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WO2018012497A1 - Modèle animal de maladie et agent thérapeutique de maladie - Google Patents

Modèle animal de maladie et agent thérapeutique de maladie Download PDF

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WO2018012497A1
WO2018012497A1 PCT/JP2017/025273 JP2017025273W WO2018012497A1 WO 2018012497 A1 WO2018012497 A1 WO 2018012497A1 JP 2017025273 W JP2017025273 W JP 2017025273W WO 2018012497 A1 WO2018012497 A1 WO 2018012497A1
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gene
disease
fus
syngapα2
brain
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Japanese (ja)
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元 祖父江
診祐 石垣
聡 横井
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Nagoya University NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • A01K67/0276—Knock-out vertebrates
    • 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
    • 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
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    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4711—Alzheimer's disease; Amyloid plaque core protein
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • 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
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates

Definitions

  • the present invention belongs to the technical field of genetically modified experimental animals and therapeutic agents for diseases.
  • INDUSTRIAL APPLICABILITY The present invention is effective for a FUS gene-specific knockout model animal in the brain that exhibits symptoms of a disease accompanied by synaptic abnormalities (for example, frontotemporal lobar degeneration), a method for producing the same, or the disease using the model animal.
  • the present invention relates to a method for screening a therapeutic agent or a therapeutic agent for the disease.
  • dementia greatly reduces the patient's quality of life, and the burden on the caring family is very large. Symptoms due to dementia range from memory impairment, visual impairment, language impairment, behavioral problems, sleep disorders, and depressive symptoms.
  • Known brain degenerative diseases that cause such dementia include Alzheimer's disease, Lewy body dementia, cerebrovascular dementia, frontotemporal lobar degeneration (FTLD), and the like.
  • ⁇ Dementia caused by FTLD is progressive dementia, second only to Alzheimer's disease, especially in the West.
  • the main feature of FTLD is that the frontal lobe and temporal lobe are degenerative sites, and histopathological findings are of three types: frontal degeneration, Pick disease, and motor neuron disease.
  • Symptoms include behavioral abnormalities due to higher brain dysfunction such as personality disorder and social behavioral abnormalities.
  • the pathological condition of FTLD is very complicated, and research on diagnostic methods and therapeutic methods has not been sufficiently advanced so far, so that a fundamental therapeutic method has not yet been established.
  • FTLD FTLD
  • a model animal system is preferably a genetically modified model animal such as a knockout mouse.
  • FTLD is said to cause neurodegeneration by abnormal aggregation / accumulation and abnormal localization of FUS and TDP-43 proteins in nerve cells. Therefore, a dementia model animal such as FTLD has been produced by overexpression of a dementia-related gene, for example, by knocking in a gene.
  • the model itself may be based on abnormal findings because it is impossible to deny the possibility that the toxicity caused by the large amount of the specific gene causing neurodegeneration.
  • Patent Document 1 discloses a model mouse of amyotrophic lateral sclerosis and / or frontotemporal lobar degeneration.
  • the model mouse is a knock-in mouse in which at least one allele of endogenous TDP-43 is replaced with mutant TDP-43.
  • the model mouse exhibits the same pathological condition as amyotrophic lateral sclerosis and frontotemporal lobar degeneration, but it is a knock-in mouse, so it is difficult to determine whether the model itself is due to abnormal findings.
  • the group of the present inventors conducted research on the above-mentioned pathological condition of FTLD using mice, and as a result, there were mice that knocked down the FUS (Fused in sarcoma) gene in the mouse brain using a shRNA-expressing lentivirus. And FTLD-like disease state (see Non-Patent Document 1). Furthermore, in cultured cells in which the FUS gene is knocked down, the amount of glutamate receptor GluA1 protein present in the synapse is significantly reduced, and FUS has a function of specifically binding to and stabilizing GluA1 mRNA, and It was shown that FTLD-like pathology of mice knocked down by the above-mentioned FUS was recovered by GluA1 gene transfer.
  • SynGAP Synthetic Ras GTPase Activating Protein
  • PSD postsynaptic thick part
  • SynGAP protein has been found to exist from nematodes to higher mammals.
  • SynGAP protein has various types of splicing consisting of a combination of multiple types of C-terminal domains ( ⁇ 1, ⁇ 2, ⁇ , ⁇ , etc.) and multiple types of N-terminal domains (A, B, C, etc.). The existence of the variant is shown, suggesting its various functions (for example, see Non-Patent Document 4 and Non-Patent Document 5).
  • a model in which the FUS gene is injected with, for example, an adeno-associated virus and knocked down has a limited effect only on the hippocampus, and it is not easy to learn a technique.
  • All reported FUS gene knockout mice are crossbred, and in particular, the behavioral analysis may have different results depending on the strain.
  • tissue-specific knockout technology using the loxP-Cre system is known, the phenotype differs depending on the choice of promoter and the expression method of Cre. ) Is not always easy to produce.
  • the main object of the present invention is to provide a novel model animal exhibiting a symptom of a disease accompanied by synaptic abnormality (for example, FTLD) and a method for producing the same.
  • Another object of the present invention is to provide a method for screening a therapeutic agent effective for a disease accompanied by synaptic abnormality using the model animal, and a therapeutic agent and a therapeutic method for the disease.
  • the present inventors have found that the above problem can be solved by conditional knockout of the FUS gene in the brain, and have completed the present invention.
  • the present inventors have also found that increasing the protein SynGAP ⁇ 2 present in the post-synaptic thick part is effective for the treatment of diseases accompanied by synaptic abnormalities.
  • Examples of the present invention include the following.
