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be dissolved in saline solution or buffer solution and formulated for injection. Additionally, dosage forms for oral administration include, but are not limited to, powders, granules, tablets, pills, and capsules.
  • the pharmaceutical composition formulated in the manner described above can be administered in an effective amount through various routes including orally, transdermally, subcutaneously, intravenously, or intramuscularly.
  • administration refers to introducing a predetermined substance into a patient by any appropriate method. This means that the substance can be administered through any general route as long as it can reach the target tissue.
  • effective amount refers to the amount that exhibits a preventive or therapeutic effect when administered to a patient.
  • the dosage of the pharmaceutical composition according to the present invention may vary depending on various factors such as the type and severity of the patient's disease, age, gender, weight, sensitivity to the drug, type of current treatment, administration method, target cells, etc., and may vary depending on the field of the art. can be easily determined by experts.
  • the pharmaceutical composition of the present invention can be administered in combination with conventional therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or multiple times.
  • the amount that can obtain the maximum effect with the minimum amount without side effects can be administered, more preferably 1 to 10,000 ⁇ g/kg of body weight/day, and even more preferably 10 to 1,000.
  • the effective dose is mg/kg body weight/day and can be administered repeatedly several times a day.
  • the invention relates to an animal model of Charcot-Marie-Tooth disease expressing HSPB8 protein containing a K141N or K141T mutation.
  • the animal model is capable of expressing K141N or K141T mutant HSPB8 protein in neurons, motor neurons, or multi-dendritic sensory neurons.
  • the animal model may be Drosophila.
  • K141N or K141T mutant HSPB8 in the neuron-specific promoter elav , motor neuron-specific promoter D42 , or multi-dendritic sensory neurons-specific promoter ppk in the animal model, K141N or K141T mutant HSPB8 in the neuron-specific promoter elav , motor neuron-specific promoter D42 , or multi-dendritic sensory neurons-specific promoter ppk.
  • Nucleic acid molecules encoding the protein can be operably linked to express the HSPB8 protein containing the K141N or K141T mutation specifically in neurons, motor cells, or multi-dendritic sensory neurons.
  • operably linked means that one nucleic acid fragment is linked to another nucleic acid fragment so that its function or expression is affected by the other nucleic acid fragment.
  • Promoters used in the present invention can be of various natures and origins and have various properties. This is because the choice of promoter used depends particularly on the gene in question. Accordingly, the promoter can be driven, for example, by a promoter that is strong or weak, ubiquitous or tissue/cell specific, or specific to a physiological or pathophysiological state (the activity may depend on the state of cell differentiation or on a specific stage of the cell cycle). being). Promoters may be of eukaryotic, prokaryotic, viral, animal, plant, artificial, human, etc. origin.
  • the present invention relates to a vector for constructing a Charcot-Marie-Tooth disease animal model comprising a nucleic acid encoding a K141N or K141T mutant HSPB8 protein and a neuron-specific promoter operably linked thereto.
  • the neuron-specific promoter may be the neuron-specific promoter elav , the motor neuron-specific promoter D42 , or the multi-dendritic sensory neuron-specific promoter ppk .
  • the present invention relates to a method of providing information necessary for diagnosing the possibility of developing Charcot-Marie-Tooth disease, including detecting the K141N or K141T mutation of the HSPB8 protein in a sample isolated from a subject.
  • the method may further include determining that the subject is likely to develop Charcot-Marie-Tooth disease when the K141N or K141T mutation of the HSPB8 protein is detected in a sample isolated from the subject. You can.
  • the present invention provides treatment of a test compound or composition to a cell line or animal model expressing HSPB8 protein containing a K141N or K141T mutation; Determining the degree of mitochondrial dysfunction in cell lines or animal models treated with the test compound or composition; and a method for screening a candidate for the treatment of Charcot-Marie-Tooth disease, comprising selecting a test compound or composition in which mitochondrial dysfunction is improved compared to a control cell line untreated with the test compound or composition.
  • the present invention provides treatment of a test compound or composition to a cell line or animal model expressing HSPB8 protein containing a K141N or K141T mutation; Confirming the expression of PINK1 or Parkin, or its activity, in cell lines or animal models treated with the test compound or composition; And a method for screening a candidate for the treatment of Charcot-Marie-Tooth disease, comprising selecting a test compound or composition with increased expression or activity of PINK1 or Parkin compared to a control cell line untreated with the test compound or composition.
  • the present invention relates to a method for diagnosing Charcot-Marie-Tooth disease, comprising detecting a K141N or K141T mutation in the HSPB8 protein.
  • the present invention provides treatment for Charcot-Marie-Tooth disease, comprising administering a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same to an individual suffering from Charcot-Marie-Tooth disease. It's about treatment methods.
