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WO2024251289A1 - Utilisation du domaine kinase m7ck de trpm7 dans la préparation d'un médicament pour le traitement de la maladie d'alzheimer - Google Patents

Utilisation du domaine kinase m7ck de trpm7 dans la préparation d'un médicament pour le traitement de la maladie d'alzheimer Download PDF

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WO2024251289A1
WO2024251289A1 PCT/CN2024/098301 CN2024098301W WO2024251289A1 WO 2024251289 A1 WO2024251289 A1 WO 2024251289A1 CN 2024098301 W CN2024098301 W CN 2024098301W WO 2024251289 A1 WO2024251289 A1 WO 2024251289A1
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m7ck
trpm7
sequence
plasmid
seq
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PCT/CN2024/098301
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Nashat Yousef Khader ABU MARIA
Wei Li
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Fudan University
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
    • 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
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11001—Non-specific serine/threonine protein kinase (2.7.11.1), i.e. casein kinase or checkpoint kinase
    • 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
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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
    • A01K2267/0312—Animal model for Alzheimer's disease
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates to the technical field of biological medicine, particularly relating to the use of a kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease.
  • AD Alzheimer's disease
  • WHO World Health Organization
  • pathogenesis is highly complex, mainly manifested by loss of neuronal cell synapses in the brain, amyloid deposition, neurofibrillary tangles, and the like.
  • the clinical manifestations are progressive memory and cognitive decline, language function and personality change and difficulty in daily activities.
  • the disease brings significant mental and economic burden to the patients themselves, their families and even the whole of society. To date, no effective radical treatment has been available.
  • amyloid deposition One of the critical targets for the development of drugs for Alzheimer's disease is directed at amyloid deposition. Therefore, targeting the elimination of amyloid to relieve the pathological characteristics of Alzheimer's disease such as synapse loss, cognitive impairment and the like has a great application prospect.
  • TRPM7 is a member of the M-type transient receptor potential family, a divalent ion channel protein widely expressed in various cell types and contains 6 transmembrane channel domains and a C-terminal kinase domain (M7CK) with both N-terminal and C-terminal directed to the cytoplasm.
  • TRPM7 is selectively permeable to divalent cations such as calcium ion, magnesium ion and zinc ion, has important effects on maintaining ion homeostasis inside and outside cells and regulating downstream signaling pathways, and is expressed in various nerve cells such as the hippocampus, cerebral cortex, cerebellum and brainstem.
  • TRPM7 cleaved kinase fragments can exist alone after being cleaved and are involved in critical cellular processes such as regulating chromatin remodeling and synaptic plasticity. Its unique dual function as an ion channel with kinase domain enables TRPM7 to be involved in a variety of cellular functions. TRPM7 is involved in neurodegenerative diseases, including Alzheimer's disease by a variety of means. However, some studies suggest that inhibition of TRPM7 may be a therapeutic strategy, while other studies suggest that activation of TRPM7 may have a protective effect.
  • TRPM7 missense mutation of TRPM7 can change the sensitivity of cells to magnesium ions, thereby affecting magnesium ion homeostasis and further causing neurodegenerative diseases and dementia.
  • presenilin protein mutants related to familial Alzheimer's disease can inhibit the channel function of TRPM7.
  • a genetic screening study in human subjects found three mutations in TRPM7 in Chinese patients with sporadic Alzheimer's and frontotemporal dementia. To date, there has been no study on the protective effect of the kinase domain M7CK of TRPM7 on Alzheimer's disease.
  • the present disclosure aims to provide the use of a kinase domain M7CK sequence of TRPM7 in the preparation of a drug for treating Alzheimer's disease and aims to solve the problems in the prior art.
  • the present disclosure provides the use of a kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease.
  • the M7CK has the amino acid sequence selected from the following:
  • polypeptide fragment having the sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%and at least 99.5%to the polypeptide of 1) on the basis of having the same biological functions as the polypeptide of 1) .
  • polypeptide sequence is shown in SEQ ID No. 3.
  • the present disclosure further provides a pAAV2/9-M7CK plasmid capable of over-expressing M7CK at sequence positions 1299-1863 of TRPM7 and constructed on the basis of a kinase domain M7CK sequence of TRPM7.
  • the present disclosure further provides a virus capable of over-expressing M7CK at sequence positions 1299-1863 of TRPM7 and constructed based on a kinase domain M7CK sequence of TRPM7, and the virus is obtained by virus packaging of the pAAV2/9-M7CK plasmid.
  • the present disclosure further provides a method for preventing or treating Alzheimer's disease, wherein the method includes administering to a subject an effective dose of the kinase domain M7CK sequence of TRPM7, the pAAV2/9-M7CK plasmid or the virus.
  • kinase domain M7CK of TRPM7 in the preparation of a drug for treating Alzheimer's disease of the present disclosure has the following beneficial effects:
  • the over-expression of M7CK in the present disclosure can relieve cognitive impairment of a mouse model with AD by increasing an MMP14 phosphorylation level, improving learning and memory ability, increasing synaptic density and density of a presynaptic vesicle protein synaptophysin and a postsynaptic density protein PSD-95, promoting degradation of A ⁇ in soluble and insoluble components, and reducing accumulation of A ⁇ amyloid plaques. Therefore, the kinase domain of TRPM7 provided by the present disclosure has a great application prospect in gene therapy of a patient with Alzheimer's disease.