  • [1] A model animal exhibiting a symptom of a disease accompanied by synaptic abnormality, which is produced by specific knockout of the FUS gene in the brain.
  • [2] The model animal according to [1] above, wherein the knockout is performed using a Cre-loxP system and a CamK2 promoter.
  • [3] The model animal according to [1] or [2] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
  • FTLD frontotemporal lobar degeneration
  • [4] The model animal according to any one of [1] to [3] above, wherein the animal is a mouse.
  • [5] The model animal according to any one of [1] to [4] above, wherein the animal is an inbred line.
  • [6] A model neuron or model neuron cell line obtained from the model animal according to any one of [1] to [5] above.
  • a method for producing a model animal exhibiting a symptom of a disease accompanied by a synaptic abnormality comprising an operation step of specifically knocking out a FUS gene in the brain.
  • the production method according to [7] above, wherein the operation step of specifically knocking out the FUS gene in the brain uses a Cre-loxP system and a CamK2 promoter.
  • the production method according to [7] or [8] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
  • FTLD frontotemporal lobar degeneration
  • a synapse comprising a step of using the model animal according to any one of [1] to [5] above, or the model nerve cell or model nerve cell line according to [6] above
  • a screening method for searching for a therapeutic drug for a disease accompanied by an abnormality [13] The screening method according to [12] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
  • FTLD frontotemporal lobar degeneration
  • a screening method for searching for a therapeutic agent for a disease associated with a synaptic abnormality comprising a step of measuring an increase or decrease of SynGAP ⁇ 2 due to application of a target drug.
  • the screening method according to [14] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
  • FTLD frontotemporal lobar degeneration
  • the method according to [18] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
  • FTLD frontotemporal lobar degeneration
  • motor neuron diseases such as frontotemporal lobar degeneration (FTLD) and motor neuron diseases
  • FTLD frontotemporal lobar degeneration
  • a drug containing a therapeutic drug found from such screening can be used for the treatment of the disease.
  • the upper diagram is a schematic diagram of a wild type gene (WT) before gene insertion
  • the middle diagram is a schematic diagram of an inserted vector gene
  • the lower diagram is a schematic diagram of a recombinant gene (KO) after gene insertion.
  • the upper figure is a schematic diagram of a wild type gene (WT)
  • the middle figure is a schematic diagram of a recombinant gene (KO) into which a vector has been inserted
  • the lower figure is a conditional knockout completed by removing the Neo cassette by crossing with FLP mice.
  • FIG. 4A a region containing exons 1 to 6 in the wild-type gene sequence of the FUS gene targeted for genetic manipulation is represented.
  • FIG. 4A a region containing exons 1 to 6 of the FUS gene.
  • FIG. 5A Represents the gene sequence of the AAV vector into which the SynGAP A ⁇ 2 gene sequence has been inserted.
  • FIG. 6A For the control mouse (Cre ⁇ ) and the FUS gene conditional knockout mouse expressing Cre (FUS-cKO mouse (Cre +)), FIG.
  • FIG. 4 shows Golgi-stained images of brain tissue sections in the hippocampal CA1 region for control mice (Cre-) and FUS-cKO mice (Cre +). It represents the total number of spines in the brain tissue section. The vertical axis represents the total number of spines per 100 ⁇ m of neurites. It represents the percentage of mature spine in the brain tissue section. The vertical axis shows the percentage (%) of mature spine in the total number of spines. The results of the open field test in control mice (Cre-) and FUS-cKO mice (Cre +) are represented.
  • the vertical axis represents the movement distance (mm) of the mouse in the arena. Elevated cruciform maze test in control mice (Cre-) and FUS-cKO mice (Cre +), figure (A) shows results with "walled road” or “wallless road” . The vertical axis represents the stay time (seconds). FIG. (B) shows the result for “road without walls”. The vertical axis represents the number of intrusions (times). For control mice (Cre ⁇ ) and FUS-cKO mice (Cre +), FIG. (A) represents the results of a novel object recognition test. The vertical axis represents search preference (%). Figure (B) represents the results of the fear conditioning test. The vertical axis shows the time (seconds) of the freezing reaction.
  • FIG. (A) shows the fluorescence immunostained image in the hippocampal CA1 region
  • FIG. (B) shows the Western blot result of the hippocampal CA1 region sample.
  • Golgi-stained images of brain tissue sections are shown for FUS-cKO mice (Cre +), FUS-cKO mice (Cre +), and control mice (Cre ⁇ ) supplemented with SynGAP A ⁇ 2. It represents the percentage of mature spine in the brain tissue section.
  • the vertical axis shows the percentage (%) of mature spine in the total number of spines.
  • the open field test results are shown for FUS-cKO mice (Cre +), FUS-cKO mice (Cre +) and control mice (Cre ⁇ ) supplemented with SynGAP A ⁇ 2.
  • the vertical axis represents the movement distance (cm).
  • the results of the elevated plus maze test are shown for FUS-cKO mice (Cre +), FUS-cKO mice (Cre +), and control mice (Cre ⁇ ) supplemented with SynGAP A ⁇ 2.
  • the vertical axis shows the number of times of intrusion (times) on a road without a wall.
  • Model animal according to the present invention A model animal according to the present invention (hereinafter referred to as “model animal of the present invention”) is produced by specific knockout of the FUS gene in the brain, and is characterized by diseases associated with synaptic abnormalities. Symptoms are present.
  • the “brain” in the present invention also exists in lower organisms, but particularly in higher organisms, mainly the cerebrum or a portion corresponding thereto, particularly the frontal lobe, temporal lobe, hippocampus and the like can be mentioned.