  • the invention relates to the diagnostic use of Charcot-Marie-Tooth disease of an agent capable of detecting the K141N or K141T mutation of the HSPB8 protein.
  • the present invention relates to the use of a vector containing a PINK1 or Parkin protein, an activator thereof, or a nucleic acid molecule encoding the same for the prevention or treatment of Charcot-Marie-Tooth disease.
  • HSPB8 small heat shock protein B8
  • HSP22 HSP22
  • elav -GAL4 a nerve (neuron)-specific GAL4 driver
  • motor neuron-specific GAL4 driver Wild-type human HSPB8 ( HSPB8 WT ), K141T mutant HSPB8 ( HSPB8 K141T ), and K141E mutant HSPB8 in Drosophila neurons using D42 -GAL4 and ppk -GAL4, a multi-dendritic sensory neurons-specific GAL4 driver, respectively.
  • Drosophila models that specifically expressed the UAS transgene HSPB8 K141E ) were created.
  • K141T and K141E mutations were induced in the cDNA of human HSPB8, respectively, using the QuikChangeTM site-directed mutagenesis kit (Agilent Technologies) using the following primer pairs: HSPB8 K141T forward primer: 5'-ctg cag gaa gct gga ttt tcg ttg tga agt tct tag aaa ca-3' and K141T reverse primer: 5'-tgt ttc taa gaa ctt cac aac gaa aat cca gct tcc tgc ag), and HSPB8 K141E forward primer: 5'-gaa gct gga ttt tct ctg tga agt tct tag aaa caa tgc cac c-3' and K141E reverse primer: 5'-ggt ggc att g g
  • the wild-type HSPB8 cDNAs and the mutant HSPB8 cDNAs were each inserted into the pACU2 vector and microinjected into y w;PBac y[+]-attP-3B VK00001 embryos.
  • elav -GAL4, ppk -GAL4, and D42 -GAL4 lines were purchased from Bloomington Stock Center.
  • Drosophila lines expressing UAS-PINK1 and UAS-Parkin were also created using the method described in Park et al., 2006.
  • genotypes of the constructed Drosophila lines are as follows: elav (elav-GAL4/+); elav HSPB8 WT (elav-GAL4/+; UAS-HSPB8 WT /+); elav HSPB K141T (elav-GAL4/+; UAS-HSPB8 K141T /+); elav HSPB K141E (elav-GAL4/+;; UAS-HSPB8 K141E /+); ppk (ppk-GAL4/+); ppk HSPB8 WT (ppk-GAL4/UAS-HSPB8 WT ); ppk HSPB K141T (ppk-GAL4/UAS-HSPB8 K141T ); ppk HSPB K141E (ppk-GAL4/UAS-HSPB8 K141E ); D42 (D42-GAL4/+); D42 HSPB8 WT (UAS-HSPB
  • HSPB8 expression in the transgenic fruit flies prepared above was confirmed by Western blot analysis, and their lifespan analysis was performed. Specifically, to confirm the expression of HSPB8, 20 10-day-old male fruit fly heads were disrupted with disruption buffer, then centrifuged, and SDS sample buffer was added and heated. Afterwards, the samples were electrophoresed on SDS-PAGE gel and transferred to a nitrocellulose membrane. The membrane was incubated with blocking solution for 30 minutes and then incubated with anti-HSPB8 antibody (1:1,000, Cell Signaling Technology, # 95357 ) or anti-beta-actin antibody (1:1,000, Santa Cruz Biotechnology, SC-1616) as the primary antibody.
  • a thermal nociception assay was performed using Drosophila larvae. Specifically, each L3 larvae (hatched) of HSPB8 WT fruit flies, HSPB8 K141N mutant fruit flies, and HSPB8 K141T mutant fruit flies that expressed the HSPB8 transgene in multi-dendritic sensory neurons using ppk-GAL4 After [AEL] 120 h) (minimum of 50 larvae) were rinsed with distilled water, placed in Petri dishes, allowed to acclimatize for 10 s, and observed under a microscope.
  • the behavior of the transgenic Drosophila lines HSPB8 WT , HSPB8 K141T , and HSPB8 K141E ( elav, elav HSPB8 WT , elav HSPB8 K141T , and elav HSPB8 K141E ) produced in Example 1, respectively. It was recorded with a digital video camera, and walking trajectory and speed were calculated with Ctrax and Matlab.
  • 5-day-old adult male transgenic fruit flies expressing HSPB8 WT (elav HSPB8 WT ), HSPB8 K141T ( elav HSPB8 K141T ), or HSPB8 K141E ( elav HSPB8 K141E ) were each spaced at 675 mm ⁇ 440 mm ⁇ 410 mm equipped with a camera. They were placed in an insulated box and acclimatized for 30 minutes at 25°C, and their free movement in a transparent, round space with a height of 2 mm and a diameter of 60 mm was recorded at 30.06 fps (frames per second). The recorded video was converted to a MATLAB file using Ctrax and the behavioral microarray toolbox was used to obtain average velocity and trajectory graphs.
  • a climbing assay was also used to determine climbing ability, which has proven useful in identifying motor deficits in fruit fly models of various neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.
  • the D42-GAL4 driver was used to express the HSPB8 WT , HSPB8 K141T or HSPB8 K141E transgene in the motor neurons of Drosophila, and then perform transformation. Motor ability as a phenotype of Drosophila was confirmed by the method described in Examples 3-1 and 3-2 above.
  • TMRM ventral nerve cord
  • TMRM tetramethylrhodamine methyl ester
  • the level of mitophagy was defined as the number of pixels with a high red/green ratio divided by the total number of pixels.
  • Mean mitophagy levels ( ⁇ SD) were calculated for 10 independent samples.
  • transgenes of PINK1 or Parkin were introduced into HSPB8 mutant Drosophila ( elav HSPB8 K141T PINK1, elav HSPB8 K141E PINK1, elav HSPB8 K141T Parkin and elav HSPB8 K141E Parkin ) Changes in mitochondrial membrane potential, mitophagy level, and exercise capacity were confirmed.
  • KR kinetin riboside
  • locomotor activity of the wild-type transgenic fruit flies appeared to be unchanged compared to the control group (vehicle administered group) due to KR administration (FIGS. 6a to 6c), confirming that there were no side effects caused by KR.
  • locomotor activity was shown to be significantly restored depending on the administered concentration of KR ( Figures 6a to c).
  • PINK1 activation can restore the phenotype induced by HSPB8 mutation and that KR can suppress HSPB8-related pathogenesis.