  • FIGs. 1A-1C show the results of analyzing the expression levels of TRPM7 in the hippocampus of patients with Alzheimer's disease, 5XFAD mice at 12 months of age and APP/PS1 mice at 15 months of age in the present disclosure;
  • FIGs. 2A-2B show the immunofluorescence signals and statistical results of synaptophysin/PSD95 in primary hippocampal neuron over-expressing M7CK and treated with A ⁇ in the present disclosure
  • FIG. 3 shows that AAV2/9-M7CK or AAV2/9-EGFP is injected into the hippocampus of WT and 5XFAD mice at 5 months of age, and then learning and memory functions thereof are tested at 6/10/15 months of age by using a novel object recognition test, a cognition wall test and a Y-maze test in the present disclosure;
  • FIGs. 4A-4F show the results of a behavioral test of mice at 6/10/15 months of age in the present disclosure
  • FIGs. 5A-5B show that before injection of M7CK in the present disclosure, 5XFAD mice at 14 months of age do not have the ability to recognize novel objects, and after over-expressing M7CK, the novel object recognition index is significantly increased in the 5XFAD mice;
  • FIGs. 6A-6B show the immunofluorescence staining and statistical results of synaptophysin and PSD95 in the hippocampus of 5XFAD mice at 15 months of age over-expressing M7CK in the present disclosure
  • FIG. 7 shows immunohistochemical staining and statistical results of A ⁇ plaques in the hippocampus of 5XFAD mice over-expressing M7CK in the present disclosure
  • FIG. 8 shows the quantitative results of ELISA of A ⁇ 1-40 and A ⁇ 1-42 in soluble and insoluble protein components in the hippocampus of 5XFAD mice over-expressing M7CK in the present disclosure
  • FIG. 9 shows that over-expressing M7CK in the hippocampus of 5XFAD mice increases the phosphorylation level of MMP14 in the present disclosure
  • FIGs. 10A-10C show that M7CK can bind and phosphorylate MMP14 in the present disclosure
  • FIGs. 11A-11B show that knock-out of TRPM7 reduces the phosphorylation level of MMP14 in the present disclosure
  • FIG. 12 shows that M7CK promotes the degradation of A ⁇ by MMP14 in the present disclosure
  • FIG. 13 shows after knock-down of MMP14, M7CK fails to restore down-regulation of synaptic density caused by A ⁇ in the present disclosure
  • FIG. 14 shows after knock-down of MMP14, M7CK fails to promote the degradation of A ⁇ in the present disclosure
  • FIG. 15 shows the structure of a TRPM7 protein in the present disclosure
  • FIG. 16 shows a pAAV2/9-EGFP plasmid in the present disclosure
  • FIGs. 17A-17F show the results of a study of the activity and specificity of a substance promoting TRPM7 ion channel part activity to promote TRPM7 cleavage to produce a free and active kinase domain (M7CK) in the present disclosure
  • FIGs. 18A-18D show the results that a chronic treatment with the TRPM7 modulator ATT which promotes the cleavage of TRPM7 to produce free and active M7CK can rescue cognitive and memory function in 5XFAD mice in the present disclosure
  • FIGs. 19A-19C show the results that a chronic treatment with the TRPM7 modulator ATT which promotes the cleavage of TRPM7 to produce free and active M7CK can protect the synaptic density and reduce A ⁇ plaques in 5XFAD mice in the present disclosure.
  • FIGs. 20A-20C show the results of altering the M7CK/TRPM7 ratio with a substance that promotes the cleavage of TRPM7 to produce M7CK in the present disclosure.
  • the present disclosure provides use of a kinase domain M7CK of TRPM7 in preparing a drug for treating Alzheimer's disease.
  • the kinase domain M7CK is a polypeptide.
  • the amino acid sequence of the M7CK is a polypeptide sequence coded by the nucleotide sequence of SEQ ID No. 1.
  • the M7CK is: a polypeptide fragment having the sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%and at least 99.5%to the polypeptide coded by the nucleotide sequence SEQ ID No. 1 on the basis of having the same biological functions as the polypeptide coded by the nucleotide sequence SEQ ID No. 1.
  • the amino acid sequence of the kinase domain M7CK of TRPM7 includes the amino acid sequence at positions 1299-1863 of TRPM7 TRPM7.
  • the amino acid sequence of the M7CK is shown in SEQ ID No. 3.
  • the kinase domain M7CK of TRPM7 may be an amino acid sequence containing a secretory tag.
  • the secretory tag directs the kinase domain of TRPM7 to the periplasmic space of a host cell.
  • a signal peptide is derived from DsbA, PelB, OmpA, TolB, MalE, lpp, TorA or HylA.
  • the secretory tag is attached to the kinase domain of TRPM7.
  • the secretory tag is cleaved from the kinase domain of TRPM7.
  • the kinase domain M7CK of TRPM7 includes a histidine tag.
  • the histidine tag or a polyhistidine tag is a sequence of 2 to 20 histidine residues attached to collagen.
  • the histidine tag includes 2 to 20 histidine residues, 5 to 15 histidine residues, 5 to 18 histidine residues, 5 to 16 histidine residues, 5 to 15 histidine residues, 5 to 14 histidine residues, 5 to 13 histidine residues, 5 to 12 histidine residues, 5 to 11 histidine residues, 5 to 10 histidine residues, 6 to 12 histidine residues, 6 to 11 histidine residues or 7 to 10 histidine residues.
  • the histidine tag can be used to purify proteins by a chromatographic method using a nickel-based chromatographic medium.
  • the drug for treating Alzheimer's disease includes an M7CK accelerator.
  • the M7CK accelerator is selected from one or more of the following: a plasmid or a virus effectively over-expressing M7CK; and/or a substance promoting the cleavage of TRPM7 to produce M7CK.
  • the plasmid or viral vector over-expressing M7CK includes a coding nucleic acid fragment of M7CK.
  • the substance promoting the cleavage of TRPM7 to produce M7CK is selected from one or more of the following compounds: resveratrol, zafirlukast, TPCA-1, ATT and glutamic acid or glutamate.
  • the coding nucleic acid fragment is shown in SEQ ID No. 1.
  • the coding nucleic acid fragment is obtained by optimizing the nucleic acid molecule shown in SEQ ID No. 1 by any one or more of the following modes: 1) compared with the nucleic acid molecule shown in SEQ ID No. 1, increasing the content of G/C; and 2) codon-optimizing the nucleic acid molecule shown in SEQ ID No. 1 to suit the species acts thereon, wherein after the codon-optimizing of the nucleic acid fragment or the content of G/C is increased, the sequence of the encoded amino acid is not altered.
  • the plasmid is selected from an adenovirus packaging plasmid, an adeno-associated virus packaging plasmid and a lentivirus packaging plasmid; and the virus is selected from an adenovirus, an adeno-associated virus and a lentivirus.
  • the plasmid is a pAAV2/9-M7CK plasmid, preferably, the sequence of the pAAV2/9-M7CK plasmid is shown in SEQ ID No. 2.
  • the pAAV2/9-M7CK plasmid is used for virus packaging of an AAV to obtain a packaged AAV.
  • a use method of the drug for treating Alzheimer's disease is injecting the packaged AAV into the brain by brain stereotaxic localization, specifically into an intracerebral hippocampus region.
  • the drug for treating Alzheimer's disease has one or more of the following functions:
  • Increasing neuronal synaptic density refers to an increase in neuronal synaptic density relative to that of patients with Alzheimer's disease; improving learning and memory ability refers to an increase in learning and memory ability relative to that of patients with Alzheimer's disease; and reducing A ⁇ plaques refers to a decrease in A ⁇ plaques relative to that of patients with Alzheimer's disease.
  • the A ⁇ plaque is a protein clump formed by aggregating A ⁇ 1-40 proteins or A ⁇ 1-42 proteins.
  • the activating the activity of MMP14 is phosphorylating the MMP14 protein.
  • the drug for treating Alzheimer's disease further includes a pharmaceutically acceptable excipient.
  • the excipient includes various excipients and diluents. These excipients are not essential active ingredients and not excessively toxic after administration.
  • the excipients include sterile water or normal saline, a stabilizer, an excipient, an antioxidant (ascorbic acid, etc. ) , a buffer (phosphoric acid, citric acid, other organic acids, etc. ) , a preservative, a surfactant (PEG, Tween, etc. ) , a chelating agent (EDTA, etc. ) or a binder.