  • FUS gene FUS gene is used for fish such as nematodes and zebrafish, amphibians such as frogs, rodents such as mice, rats and guinea pigs, rabbits, ferrets, dogs, cats, pigs, sheep, goats, cattle, It is a gene that exists widely in horses and higher mammals such as monkeys and humans.
  • amphibians such as frogs
  • rodents such as mice, rats and guinea pigs, rabbits, ferrets, dogs, cats, pigs, sheep, goats, cattle
  • It is a gene that exists widely in horses and higher mammals such as monkeys and humans.
  • the base sequence information of the FUS gene in each animal can be obtained from, for example, Genbank, which is a gene information database.
  • Genbank which is a gene information database.
  • the FUS gene of the target animal is genetically manipulated using data such as a gene information database and specifically knocked out.
  • the model animal of the present invention is produced by specifically knocking out the FUS gene of the animal in the brain.
  • a conditional knockout method which is a method of knocking out a target gene of a target model animal in terms of time and space, can be mentioned.
  • a recombinant enzyme Cre (Cre recombinase) derived from bacteriophage P1 and its target sequence
  • a loxP sequence system (Cre-loxP system)
  • budding yeast Sacharomyces cerevisiae
  • soy sauce yeast Zaygosaccharomyces rouxii
  • bacteriophage Mu-derived recombinant enzyme Gin and its target Examples include Gix array systems and tetracycline ON-OFF systems. Of these, the Cre-loxP system is preferable.
  • the Cre-loxP system is a system using Cre recombinase, which is a gene recombination enzyme, and loxP sequence, which is a recognition sequence thereof.
  • the system is usually a genetically engineered model animal in which Cre recombinase is linked to a promoter that functions in a time-specific and / or site-specific manner, and the target gene is sandwiched between loxP sequences. Cre recombinase expressed in a manner causes deletion of the target gene flanked by loxP sequences.
  • the FUS gene can function normally without being knocked out at the stage of fertilized egg or early development, and the FUS gene can be specifically knocked out in the brain of the model animal at the stage of growth to adulthood.
  • the gene of interest is manipulated in the fertilized egg or the early stage of development of the model animal, for example, from the 2 cell stage to the blastocyst, the blastocyst, and the FUS gene conditional knockout which is the model animal of the present invention
  • a model animal (FUS-cKO model animal) is created.
  • the promoter into which the Cre recombinase gene is incorporated is not particularly limited as long as it specifically functions in the adult brain of the model animal, and examples thereof include CamK2, Arc, and Chat. Of these, the CamK2 promoter is preferred.
  • the mouse CamK2 promoter is a promoter in which significant gene expression is observed in the mouse brain after 2 months of age, and can specifically knock out the FUS gene in the adult brain.
  • FTLD frontotemporal lobar degeneration
  • motor neuron diseases examples include frontotemporal lobar degeneration (FTLD) and motor neuron diseases.
  • the model animal of the present invention develops degeneration mainly in the frontal and temporal lobes, and exhibits histopathological findings of frontal lobe degeneration and motor neuron disease.
  • Symptoms include, for example, marked atrophy of frontal and temporal lobes or behavioral abnormalities caused by higher brain dysfunction.
  • Examples of the synaptic abnormality exhibited by the model animal of the present invention include an abnormality of spine of dendrites existing in the cell body of nerve cells.
  • the form of spine present in the post-synapse changes to a mushroom type when the synapse formation matures, and it is said that the increase in the surface area in contact with the pre-synapse causes synaptic enhancement, which affects behavioral physiology (Hippocampus 2000; 10: 501, JCB 2010; 189: 619).
  • changes in dendritic spines, which are post-synaptic may be involved in the pathological condition (Annu Rev Pathol 2016; 11: 221, Neurosci Biobehav Rev 2015; 59: 208).
  • Examples of the spine abnormality exhibited by the model animal of the present invention include a decrease in the number of spines, a decrease in the number of mature spines, and an abnormality in the spine form.
  • an abnormality of a protein present in the synapse is caused along with the synapse abnormality.
  • Examples of abnormalities in the protein present at the synapse include abnormal localization of PSD-95 and a decrease in SynGAP ⁇ 2.
  • the disease accompanied by synaptic abnormality exhibited by the model animal of the present invention may show higher brain dysfunction as a result.
  • the higher brain dysfunction include hyperactivity (abnormal amount of behavior), anxiety behavior, memory impairment, depression-like behavior, personality disorder, social behavior abnormality, and pain sensitivity abnormality.
  • behavior tests for abnormal behavioral quantities include an open field test and a home cage activity analysis test.
  • anxiety behavior test include an elevated plus maze test (Elevated plus maze test) and a light / dark selection box test (Light / dark transition test).
  • Examples of the memory impairment behavior test include a novel object recognition test, a fear conditioning test, and a Morris water maze test.
  • Examples of the behavior test for depression-like behavior include a porsol forced swim test and a tail suspension test.
  • Examples of the behavioral test for abnormal social behavior include a social behavior test and a 24-hour home cage social behavior test.
  • Examples of tests for abnormal pain sensitivity include a hot plate test and a formalin test.
  • the animal species used for the production of the model animal of the present invention is not particularly limited as long as it is usually used as an experimental animal.
  • nematodes fish such as zebrafish, frogs, etc.
  • Amphibians rodents such as mice, rats and guinea pigs, primates such as monkeys and marmosets, rabbits, ferrets, dogs, cats, pigs, sheep, goats, cows and horses.
  • rodents such as mice, rats and guinea pigs, or primates such as monkeys and marmosets, and more preferred are mice.
  • Many of these animal strains are readily available from laboratory animal vendors.