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Abstract

La présente invention concerne un modèle animal de la maladie de Charcot-Marie-Tooth provoquée par des mutations K141N ou K141T dans HSPB8. Dans ce modèle animal exprimant la protéine HSPB8 mutante K141N ou K141T, des symptômes de la maladie de Charcot-Marie-Tooth, tels qu'un déficit moteur, un dysfonctionnement mitochondrial et des niveaux réduits de mitophagie, ont été observés, confirmant que la maladie de Charcot-Marie-Tooth peut être induite par des mutations K141N ou K141T de la protéine HSPB8. Par conséquent, le modèle animal exprimant la protéine HSPB8 mutante K141N ou K141T peut être utilisé comme modèle animal de la maladie de Charcot-Marie-Tooth. Les symptômes de la maladie de Charcot-Marie-Tooth ont été considérablement réduits par l'expression et l'activation de PINK1 ou de la parkine, faisant de ce modèle animal un outil pour les applications thérapeutiques contre la maladie de Charcot-Marie-Tooth.
PCT/KR2023/009373 2022-08-01 2023-07-04 Modèle animal de maladie de charcot-marie-tooth Ceased WO2024029755A1 (fr)

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Citations (2)

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WO2019068865A1 (fr) * 2017-10-06 2019-04-11 Universiteit Antwerpen Biomarqueurs pour maladies neurodégénératives et/ou neuromusculaires

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US20070101447A1 (en) * 2003-11-13 2007-05-03 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Diagnostic tests for the detection of motor neuropathy
WO2019068865A1 (fr) * 2017-10-06 2019-04-11 Universiteit Antwerpen Biomarqueurs pour maladies neurodégénératives et/ou neuromusculaires

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HAN, J. E. ET AL.: "Small heat shock protein HSPB8 mutation recapitulates features of Charcot-Marie-Tooth disease (CMT) accompanying with mitochondria dysfunction in Drosophila", THE 51ST ANNUAL MEETING OF KOREAN DROSOPHILA SOCIETY, poster-44, 4 July 2022 (2022-07-04) *
KANG KYONG-HWA, HAN JI EUN, KIM HYUNJIN, KIM SOHEE, HONG YOUNG BIN, YUN JEANHO, NAM SOO HYUN, CHOI BYUNG-OK, KOH HYONGJONG: "PINK1 and Parkin Ameliorate the Loss of Motor Activity and Mitochondrial Dysfunction Induced by Peripheral Neuropathy-Associated HSPB8 Mutants in Drosophila Models", BIOMEDICINES, MDPI, BASEL, vol. 11, no. 3, Basel , pages 832, XP093127990, ISSN: 2227-9059, DOI: 10.3390/biomedicines11030832 *
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