  • the excipients further include other low molecular weight polypeptides, serum albumin, glycine, glutamine, asparagine, arginine, polysaccharides, monosaccharides, mannitol or sorbitol.
  • the excipients are selected from normal saline, a glucose isotonic solution, a D-sorbitol isotonic solution, a D-mannose isotonic solution, a D-mannitol and sugar alcohol isotonic solution when used in an injectable aqueous solution.
  • the injectable aqueous solution includes a solubilizer.
  • the solubilizer is selected from alcohol (ethanol) , polyalcohol (propylene glycol or PEG) and a nonionic surfactant (Tween 80 or HCO-50) .
  • the kinase domain M7CK sequence of TRPM7 may be a single effective component or may be combined with one or more other active ingredients useful for treating Alzheimer's disease to form a combined preparation.
  • the active ingredients are other various drugs for treating Alzheimer's disease.
  • the content of the active ingredients in a pharmaceutical composition is a safe and effective amount which can be adjusted by those skilled in the art.
  • the administration amount of the kinase domain M7CK sequence of TRPM7 and the active ingredients of a therapeutic product of Alzheimer's disease depends on the body weight of patients, the type of application and the condition and severity of the disease, for example, the administration amount of the pharmaceutical composition as an active component is 1-1,000 mg/kg/day, 1-3 mg/kg/day, 3-5 mg/kg/day, 5-10 mg/kg/day, 10-20 mg/kg/day, 20-30 mg/kg/day, 30-40 mg/kg/day, 40-60 mg/kg/day, 60-80 mg/kg/day, 80-100 mg/kg/day, 100-200 mg/kg/day, 200-500 mg/kg/day, or greater than500 mg/kg/day.
  • the present disclosure further provides a pAAV2/9-M7CK plasmid capable of effectively over-expressing M7CK at sequence positions 1299-1863 of TRPM7 and constructed on the basis of a sequence of kinase domain M7CK of TRPM7.
  • the amino acid sequence of the M7CK is a polypeptide sequence encoded by the nucleotide sequence of SEQ ID No. 1.
  • the M7CK is: a polypeptide fragment having the sequence identity of at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%and at least 99.5%to the polypeptide coded by the nucleotide sequence SEQ ID No. 1 on the basis of having the same biological functions as the polypeptide coded by the nucleotide sequence SEQ ID No. 1.
  • the amino acid sequence of the M7CK includes the amino acid sequence at positions 1299-1863 of TRPM7.
  • the amino acid sequence of the M7CK is shown in SEQ ID No. 3.
  • the plasmid or viral vector over-expressing M7CK includes a coding nucleic acid fragment of M7CK.
  • the coding nucleic acid fragment is shown in SEQ ID No. 1.
  • the coding nucleic acid fragment is obtained by optimizing the nucleic acid molecule shown in SEQ ID No. 1 by any one or more of the following modes: 1) compared with the nucleic acid molecule shown in SEQ ID No. 1, increasing the content of G/C; and 2) codon-optimizing the nucleic acid molecule shown in SEQ ID No. 1 to suit the species acts thereon, wherein after the codon-optimizing of the nucleic acid fragment or the content of G/C is increased, the sequence of the encoded amino acid is not altered.
  • nucleotide sequence of the pAAV2/9-M7CK plasmid is shown in SEQ ID No. 2.
  • the present disclosure further provides a virus capable of over-expressing M7CK at sequence positions 1299-1863 of TRPM7 and constructed on the basis of a sequence of kinase domain M7CK of TRPM7, which is obtained by virus packaging of the pAAV2/9-M7CK plasmid.
  • the virus may be selected from one or more of an adenovirus, an adeno-associated virus and a lentivirus.
  • the plasmid backbone of the virus may be any one or more of pLKO. 1-CMV-tGFP, pLKO. 1-puro-CMV-tGFP, pLKO. 1-CMV-Neo, pLKO. 1-Neo, pLKO. 1-Neo-CMV-tGFP, pLKO. 1-puro-CMV-TagCFP, pLKO. 1-puro-CMV-TagYFP, pLKO. 1-puro-CMV-TagRFP, pLKO. 1-puro-CMV-TagFP635, pLKO. 1-puro-UbC-TurboGFP, pLKO.
  • the pAAV2/9-M7CK plasmid or virus is obtained by introducing the M7CK sequence into an engineered cell and performing intracellular replication or assembly.
  • the engineered cell may be one or more of escherichia coli, lactobacillus, bacillus licheniformis, bacillus subtilis, streptomyces, saccharomyces cerevisiae, candida, torulopsis, rhodotorula, hansenula, pichia, kluyveromyces, 293, 293T, HUCCT1, HCCC-9810and RBE.
  • the present disclosure further provides a method for preventing or treating Alzheimer's disease, specifically a method for preventing or treating a subject suffering from Alzheimer's disease, including administering to a subject an effective dose of TRPM7 modulator, M7CK accelerator, pAAV2/9-M7CK plasmid or the virus in the use.
  • the administration amount is 1-1,000 mg/kg/day. Specifically, the administration amount is1-3 mg/kg/day, 3-5 mg/kg/day, 5-10 mg/kg/day, 10-20 mg/kg/day, 20-30 mg/kg/day, 30-40 mg/kg/day, 40-60 mg/kg/day, 60-80 mg/kg/day, 80-100 mg/kg/day, 100-200 mg/kg/day, 200-500 mg/kg/day, or 500-1,000 mg/kg/day.
  • the subject of the method may be a mammal, preferably a human.
  • the term “identity” is generally a relationship between the sequences of two or more polypeptides or polynucleotides, as determined by comparing the sequences. In the art, the identity also means the degree of sequence correlation between them, as determined by the number of matches between series sections of two or more amino acid residues or nucleotide residues. The identity measures the percentage of identical matches between two or more sequences compared to a smaller sequence, wherein gap alignments are processed by a particular mathematical model or computer program (e.g., an “algorithm” ) . The identity of the related peptides may be readily calculated by a known method.
  • identity% is defined as the percentage of residues (amino acid residues or nucleic acid residues) in a candidate amino acid or nucleic acid sequence that are identical to the residues in an amino acid sequence or nucleic acid sequence of a second sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Methods and computer programs for alignment are well-known in the art. It should be understood that identity depends on the calculation of percent identity but can differ in value due to gaps and penalties introduced in the calculation.
  • variants of specific polynucleotides or peptides have at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%but less than 100%of sequence identity with that specific reference polynucleotides or peptides, as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
  • phosphorylation generally refers to a process in which a phosphate group is added to a specific amino acid residue of a protein in an enzymatic reaction, such as serine, threonine, tyrosine or histidine. Such modifications may result in an increase or decrease in the activity of the protein or alter its interaction with other molecules.