  • the strain of the animal may be a hybrid or inbred, and is not particularly limited. However, in order to minimize individual differences in the results of animal experiments, an inbred strain with a uniform genetic background is preferable. Inbred experimental animals are usually obtained by inbred mating of 20 generations or more in the case of mice, and therefore have 0.01% or less heterozygous genes. It can be regarded as the same individual. Examples of general inbred experimental animals include the Wister system in rats and the DBA / 2 system, C57BL / 6 system, and BALB / c system in mice. These inbred experimental animals can be easily obtained from experimental animal manufacturers. It can also be established using an inbred animal production service such as the MAX-BAX program provided by an experimental animal company such as Charles River.
  • an inbred model animal especially a mouse suitable for experiments is also provided.
  • the present invention is a model neuron obtained from the model animal of the present invention (hereinafter referred to as “the model neuron of the present invention”) or a model neuron cell line (hereinafter “model neuron cell”). And “the model neuronal cell line of the present invention”).
  • the model neuron of the present invention is a primary cultured neuron obtained from the model animal of the present invention, and can be obtained and cultured by a known method used for primary culture of neurons.
  • the model nerve cells of the present invention are usually obtained from the brain of the model animal of the present invention or the nerve cells of the nervous system.
  • the model neuronal cell line of the present invention is a cell line (established cell) established by repeating subculture from the above-described model neuronal cell of the present invention.
  • the model neuron of the present invention and the model neuron cell line of the present invention have the advantage that they can be easily handled as compared with the model animal of the present invention, and are stored frozen in liquid nitrogen or the like, and thawed as necessary. You can also.
  • Known methods in the technical field of cell culture can be used for culturing, subculturing, cryopreserving, thawing and the like of the model neuron of the present invention.
  • Known methods in the technical field of cell culture can also be used for research, experiments, and tests using the model nerve cells of the present invention.
  • the “cell line” refers to a cell that has been maintained by in vitro subculture for a long period of time and has reached a certain stable property.
  • the present invention comprises a method for producing a model animal exhibiting a symptom of a disease accompanied by a synaptic abnormality characterized by having an operation step of specifically knocking out a FUS gene in the brain (hereinafter referred to as “production of the present invention”).
  • Method “FUS gene”, “disease with synaptic abnormality”, “animal species” and the like are as defined above.
  • Examples of the operation step for specifically knocking out the FUS gene in the brain according to the production method of the present invention include the conditional knockout method described in the section “1.2 Specific knockout of FUS gene in the brain”.
  • a system used in such a conditional knockout method for example, the same system as that shown in the same section can be cited, among which the Cre-loxP system is preferable.
  • the knockout operation it is preferable to use the Cre-loxP system and the CamK2 promoter.
  • a specific gene is genetically manipulated at the developmental stage of the target animal.
  • the method for performing such genetic manipulation include a microinjection method in which DNA is directly injected into the pronucleus of a fertilized egg, a method using a retroviral vector, a method using an ES cell, and a method using an iPS cell. be able to.
  • a gene injected into a fertilized egg or the like is integrated into a chromosome by, for example, homologous recombination.
  • Cre gene a FUS gene sandwiched between loxP sequences and a sequence encoding the full length of the Cre recombinase gene
  • Cre gene a sequence encoding the full length of the Cre recombinase gene
  • a fertilized egg is first collected from the oviduct of a female mouse in which mating has been confirmed, and after culturing, a desired DNA construct is injected into its pronucleus.
  • a DNA construct containing a Cre gene is used.
  • the CamK2 promoter which is a promoter sequence that enables time-specific expression of the transgene, can be used.
  • a FUS gene having a loxP sequence is inserted into the chromosome by homologous recombination.
  • the fertilized egg that has completed the injection operation is transplanted into the oviduct of a pseudopregnant mouse, and the transplanted mouse is bred for a predetermined period to obtain a pup mouse (F0).
  • the Cre gene and / or the FUS gene having the loxP sequence are properly integrated into the chromosome of the pup, extract DNA from the tail of the pup and use a primer specific to the transgene.
  • the appropriate recombinant mouse is selected by the conventional PCR method or dot hybridization method using a probe specific to the transgene.
  • the evaluation of whether or not the conditional knockout has been properly performed can be confirmed by examining the target tissue, that is, the brain tissue.
  • test method in a test for FUS protein, for example, immunostaining of a tissue section, Western blot of extracted protein, and for a test for FUS mRNA, for example, quantitative PCR, Northern hybridization, in situ hybridization is mentioned.
  • the present invention searches for a therapeutic agent for diseases associated with synaptic abnormalities, comprising the step of using the model animal of the present invention, or the model neuronal cell of the present invention or the model neuronal cell line of the present invention.
  • Screening method (hereinafter referred to as “the screening method of the present invention”).
  • the screening method of the present invention is usually applied to the model animal of the present invention that exhibits symptoms such as diseases associated with synaptic abnormalities such as frontotemporal lobar degeneration (FTLD) and motor neuron disease. This is done by administering a drug and assessing whether its characteristic symptoms, pathological findings, or higher brain dysfunction is alleviated and treated.
  • FTLD frontotemporal lobar degeneration
  • the target drug to the model animal of the present invention, when the characteristic symptoms, pathological findings or higher brain dysfunction is alleviated and treated, it is evaluated that the drug may treat or alleviate the disease can do.
  • the target drug is administered to the model animal of the present invention.
  • a step of measuring a change (a change before and after administration) of a protein or mRNA of a gene associated with a disease accompanied by a synaptic abnormality, particularly a brain, caused as a result thereof can be mentioned.