  • SEQ ID NO. 1 (M7CK sequence) :
  • TRPM7 The expression level of TRPM7 in the hippocampus of patients with Alzheimer's disease and mice in transgenic models of Alzheimer's disease (5XFAD and APP/PS1) was significantly reduced.
  • the inventors of the present application observed that the expression level of a TRPM7 protein in the hippocampus of patients with Alzheimer's disease was obviously reduced compared with that of a control group by using a western blotting method. Further, a significant reduction in the expression level of TRPM7 was observed in the hippocampus of 5XFAD mice at 12 months ofage and APP/PS1 mice at 15 months ofage compared to the same-age wild-type mice by using the same method as shown in FIGs. 1A-1C.
  • the hippocampal sample of the patients with Alzheimer's disease used in this example was from the National Health And Disease Human Brain Tissue Resource Center (China Human Brain Center, School of Medicine, Zhejiang University) .
  • the 5XFAD and APP/PS1 mice purchased from Nanjing university&Model Animal Research Center, Nanjing Biomedical Research Institute) were widely used mouse models with Alzheimer's disease.
  • the method of western blotting analysis was as follows:
  • Cryopreserved hippocampal tissues of the patients with Alzheimer's disease, fresh hippocampal tissues of the 5XFAD and APP/PS1 mice, and hippocampal tissues of the control group were taken, placed in EP tubes, proteins were extracted by using a RIPA lysate (100 ⁇ L lysate/10 mg tissue) containing a phosphatase inhibitor and a protease inhibitor, and protein concentration was quantified by using a BCA kit. A sampling buffer solution was added into the protein and the protein was fully denatured at 100°Cfor 5 min. Sampling was performed on a 10%SDS-PAGE gel in the amount of 20 ⁇ g.
  • Electrophoresis was started for 30 min at 80 V on an upper layer glue and 50 min at 120 V on a lower layer glue.
  • a PVDF membrane was cut into the same size as the SDS-PAGE gel and put into methanol for activation for several seconds, then the gel blocks and the PVDF membrane were clamped between multiple layers of filter paper of a transfer clamp in a membrane transfer liquid after precooled at 4°C (the gel faced a black cathode and the membrane faced a red anode) to perform membrane transfer at a constant pressure of 100 V for 70 min.
  • the membrane was blocked in a blocking solution made of 5%skim milk powder for 2 h and then transferred to a primary antibody (TRPM7 antibody, Alomone, ACC-047, 1: 750; GAPDH antibody, Proteitech, 1: 10,000) diluted with the blocking solution for overnight incubation, and the membrane was washed three time with PBST, incubated with a secondary antibody for 1.5 h and was washed three times with PBST. A freshly prepared ECL developing solution was dripped for developing in a Tanon developing instrument, proper exposure time was selected and pictures were stored. The strips were subjected to an optical density quantitative analysis by using an Image Pro Plus software. The relative expression level of TRPM7 was calculated by using GAPDH as an internal control.
  • An AAV2/9-M7CK adeno-associated virus capable of efficiently over-expressing M7CK at positions 1299-1863 of TRPM7 was constructed.
  • the PCR product was subjected to electrophoresis by using 1%agarose gel, a strip of about 1.7 kb was cut, and the M7CK fragment was collected according to instructions of a gel recovery kit: the gel at the position of the target strip was cut in a nucleic acid developer, collected in an EP tube and weighed, a PN solution (1 ⁇ L/mg gel block) was added, the gel was dissolved in water bath at 50°C and shaken continuously until the gel was completely dissolved; 500 ⁇ L of a BL solution was added into an adsorption column in advance at 12,000rpm for 1 min, and waste liquid in a collection tube was discarded; the completely dissolved gel block solution was restored to room temperature, added into the adsorption column, stood at room temperature for 2 min and centrifuged at 12,000rpm for 1 min, and waste liquid was removed; 600 ⁇ L of PW was added, the solution was stood for 4 min and centrifuged at 12,000rpm for 1 min, the waste liquid was removed; 600
  • Target gene fragment ( ⁇ 50ng/ ⁇ L) : supplemented to 20 ⁇ L
  • the corresponding bacterial liquid was added into a 300-mL culture flask for amplification culture for 12 h and then the plasmid was extracted according to the plasmid extraction steps (Vazyme, #DC202-01) : the overnight cultured bacterial liquid was transferred into a 50-mL centrifuge tube and centrifuged at 11,000 rpm for 2 min, and the supernatant was discarded; 7.5 mL of a P1 solution was added into the bacterial precipitate and evenly mixed by vortex oscillation; 7.5 mL of a P2 solution was continuously added and mixed evenly by gently turning upside down, and the solution was stood at room temperature for 5 min; 7.5 mL of a P4 solution was added, the solution was immediately turned upside down for about 8 times, a white flocculent precipitate generated at this time, and the solution was centrifuged at 11,000 rpm for 10 min, and the supernatant (avoiding sucking the white precipitate) was sucked to a new50-mL round bottom
  • AAV2/9 virus packaging 293 cells (six 15-cm dishes) transfected (VigoFect transfection reagent) with helper and target plasmids 60 hours were taken out from an incubator, the culture solution was removed, the cells were washed once by gently adding PBS, then fresh PBS (25 mL/dish) was added and the cells were collected in three 50-mL tubes by using a cell scraper; the cells were centrifuged at 400 g at room temperature for 10 min; the supernatant was removed, 10 mL of a salt solution (150 mM NaCl and 20 mM Tris-HCI) was added into each tube of the precipitate, the cell precipitate was shaken and resuspended, and collected in a 50-mL tube; 1.5 mL of 10%sodium deoxycholate (prepared when used and working concentration of 0.5%) was prepared and all added into 30 mL of the cell suspension, a Benzonase nuclease (working concentration of
  • AAV2/9-M7CK or AAV2/9-EGFP was added by the inventors of the present application when the primary hippocampal neurons were cultured in vitro to the 7th day and A ⁇ was added on the 12nd day.
  • the synaptic density marked by synaptophysin and PSD95 was observed to be remarkably reduced in an A ⁇ group and significantly recovered in an A ⁇ group over-expressing the M7CK (FIGs. 2A-2B) . Therefore, over-expressing the M7CK had a protective effect on synaptic toxicity induced by A ⁇ .
  • the cells After cultured for 1.5-2 h in a constant-temperature incubator, the cells basically adhered to the wall, at this time, 1 mL of the plating culture medium/well was added, the culture medium was changed into a serum-free culture medium after two days, and half of the culture medium was changed at an interval of 3-4 days.
  • the cell-growing slide was air-dried. After immunofluorescent staining of the in-vitro cultured neurons was completed, the slide was placed under a confocal microscopy and shot using a 60 ⁇ oil immersion lens (digital magnification 3 times at the same time) .
  • the body of the neurons was arranged at the edge of a visual field and then synaptophysin or PSD-95 signals on dendrites were shot with the shooting thickness of 7 ⁇ m and the interval of 1 ⁇ m/layer.
  • the number of synaptopphysin/PSD-95 puncta on the dendrites 10 ⁇ m away from the cell body was selected for counting and the number of the puncta on the dendrites per 10 ⁇ m was taken as the dendrite density.
  • this example determined the median effective concentration (EC50) of these compounds (resveratrol: 28.1 ⁇ M; zafirlukast: 1.4 ⁇ M; TPCA-1: 9.0 ⁇ M; ATT: 15.7 ⁇ M, FIG. 17C) .
  • EC50 median effective concentration
  • this example identified four substances that promote the cleavage of TRPM7 to produce M7CK and confirmed their ability to activate the TRPM7 channel.
  • this example examined substances that have been determined to promote the cleavage of TRPM7 to produce M7CK. This example evaluated their effects on other TRPM ion channels, such as TRPM2, TRPM6 and TRPM8.
  • the TRPM2 only the TPCA-1 showed an excitatory effect on the activity of an ion channel (FIG. 17D) .
  • the TRPM6 the resveratrol and zafirlukast activated the ion channel; and at the same time, the ATT inhibited the activity of the channel (FIG. 17E) .
  • the TRPM8 the resveratrol, zafirlukast and ATT inhibited the activity of the ion channel (FIG. 17F) . Therefore, in the determined regulators, only ATT appeared to specifically activate the TRPM7; and at the same time, other determined regulators activated other TRPM ion channels. In contrast, the ATT may inhibit two TRPM ion channels, TRPM6 and TRPM8.
  • An electrophysiological experiment in this example specifically included the following steps: whole-cell patch clamp recording was performed at room temperature by using an EPC-10 amplifier and a Patch Master software (HEKA, Germany) . The current was digitized at a sampling rate of 10 kHz and low-pass filtered at 2.0 kHz. Needle tubes were prepared from glass capillaries (World Precision Instruments, USA) with the electrode resistance between 2 M ⁇ and 4 M ⁇ . A needle tube solution free of Mg 2+ contained (in mM) : 145CsCl, 8NaCl, 10HEPES and10 EGTA (adjusted to pH 7.2 with CsOH) .
  • a bath solution contained (in mM) : 140 NaCl, 5 KCl, 2 CaCl 2 , 20HEPES and 10 glucose (adjusted to pH 7.4 with NaOH) .
  • a 300 ms voltage ramp injection from -100 mV to +100 mV was performed in a manner repeated every 5 s, and the TRPM7 current was recorded.
  • the TRPM7 channel current at +100 mV and -100 mV were obtained.
  • this Example found that promoting neuronal activity by increasing the extracellular glutamate concentration (for 1 hr) promotes the cleavage of TRPM7 in a dose-dependent manner, as indicated by the gradual increase in the M7CK/TRPM7ratio (FIG. 20A) .
  • the minimum effective glutamate concentration (40 ⁇ M) was used to determine the time course of the cleavage process.
  • This embodiment found that glutamate-induced cleavage started after 30 min of incubation with glutamate, peaked after 1 h, and returned to baseline after 3 h (FIG. 20B and FIG. 20C) .
  • FIGs. 17A-17F were specifically described as follows:
  • Right figures A representative current-voltage relationship of the substances promoting the cleavage of TRPM7to produce M7CK.
  • FIG. 17B Representative time course of the substances promoting the cleavage of TRPM7to produce M7CK (resveratrol, zafirlukast, TPCA-1 and ATT) for the TRPM7 channel.
  • FIG. 17C A dose-response curve of the substances promoting the cleavage of TRPM7 to produce M7CK (resveratrol, zafirlukast, TPCA-1 and ATT) .
  • the analytical methods were the Mann-Whitney test (resveratrol) and the unpaired t test (zafirlukast, TPCA-1 and ATT) .
  • Right figures A representative current-voltage relationships of the effect of a TRPM7 agonist.
  • the AAV2/9-M7CK adeno-associated virus constructed by the present disclosure can effectively restore the impaired learning and memory functions of 5XFAD mice, restore the synaptic density and reduce the A ⁇ level.
  • the constructed AAV2/9-M7CK virus was injected into the bilateral hippocampus of 5XFAD mice at5 months of age by using a mouse brain stereotaxic localization technology by the inventors of the present application. It was discovered that over-expressing M7CK can restore the impaired learning and memory functions of the 5XFAD mice, by behavioral analysis (novel object recognition, Y-maze and cognition wall tests, and the results were shown in FIGs. 3-5B) at 6 months of age, 10 months of age and 15 months of age.
  • AAV2/9-M7CK and AAV2/9-EGFP viruses were prepared and respectively injected into the bilateral hippocampus of the WT and5XFAD mice at 5 months of age.
  • the experiments were divided into 4 groups: WT mice given AAV2/9-EGFP; WT mice given AAV2/9-M7CK; 5XFAD mice given AAV2/9-EGFP; and 5XFAD mice given AAV2/9-M7CK.
  • WT mice given AAV2/9-EGFP WT mice given AAV2/9-M7CK
  • 5XFAD mice given AAV2/9-EGFP
  • 5XFAD mice given AAV2/9-M7CK
  • mice were anesthetized with 2%isoflurane; after the mice were successfully anesthetized, the heads of the mice were firmly fixed by using ear rods and nose rods; the left, right, front and back were adjusted to enable the back sides of the brains of the mice to be positioned on the same horizontal plane; after the surface skin of the heads of the mice were wiped with alcohol and disinfected, the skin was cut with scissors to fully expose the skull, and surface bloodstains were wiped off with a cotton swab stained with iodophors; and holes were drilled according to the position of the hippocampus in the brain atlas of the mice and the following coordinates. 2 holes were drilled on the left side and the right side respectively.
  • an injection needle was slowly inserted into the brain region corresponding to the coordinates, the viruses were started to be injected at the speed of 1 nl/s at the volume of 300 nl per point. After the injection, the needle was kept for 10 min and slowly leaved. The scalp was sutured and the mice were placed in a warm environment to wait for recovery from anesthesia.
  • Novel object recognition test the mice were changed from being fed in one cage with multiple mice into one cage with one mouse in one week before the start of the test until the end of the test.
  • a test device consisted of a colorless, tasteless and opaque box body with the top hung with a camera and the length, width and height of 50 cm, a computer connected with the camera and objects A, B and C.
  • a mouse was placed in a box (without any object) and allowed to explore freely for10 min to adapt to the environment. Before the next mouse was placed in the box, the box body required to be cleaned with a 30%odorless diluted hand sanitizer to avoid the smell left by the last mouse to affect the behaviors of the next mouse.
  • Cognition wall test the test was performed by using a PhenoTyper model 3000 device from the Noldus company and a fully automated record analysis software.
  • a mouse was put into a PhenoTyper cage to adapt for 11 h, fasted, but free to water.