  • genes associated with diseases associated with such synaptic abnormalities include SynGAP ⁇ 2, Tau, and GluA1. Among these, it is preferable to measure increase / decrease in SynGAP ⁇ 2 protein or mRNA.
  • the drug when the amount of SynGAP ⁇ 2 protein or mRNA in the diseased part is increased before administration or compared to the control group, the drug may treat or alleviate the disease Can be evaluated.
  • the increase or decrease of SynGAP ⁇ 2 can be confirmed by measuring the amount of mRNA encoding SynGAP ⁇ 2 or the amount of SynGAP ⁇ 2 protein by a conventional method.
  • the mRNA measurement method include quantitative RT-PCR and Northern hybridization.
  • protein measurement methods include Western blotting and immunostaining.
  • the evaluation of the effectiveness by the above-mentioned measurement is performed using, for example, a tissue extract of a disease site or a tissue section.
  • a disease accompanied by synaptic abnormality eg, frontotemporal lobar degeneration (FTLD), motor neuron diseases, etc.
  • FTLD frontotemporal lobar degeneration
  • motor neuron diseases etc.
  • Such a screening method is also included in the present invention (hereinafter also referred to as “the screening method of the present invention” including the screening method).
  • Increase / decrease of SynGAP ⁇ 2 can be measured by a conventional method. As a result of application of the target drug, when SynGAP ⁇ 2 increases before application or compared to the control group, it can be evaluated that the drug may treat or alleviate the disease.
  • the increase or decrease of SynGAP ⁇ 2 in the brain of the animal used is usually measured.
  • the increase or decrease of SynGAP ⁇ 2 in cells or the like to be used is usually measured.
  • the animal species that can be used are not particularly limited as long as they are non-human animals that are usually used as experimental animals.
  • non-human animals that are usually used as experimental animals.
  • primates monkeys, marmosets
  • rodents Mouse, rat, guinea pig, etc.
  • rabbit, dog, cat, pig, cow, sheep, horse in the in vitro screening method described above.
  • cultured cells of the brain or nerves of humans and non-human animals used as experimental animals as described above are usually used.
  • Examples of cultured human cells include NSC-34, which is a cultured cell derived from motor nerves.
  • Examples of cultured cells of non-human animals include mouse primary cultured neurons, Neuro2A. It is also possible to use iPS cells prepared from cells of humans or non-human animals with or without the above-mentioned diseases that exhibit synaptic abnormalities.
  • the target drug (therapeutic drug candidate) applicable to the screening method of the present invention is not particularly limited as long as it is usually used as a drug.
  • a drug for example, low molecular organic compounds, inorganic compounds, peptides, proteins, glycoproteins, Mention may be made of lipids, glycolipids, sugars and nucleic acids. In addition, it may be an extract or culture supernatant of plants, microorganisms, cells and the like.
  • the application method (administration method) of the target drug to the model animal of the present invention can include a normal administration method to a laboratory animal.
  • oral administration intravascular Injection, administration using a catheter, application to the epidermis, subcutaneous administration, intraperitoneal administration, intrathecal administration, intraventricular administration, brain parenchymal injection, but since the target site is a diseased part of the brain, oral Administration, intravascular injection, administration using a catheter to a diseased site, intrathecal administration, intraventricular administration, and brain parenchymal injection are preferred.
  • the present invention is characterized by containing as an active ingredient a gene encoding SynGAP ⁇ 2 or a SynGAP ⁇ 2 protein, or a drug that promotes the expression of the SynGAP ⁇ 2 gene in the brain or a drug that can increase the SynGAP ⁇ 2 protein.
  • a therapeutic agent for diseases associated with synaptic abnormalities hereinafter referred to as “the therapeutic agent of the present invention”.
  • “treatment” includes not only directly or indirectly improving, alleviating or curing the target disease, symptoms or associated symptoms, but also, for example, preventing such diseases and the like. It is a term.
  • SynGAP ⁇ 2 in the present invention has an ⁇ 2 domain on the C-terminal side among the Splicing variants of SynGAP, and the domain on the N-terminal side may be any of A, B, C, and the like.
  • the present inventors have confirmed that mature spines in brain neurons are significantly reduced while investigating the involvement of FUS in the pathophysiology using the model animals of the present invention, and at the same time, synGAP2 ⁇ in brain neurons. It has also been confirmed that the expression of this gene is decreased, that the selective phenotype of SynGAP2 ⁇ has the same phenotype as that of the model animal of the present invention, and that mature spine has been restored by supplementation with the SynGAP2 ⁇ gene. Therefore, a drug capable of increasing SynGAP ⁇ 2 in the brain may be effective for treating diseases associated with synaptic abnormalities such as FTLD.
  • Examples of such a drug include a drug containing a gene encoding SynGAP ⁇ 2 or a SynGAP ⁇ 2 protein, a drug that promotes the expression of the SynGAP ⁇ 2 gene in the brain, or a drug that can increase the SynGAP ⁇ 2 protein as an active ingredient.
  • the nucleotide sequence of the human SynGAP ⁇ 2 gene and the amino acid sequence of the protein encoded by the gene are represented by SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the nucleotide sequence of the mouse SynGAP ⁇ 2 gene and the amino acid sequence of the protein encoded by the gene are represented by SEQ ID NO: 3 and SEQ ID NO: 4, respectively.
  • SynGAP ⁇ 2 DNA contained in the therapeutic agent of the present invention hybridizes under stringent conditions with DNA having a base sequence complementary to the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3.
  • DNA to be soybean base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3, BLAST (Basic Local Alignment Search at the National Center for Biological Information) (National Biological Information Center Basic Local Alignment Search Tool), etc. (E.g., using default or default parameters) and at least 85%, preferably 90%, more preferably 95%, especially preferred.
  • DNA having a homology of 97% or more or the amino acid sequence of the protein encoded by the DNA.
  • stringent conditions are, for example, conditions of about “1XSSC, 0.1% SDS, 37 ° C.”, and more severe conditions are “0.5XSSC, 0.1% SDS, 42 ° C.” The condition is about “0.2XSSC, 0.1% SDS, 65 ° C.”.
  • isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe.
  • the combination of the above SSC, SDS, and temperature conditions is an example, and the necessary stringency can be realized by appropriately combining the probe concentration, probe length, hybridization reaction time, and the like. is there.
  • the human SynGAP ⁇ 2 gene can be obtained from human cells, human tissues and the like based on the sequence information of SEQ ID NO: 1.
  • the mouse SynGAP ⁇ 2 gene can be obtained from mouse cells, mouse tissues and the like based on the sequence information of SEQ ID NO: 3.
  • the therapeutic agent of the present invention also includes a vector containing the SynGAP ⁇ 2 gene. By introducing the vector into a subject, the SynGAP ⁇ 2 protein is expressed in the subject and can exert an effect of promoting mature spine formation. Such introduction of the target gene into the subject in the administration of the therapeutic agent of the present invention can be performed by a known method.
  • viral vectors used in the therapeutic agent of the present invention include viral vectors such as adenovirus, adeno-associated virus (AAV), retrovirus, detoxified retrovirus, herpes virus, vaccinia virus, poxvirus, poliovirus, syn Examples thereof include DNA viruses or RNA viruses such as bisvirus, Sendai virus, SV40, and immunodeficiency virus (HIV). Of these, adenovirus and adeno-associated virus (AAV) are preferred. It is possible to introduce a gene into a cell by introducing the gene of interest into the above viral vector and infecting the cell with a recombinant virus.
  • AAV adeno-associated virus
  • an adeno-associated virus vector (AAV vector) is preferably used.
  • the AAV vector may contain regulatory elements for efficiently expressing the target DNA, such as a promoter, enhancer, transcription terminator, etc., and insert a translation start codon, a translation stop codon, etc. as necessary. Also good.
  • AAV vectors can be used for gene transfer into both proliferating / non-proliferating cells, and in particular, non-dividing cells can express the target gene for a long period of time. Compared to adenovirus vectors and retrovirus vectors, it is less immunogenic and suitable for gene transfer into animals. In addition, since it is a non-pathogenic virus, it can be handled in P1 level facilities and is widely used as a research virus vector that is safe and easy to handle. In addition, there are more than 100 serotypes in AAV, and it is known that the characteristics of host ranges and viruses differ depending on the serotype. Serotype 2 (AAV2) is one of the serotypes that has been extensively studied since ancient times, and is known to have a very wide host range.
  • Serotype 1 (AAV1), serotype 5 (AAV5) and serotype 6 (AAV6) are serotypes with higher tissue orientation, AAV1 is muscle, liver, airway, central nervous system, etc.
  • AAV5 is AAV6, such as the central nervous system, liver, and retina, is said to have high gene transfer efficiency into the heart, muscle, liver, and the like, and can be used properly according to the target tissue.
  • serotype 9 (AAV9) was used.
  • Non-Patent Document 1 has a track record of using a virus prepared from a plasmid having the same backbone as AAV9 used in this experiment.
  • the AAV vector used for the therapeutic agent of the present invention can be prepared by standard methods well known in the art.
  • US Pat. No. 5,858,351 and references cited therein describe various recombinant AAV suitable for use in gene therapy, as well as methods for making and propagating these vectors ( For example, Kotin (1994) Human Gene Therapy 5: 793 ⁇ 801 or Berns “Parvoviridae and their Replication” Fundamental Virology, 2nd edition, edited by Fields & ipeKnipe).
  • a plasmid is produced by leaving the ITRs at both ends of wild-type AAV and inserting a target gene between them (AAV vector plasmid).
  • AAV vector plasmid that expresses the Rep gene (a gene that encodes a replicative protein) and a Cap gene (a gene that encodes the cranial protein of a virus), and a plasmid that expresses each of the adenovirus genes E2A, E4, and VA
  • These three plasmids are then simultaneously transfected into packaging cells that express the E1 gene, eg, HEK293 cells, and the cells are cultured.
  • E1 gene eg, HEK293 cells
  • the non-viral vector used in the therapeutic agent of the present invention is not particularly limited as long as it is a vector that can express the target gene in vivo.
  • pCAGGS Gene 108, 193-200 (1991)
  • pBK -Expression vectors such as CMV, pcDNA3, 1, pZeoSV (Invitrogen, Stratagene), pVAX1 and the like.
  • pVAX1 pVAX1
  • Examples of such methods include lipofection method, phosphate-calcium coprecipitation method, DEAE-dextran method, direct DNA injection method using micro glass tube, gene transfer method using internal liposome, electrostatic liposome. (Electrostatic type liposome) gene transfer method, HVJ-liposome method, improved HVJ-liposome method (HVJ-AVE liposome method), method using HVJ-E (envelope) vector, receptor-mediated gene transfer method, particle gun And a method of transferring a DNA molecule together with a carrier (metal particles) into a cell, a direct introduction method of naked-DNA, an introduction method using various polymers, and the like.
  • the vector containing the SynGAP ⁇ 2 gene may contain a promoter or enhancer for appropriately transcription of the gene, a poly A signal, a marker gene for labeling and / or selecting a cell into which the gene has been introduced, and the like.