  • 8: 00 a. m a feeding device was communicated with the cage and placed in a learning box, about 10 grains of sugar beans were manually given to the mouse and then a video tracking software was started. In the process, the software tracked the shuttling times of the mouse in the learning box in real time and the sugar beans were awarded according to the following rules.
  • a single sugar bean was given as a reward if the mouse entered the learning box from a left hole 5 consecutive times without interruption. If the mouse entered the left hole 24 out of 30, it can be considered that the mouse had reached a learning standard, i.e. 80%criteria. A learning curve of each group of mice can be drawn according to the required hole-entering times when the mice reached the 80%criteria. The number of the remaining sugar beans in the feeding device was counted before and after the test, and was compared with the feeding number automatically calculated by the software. Whether software or hardware faults occurred or not was checked in time.
  • a spontaneous alternation e.g., ABC/BCA/CAB, etc.
  • mice were anesthetized with 2%isoflurane; after the mice entered a complete anesthesia state, four limbs of the mice were fixed on a foam board, the chest skin was cut open to fully expose the heart, the right auricle was cut open, and the needle tip of an injector was inserted into the left ventricle, which was perfused once with 20 mL of precooled normal saline firstly and then perfused with 20 mL of 4%PFA once.
  • the brain tissues of the mice were carefully peeled off, soaked in 4%PFA overnight, transferred to 20%and 30%sucrose solutions in sequence until the brain tissues were dehydrated and sunk to the bottom of a tube; and the dehydrated brain tissues were embedded in an OCT embedding medium, placed in a freezing microtome, and started to perform frozen sectioning after the brain tissues were completely frozen on a freezing head.
  • the slice thickness was 16 ⁇ m.
  • the cut brain slices were stored in PBS, carefully stuck on a glass slide by using a brush, and air-dried at room temperature for 2-4 h until the brain slices were firmly stuck on the glass slide; closed circles were coated around the brain slices with an immunohistochemical pen and air-dried for 30 min; the brain slices were washed in PBS for 3 times with5 min/time; the brain slices were blocked in a 5%goat serum (in 0.2%Triton-PBS) for 2 h in a wet box at room temperature; a primary antibody (synaptophysin, SYSY, 101011, 1: 500; PSD95, CST, 3450, 1: 200) was diluted in a blocking liquid and the brain slices were incubated overnight at 4°C; the brain slices were washed in PBS for 3 times with5 min/time; a secondary antibody was diluted in0.2%Triton-PBS (1: 500) and the brain slices were incubated at room temperature for 2 h; the brain slices were washed in P
  • the slide was placed under the confocal microscope and photographed using a 60 ⁇ lens (digital magnification 3 times at the same time) .
  • the shooting thickness wa s9 ⁇ m at the interval of 1 ⁇ m per layer.
  • the three layers of brightest signals in the photographed pictures were synthesized into a picture of a tiff format with a derived resolution of 1,024*1,024.
  • the derived pictures were subjected to puncta automatic counting by using Image Pro Plus: firstly, a picture was firstly subjected to a HiGauss enhanced filter twice; most typical puncta areas in the picture were measured and the appropriate threshold range (puncta greater than the maximum threshold will be segmented; puncta less than the minimum threshold will not be counted) was set; and finally, a measurement button was clicked to obtain the number of the puncta in the picture. Finally, according to the scale, the number of puncta contained in each 1,000 ⁇ m 2 was calculated to be used as the synaptic density index.
  • a ⁇ ELISA the soluble fraction of an A ⁇ protein was extracted in the same method as described above for western blotting. 5M guanidine hydrochloride (dissolved in 50 mM Tris-HCI, pH 8.0) was added into the precipitate after RIPA lysis and centrifugation, the precipitate was dissolved in a shaker at room temperature for 4 h, the solution was centrifuged at 12,000 rpm at 4°C for 30 min, at this time, almost no precipitate was seen, and the supernatant was taken as an insoluble fraction; a soluble fraction of the protein was diluted by 100 times and 50 times by using a dilution buffer in a kit for detecting A ⁇ 1-42 and A ⁇ 1-40 respectively; an insoluble fraction of the protein was diluted 20,000 times and 2,000 times respectively for detecting A ⁇ 1-42 and A ⁇ 1-40 ; the ELISA kit (Wako, 292-62301, 298-62401) and the related liquid were taken out from a 4°C refrigerator and restored to room temperature for
  • the liquid in the wells needed to be poured as completely as possible (the plate was patted on absorbent paper) during each washing; and an HRP-conjugated secondary antibody was added at 100 ⁇ L/well.
  • the samples were incubated for 1 h at the 4°Cr efrigerator; the secondary antibody in the ELISA plate was discarded, and the samples were washed 5 times with the rinsing solution (200 ⁇ L/well) ; 100 ⁇ L/well of a TMB solution (the samples were ensured to be added into all the wells in short time as much as possible) , and the samples were incubated at room temperature in a dark place for 30 min; a stop solution was added to stop the reaction and the light absorption value of 450 nm was read (the reading needed to be finished within 30 min after the stop solution was added) ; and standard curves were drawn according to the concentrations of the standard substances and the corresponding absorbance values, and thus the concentrations of the A ⁇ 1-42 and the A ⁇ 1-40
  • ATT treatment can protect memory, preserve synaptic density, and reduce A ⁇ plaques in a5XFAD AD mouse model.
  • This example investigated whether the treatment with the substances promoting the cleavage of TRPM7 to produce M7CK can prevent AD pathology in the 5XFAD mouse model.
  • Pharmacological and high performance liquid chromatography studies showed that TPCA-1cannot cross the blood-brain barrier, since no TPCA-1 or its metabolites can be detected from the brain tissue and cerebrospinal fluid samples of the treated mice. Therefore, ATT was selected for in-vivo studies in this example.
  • This example prepared two groups of to-be-treated mice injected daily (i. p. ) with two doses (50 mg/kg or 75 mg/kg) of ATT. Another group of control mice was provided to receive a similar treatment regimen using the equal amount of a vehicle.
  • an open field test showed that the drug treatments did not affect the movement or exploratory activity of the mice (FIG. 18A) .
  • the nesting behavior of the 5XFAD mice treated by the vehicle was impaired.
  • the nesting behavior of the mice treated by the low dose (50 mg/kg) of ATT was also affected.
  • the high dose (75 mg/kg) of ATT successfully rescued the nesting behavior of the mice (FIGs. 18B and C) .
  • the memory function was assessed by a novel object recognition test (NORT) .
  • This example further investigated whether the substances promoting the cleavage of TRPM7to produce M7CK, namely ATT, can protect the synaptic density and reduce ⁇ plaques in the brain of the 5XFAD mice.
  • the mice were perfused in this example and the brains were collected for histological analysis.
  • This example showed that the synaptophysin puncta density of in a CA1 stratum radiatum of the hippocampus of the 5XFAD mice without ATT treatment was significantly reduced.