  • a promoter in this case, a known promoter can be used.
  • the protein encoded by the SynGAP ⁇ 2 gene contained in the therapeutic agent of the present invention has an amino acid sequence substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, and induces the formation of mature spine A protein having activity can be used.
  • the substantially identical amino acid sequence one or more or several (1 to 10, preferably 1 to 5, more preferably 1 or 2) amino acids are included in the amino acid sequence.
  • Substitution, deletion and / or addition amino acid sequence or the amino acid sequence, and BLAST Basic Local Alignment Search at the National Center for Biological Information
  • BLAST Basic Local Alignment Search at the National Center for Biological Information
  • BLAST Basic Local Alignment Search at the National Center for Biological Information
  • BLAST Basic Local Alignment Search tool of the National Center for Biological Information
  • having a homology of at least 85%, preferably 90%, more preferably 95%, particularly preferably 97% or more is something that can be cited.
  • Incorporation of the SynGAP ⁇ 2 protein into the nerve cell may be performed, for example, by combining the SynGAP ⁇ 2 protein with a peptide capable of passing through the cell membrane and administering it to the brain tissue site.
  • a peptide capable of passing through the cell membrane Various peptides are known as peptides that can pass through the cell membrane, and these known peptides can be used.
  • HIV-1 TAT cell membrane domain Drosophila homeobox protein Antennapedia's transmembrane domain, C-terminal (267-300) peptide of VP22, HIV-1 / Rev (34-50) peptide, FHV / coat (35-49) peptide, N-terminal of K-FGF (7-22 ) Hydrophobic region.
  • it may be administered locally to the brain tissue site, or by oral administration or the like, and the SynGAP ⁇ 2 protein may be delivered to nerve cells.
  • Examples of such a compound capable of specifically binding to a nerve cell include a homing signal peptide that binds to a receptor expressed on the surface of the nerve cell.
  • a DNA encoding the SynGAP ⁇ 2 protein and a DNA encoding the above-mentioned cell membrane-passing peptide or homing signal peptide are linked in-frame, and known genetic engineering is performed. It may be produced as a fusion protein by technology.
  • the SynGAP ⁇ 2 protein or the SynGAP ⁇ 2 gene contained in the therapeutic agent of the present invention is a fragment peptide consisting of a partial amino acid sequence of the amino acid sequence of the protein, a peptide having the activity of promoting the formation of mature spine, and the nucleotide sequence of the DNA
  • a fragment nucleotide consisting of a partial base sequence and a nucleotide encoding a peptide having activity for promoting the formation of mature spine is also included.
  • Such a fragment peptide or fragment nucleotide can be easily obtained by cleaving full-length protein or full-length DNA at an appropriate site and determining whether it has activity to promote formation of mature spine.
  • the therapeutic agent of the present invention is a pharmacologically acceptable carrier in addition to a SynGAP ⁇ 2 protein or a fusion protein of a SynGAP ⁇ 2 protein and a cell membrane-passing peptide or a homing signal peptide, or a SynGAP ⁇ 2 gene DNA or a vector containing the DNA, Diluents or excipients can be included.
  • the therapeutic agent of the present invention can be administered in various forms, orally by tablets, capsules, granules, powders, syrups, etc., or injections, drops, suppositories, sprays, eye drops, nasal Parenteral administration by administration agent, patch, etc. can be mentioned.
  • the therapeutic agent of the present invention includes a carrier, a diluent, and an excipient that are usually used in the pharmaceutical field.
  • lactose and magnesium stearate are used as carriers and excipients for tablets.
  • aqueous solution for injection isotonic solutions containing physiological saline, glucose and other adjuvants are used.
  • Suitable solubilizers such as polyalcohols such as alcohol and propylene glycol, nonionic surfactants and the like You may use together.
  • As the oily liquid, sesame oil, soybean oil or the like is used, and as a solubilizing agent, benzyl benzoate, benzyl alcohol or the like may be used in combination.
  • the therapeutic agent of the present invention is usually administered in an animal (living body).
  • the target animal that can be administered is not particularly limited, and examples thereof include human or non-human animals.
  • Non-human animals are not particularly limited, but mammals other than humans, specifically, primates (monkeys, marmosets), rodents (mouse, rats, guinea pigs, etc.), rabbits, dogs, cats, pigs, cows , Sheep and horses.
  • the method of administering the therapeutic agent of the present invention to an animal (in vivo) is not particularly limited and can be appropriately determined by those skilled in the art depending on the treatment method and the like.
  • the administration method may be systemic administration or local administration.
  • Administration of the therapeutic agent of the present invention can be performed by any of injection (needle-type, needle-free), administration using a catheter, internal use, and external use.
  • As the administration route it is preferable to select an effective route for treatment or the like.
  • parenteral administration such as intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, and tissue administration in addition to oral administration.
  • the target site of the therapeutic agent of the present invention is mainly a diseased part of the brain, oral administration, intravascular injection, administration using a catheter to the diseased site, intrathecal administration, intraventricular chamber Administration and brain parenchymal injection are preferred.
  • the dosage of the therapeutic agent of the present invention can be appropriately selected according to the type of active ingredient, the type of disease, the patient's condition, age, weight, sex, type of carrier, additive and the like.
  • the amount of the active ingredient is suitably in the range of 0.1 ng to 1000 mg per day for an adult, preferably in the range of 1 ng to 500 mg, more preferably in the range of 10 ng to 200 mg. There may be cases where more are needed, and there are cases where less than this is sufficient. Further, such a dose can be administered once a day or divided into a plurality of times.