  • the ATT treatment counteracted the reduction of the synaptic density in a dose-dependent manner.
  • the synaptic density remained unchanged only at the dose of 75 mg/kg, and changed at the dose of 50 mg/kg (FIGs. 19A and B) .
  • both doses of the ATT treatment significantly reduced the ⁇ plaque density in the hippocampus and cortex in the brain sections of the5XFAD mice (FIG. 19C) . Therefore, the substance promoting the cleavage of TRPM7 to produce M7CK, namely ATT, can retain the synaptic density and reduce A ⁇ plaque deposition in the5XFAD mice.
  • the data of this example showed that the activation of TRPM7 may mitigate AD pathology by protecting synapses and through a disease regulatory mechanism.
  • the steps of the behavioral experiment or histological staining in this example can be referred to other examples of the present disclosure that include the behavioral experiment or histological staining.
  • FIGs. 18A-18D The detail description of FIGs. 18A-18D was as follows:
  • FIG. 18A Measurement of the total distance that the wild type (WT) mice and 5XFAD mice treated by the vehicle and ATT (50 mg/kg and 75 mg/kg) were free to move for 5 min in an open field.
  • N at least 10 mice per group, and the data was analyzed by a one-way analysis of variance and a Kruskal-Wallis test.
  • FIG. 18B Representative images of the nesting social behavior of the WT and 5XFAD mice at6 months of age, wherein 5XFAD mice were respectively treated by the vehicle and ATT (50 mg/kg and 75 mg/kg) .
  • FIG. 18C Nesting scores based on the nesting social behavior of the WT and 5XFAD mice at6 months of age, wherein 5XFAD mice were respectively treated by the vehicle and ATT (50 mg/kg and 75 mg/kg) .
  • N at least 10 mice per group, and the data was analyzed by a one-way analysis of variance and a Kruskal-Wallis test.
  • FIG. 18D The upper figure was a schematic diagram of an experimental design of a novel object recognition test.
  • the lower figure showed the recognition index of the familiar and novel objects of the WT mice and 5XFAD mice, wherein5XFAD mice were treated by the vehicle and ATT (50 mg/kg and 75 mg/kg) , the left drawing corresponded to an NORT training period, the middle drawing corresponded to a short-term memory test two hours after the NORT training, and the right drawing corresponded to a long-term memory test twenty-four hours after the NORT training.
  • N at least 8 mice per group, and the data was analyzed by a two-way analysis of variance, followed by a Bonferroni post-test.
  • FIGs. 19A-19C The detail description of FIGs. 19A-19C wasas follows:
  • FIG. 19A Representative images of the synaptophysin puncta density of a stratum radiatum in a CA1 region of the wild type (WT) and 5XFAD mice at 6 months of age, wherein 5XFAD mice were treated by the drug and ATT (50 mg/kg and 75 mg/kg) .
  • FIG. 19B Quantitative analysis of the synaptophysin puncta density of a stratum radiatum in a CA1 region of the WT and 5XFAD mice at 6 months of age, wherein 5XFAD mice were treated by the drug and ATT (50 mg/kg and 75 mg/kg) .
  • N at least 22 brain sections of 6 mice per group, and the data was analyzed by a one-way analysis of variance, followed by a Bonferroni post-test.
  • FIG. 19C The upper figure was representative images of hippocampal and cortical amyloid plaques of the 5XFAD mice treated by the vehicle and ATT (50 mg/kg and75 mg/kg) .
  • the lower figure was quantitative analysis of the hippocampal and cortical amyloid plaques of the 5XFAD mice treated by the vehicle and ATT (50 mg/kg and 75 mg/kg) .
  • N at least 18 brain sections of 6 mice per group, and the data was analyzed by a one-way analysis of variance, followed by a Bonferroni post-test. At least three brain sections per mouse were used for immunohistochemical quantitation.
  • the content of MMP14 in the WT and 5XFAD mice in example 4 was detected. It was observed that the expression level of the A ⁇ -degrading enzyme MMP14 was up-regulated in the 5XFAD mice compared with the WT mice, and the expression level was not affected by over-expressing M7CK. Compared with the 5XFAD-EGFP group, the phosphorylation level of MMP14 was significantly up-regulated in the brains of the 5XFAD-M7CK mice. The results were shown in FIG. 9. The results of protein co-immunoprecipitation in this example illustrated that MMP14 had an interaction with M7CK.
  • Mouse hippocampal cDNA was used as a template and a Forward-CCGCTCGAGATGTCTCCCGCCCCTCGACC and a Reverse-TGCTCTAGAGACCTTGTCCAGCAGCGAACG were used as primers (two ends of the primers included two restriction enzyme sites XhoI and XbaI and corresponding protection bases) for cloning a coding (CDS) region of MMP14.
  • the PCR reaction system was as follows:
  • the gel at the position of the target band was cut into small pieces in a nucleic acid developer, collected in an EP tube, and weighed, a PN solution (1 ⁇ L/mg gel) was added, the gel was placed in a water bath at50°Cand shaken continuously until the gel was completely dissolved;
  • the column was subjected to centrifugation at 12,000 rpm for 2 min to remove any remaining liquid and then air-dried at room temperature for 9 min with the lid was opened;
  • reaction were inactivated in 65°C water bath for 20 min, a sampling buffer was added, the mixture was subjected to agarose gel electrophoresis for 30 min at 120 V, and the fragment and vector of corresponding size were recovered from the gel.
  • Target gene fragment ( ⁇ 50ng/ ⁇ L) : up to 20 ⁇ L
  • the mixture was centrifuged at 11,000 rpm and at room temperature for 10 min, the upper layer water phase (containing DNA) was transferred to a new tube, isopropanol at the volume of 0.5 time was added, and the solution was transferred into the adsorption column in multiple times and centrifuged at 11,000 rpm for 1 min, and the waste liquid in the collection tube was discarded;
  • the column was washed by using 10 mL of a rinsing liquid PW and the obtained solution was centrifuged at 11,000 rpm for 1 min, the waste liquid was discarded, and the operation was repeated twice;
  • a bacterial lysate (50 mM Tris HCI pH 8.0, 100 mM NaCl, 10 mM MgCl 2 , 1 mM EDTA, 10 mM mercaptoethanol, 20%glycerol, 1 mg/ ⁇ L lysozyme, 8 M urea, one tablet of a Roche protease inhibitor) was added into the bacterial precipitate and incubated on ice for 30 min.
  • the supernatant was added to an amicon ultra15ML-30K protein ultrafiltration centrifuge tube to be centrifuged at 4,200 rpm and 4°C for 60 min, and this operation was repeated several times until the remaining liquid was less than 1 mL.
  • the liquid contained a concentrated protein and was used for a subsequent in-vitro enzyme activity test.
  • Co-IP Protein co-immunoprecipitation
  • MMP14 and M7CK recombinant protein were purified from escherichia coli by the above method.
  • 80 nM MMP14/M7CK was mixed with 2 ⁇ M A ⁇ 1-42 , a zymogen buffer (50 mM tris HCl, 200 mM NaCl, 5 mM CaCl 2 , pH 7.5) and10 mM ATP were added, and a total volume of the system was 50 ⁇ L, and the system was incubated at 37°C for 10h. The remaining A ⁇ content was detected by using western blotting. Lower residual A ⁇ content indicated higher degradation efficiency.
  • the cells After cultured for 1.5-2 h in a constant-temperature incubator, the cells basically adhered to the wall, at this time, 1 mL of the plating culture medium/well was added, the culture medium was changed into a serum-free culture medium after two days, and half of the culture medium was changed at an interval of 3-4 days.
  • a synthesized TRPM7-shRNA sequence (coding sequence 5153-5172 5'-AAT GCA TGA CTG GGG AAT TTC AAG AGA ATT CCC CAG TCA TGC at-3') was inserted into an AAV-CAG-mCherry plasmid, and the plasmid was packaged into an AAV virus.
  • the AAV virus (1 ⁇ l/ml of a culture medium) was added when the primary hippocampal neurons of the mice were cultured to the seventh day.
  • the cells After cultured for 1.5-2 h in a constant-temperature incubator, the cells basically adhered to the wall, at this time, 1 mL of the plating culture medium/well was added, the culture medium was changed into a serum-free culture medium after two days, and half of the culture medium was changed at an interval of 3-4days.
  • a synthesized MMP14-shRNA sequence (#1 coding sequence 1026-1046, 5'-ACC GGG GGG GTG AGG AAT AAC CAA GTC TCG AGA CTT GGT TAT TCC TCA CCC GCT TTT T-3'. #2 coding sequence 788-808, 5'-ACC GGG GAT GGA CAC AGA GAA CTT CGC TCG AGC GAA GTT CTC TGT GTC CAT CCT TTT T-3') was inserted into an AAV-CAG-mCherry plasmid, and the plasmid was packaged into an AAV virus.
  • the AAV virus (1 ⁇ l/ml of a culture medium) was added when the primary hippocampal neurons of the mice were cultured to the seventh day.
  • TRPM7 was down-regulated in the hippocampus of the patients with AD and both AD mouse models; over-expressing the kinase domain of TRPM7 in the primary hippocampal neurons cultured in vitro can counteract the reduction in the synaptic density induced by ⁇ , whereas the channel domain of TRPM7 had no such protective effect.
  • over-expressing M7CK in the hippocampus of the 5XFAD mice can relieve cognitive impairment, increase the synaptic density and reduce the accumulation of amyloid plaques induced by A ⁇ . Meanwhile, M7CK further promotes the degradation of ⁇ in the soluble and insoluble components, and M7CK did not influence the generation process of A ⁇ .
  • MMP14 a member of the MMP family, is a specific MMP located on the membrane and has activity itself.
  • the experimental results of the above examples showed that MMP14 had an interaction with M7CK, while MMP2 and MMP9 had no interaction with M7CK.
  • MMP14 coexists with M7CK
  • the activity of MMP14 was further improved, and the protective effect of M7CK against A ⁇ toxicity also depended on MMP14 rather than MMP2 and MMP9.
  • MMP14 can degrade exogenous A ⁇ 40 and A ⁇ 42 : under in-vitro suitable conditions, after the purified recombinant MMP14 protein was mixed and incubated with the A ⁇ peptide fragment for 10 h, the A ⁇ level was reduced by 50%, which was consistent with the literature report.
  • the above examples showed that in the hippocampus of the 5XFAD mice at 6 months of age, the MMP14 protein level was higher than that of the wild-type mice in the same age, which indicated that merely increasing the MMP14 expression level was not sufficient to alleviate AD-related pathological features.
  • over-expressing M7CK can promote degradation of A ⁇ , increase the synaptic density, and improve the cognitive function of the 5XFAD mice by increasing the MMP14 phosphorylation level, indicating that regulating the MMP phosphorylation level had a more significant impact on relieving the AD pathology compared to regulating the MMP14 expression level, with no change in the production process of APP (APP, ⁇ CTF) .
  • the knock-out of MMP14 in the hippocampal primary neurons reduced the synaptic density, suggesting that the MMP14 itself played an important regulatory role in the synaptic function.
  • the present disclosure will fill the major gap in the prior art for understanding the mechanism of action of the TRPM7 kinase domain in the central nervous system.
  • This project will reveal new signaling pathways and mechanisms of TRPM7 kinase domain regulation.
  • the present disclosure will demonstrate that TRPM7 uses physical and structural changes (cleavage and transport of peptides) rather than chemical modifications (phosphorylation) as triggers for the release and activation of kinases.
  • structural changes of CaMKII rather than phosphorylation have been shown to trigger important effects on synaptic and cognitive functions. Therefore, the present disclosure is introducing entirely new concepts and discoveries that will expand the understanding of the prior art in cellular, molecular, and cognitive neuroscience.
  • the present disclosure combines computational, molecular, systematic, and biomedical research to achieve a transformation from basic science to transformation science.
  • the new mechanisms are expected to be converted into potential drug targets so as to develop new drugs for treating neurodegenerative diseases (particularly Alzheimer's disease) .
  • the present disclosure provides the evidence for the feasibility of TRPM7 as a target for treating Alzheimer's disease.
  • a novel gene therapy method for AD is provided: over-expressing the TRPM7enzymatic domain mediated by viruses.
  • the present disclosure explores in detail the role that TRPM7 may play in the course of AD and the specific action mechanism thereof.
  • the present disclosure provides a potential target with important value for the research and development of drugs for AD.

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Abstract

La présente invention concerne le domaine technique de la médecine biologique, en particulier l'utilisation d'un domaine kinase M7CK de TRPM7 dans la préparation d'un médicament pour le traitement de la maladie d'Alzheimer. La surexpression de M7CK peut atténuer une déficience cognitive d'un modèle de souris atteint de la MA en augmentant un niveau de phosphorylation de MMP14, en améliorant la capacité d'apprentissage et de mémoire, en augmentant la densité synaptique et la densité d'une synaptophysine de protéine de vésicule présynaptique et d'une protéine de densité postsynaptique PSD-95, en favorisant la dégradation de Aβ dans des composants solubles et insolubles, et en réduisant l'accumulation de plaques amyloïdes Aβ. Par conséquent, le domaine kinase de TRPM7 fourni présente une grande perspective d'applications dans la thérapie génique d'un patient atteint de la maladie d'Alzheimer.
PCT/CN2024/098301 2023-06-09 2024-06-08 Utilisation du domaine kinase m7ck de trpm7 dans la préparation d'un médicament pour le traitement de la maladie d'alzheimer WO2024251289A1 (fr)

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