  • the active ingredient is SynGAP ⁇ 2 protein
  • it is preferably about 0.001 mg to 100 mg per day for oral administration, and is preferably administered once or divided into several times.
  • 0.001 mg to 100 mg per administration is preferably administered by subcutaneous injection, intramuscular injection, or intravenous injection.
  • the active ingredient is SynGAP ⁇ 2 gene DNA inserted into an expression vector to be translated in the subject
  • 0.001 mg to 100 mg is subcutaneously or intramuscularly injected once every several days, weeks or months Or by intravenous injection.
  • the therapeutic agent of the present invention may contain a drug that promotes the expression of the SynGAP ⁇ 2 gene in the brain or a drug that can increase the SynGAP ⁇ 2 protein as an active ingredient.
  • the drug include low molecular weight organic compounds, inorganic compounds, peptides, proteins, glycoproteins, lipids, glycolipids, sugars, nucleic acids, plants, and the like, as long as they are found by the screening method of the present invention and have the relevant effects. There are no particular restrictions on the extract of microorganisms, cells, etc., and the culture supernatant.
  • a pharmaceutically acceptable carrier or the like can be included as long as the effects of the present invention are not impaired.
  • the carrier include solid, semi-solid or liquid diluents, fillers, and other formulation aids, such as edible carbohydrates such as starch and mannitol.
  • the amount of these carriers and the like is suitably in the range of 0.5 to 99.999% in the preparation, and more preferably in the range of 10 to 99.99% by weight.
  • the therapeutic agent of the present invention containing the drug as an active ingredient can be blended with pharmaceutically acceptable additives, excipients and the like in addition to the above carriers as long as the effects of the present invention are not impaired.
  • additives or excipients include, for example, emulsification aids (eg, fatty acids having 6 to 22 carbon atoms and pharmaceutically acceptable salts thereof, albumin, dextran), stabilizers (eg, cholesterol, phosphatidic acid).
  • Isotonic agents eg, sodium chloride, glucose, maltose, lactose, sucrose, trehalose
  • pH adjusters eg, hydrochloric acid, sulfuric acid, phosphoric acid, acetic acid, sodium hydroxide, potassium hydroxide, triethanolamine
  • the content of the additive and the like in the therapeutic agent of the present invention is suitably 90% by weight or less, preferably 70% by weight or less, and more preferably 50% by weight or less.
  • the dosage form of the therapeutic agent of the present invention containing the drug as an active ingredient is not particularly limited and is appropriately selected depending on the administration method and the like.
  • a preparation for parenteral administration for example, an injection, an ointment
  • examples include gels, creams, patches, liniments, suppositories, sprays, inhalants, sprays, eye drops, and nasal drops.
  • an injection is preferable.
  • preparations for oral administration include powders, granules, tablets, capsules, syrups, and liquids.
  • the administration subject, administration subject, dosage and the like are the same as described above.
  • the present invention includes a method for treating a disease accompanied by synaptic abnormality (hereinafter referred to as “the treatment method of the present invention”), which comprises a step of supplementing or increasing SynGAP ⁇ 2 in the brain.
  • the treatment method of the present invention includes frontotemporal lobar degeneration (FTLD) and motor neuron disease.
  • a gene encoding SynGAP ⁇ 2 or a SynGAP ⁇ 2 protein for example, a gene encoding SynGAP ⁇ 2 or a SynGAP ⁇ 2 protein, a drug that promotes the expression of SynGAP ⁇ 2 gene in the brain, or a drug that can increase the SynGAP ⁇ 2 protein Can be achieved directly or indirectly.
  • Examples of the drug that promotes the expression of the SynGAP ⁇ 2 gene in the brain or the drug that can increase the SynGAP ⁇ 2 protein include, for example, low molecular organic compounds, inorganic compounds, peptides, Extracts and culture supernatants of proteins, glycoproteins, lipids, glycolipids, sugars, nucleic acids, plants, microorganisms, cells and the like are not particularly limited.
  • the preparation for administering the active ingredient, the administration subject, the dosage, etc. are the same as described above.
  • Example 1 Preparation of model animal of the present invention (FUS-cKO mouse (Cre +))
  • a model animal of the present invention was prepared using the Cre-loxP system.
  • a vector having a gene sequence in which a 5 ⁇ UTR of the FUS gene and a loxP sequence in the same direction were inserted into intron 3 was designed together with a neomycin cassette having an Frt sequence (see FIG. 1), and a vector having the sequence was prepared.
  • Schematic representation of exon 1-6 region of wild type (WT) gene FIGGS. 4A and 4B, SEQ ID NO: 5
  • vector, and gene into which the vector has been inserted FIGS. 5A and 5B, SEQ ID NO: 6
  • the figure is shown in FIG.
  • the DNA was subjected to restriction enzyme treatment with BamHI or HindIII and subjected to Southern blotting to confirm the introduction of the target gene.
  • iTL BA1 ES cells C57BL / 6J ⁇ 129 / SvEv
  • the ES cell into which the gene was introduced was confirmed by amplifying the target sequence of the DNA by PCR.
  • the produced ES cell having the inserted vector was inserted into a blastocyst of a mouse of C57BL / 6J strain by microinjection.
  • Newborn pups were mated with C57BL / 6J FLP mice to remove the neomycin cassette (the above-mentioned process was commissioned to inGenetic Targeting Laboratories).
  • a schematic diagram of the WT gene, KO gene, and gene from which the neomycin cassette has been removed (Neo deletion) is shown in FIG.
  • the mouse DNA was confirmed by PCR for detection of mice with Neo deletion containing the LoxP sequence. Since the WT is 286 bp and the Neo deletion sequence is 451 bp, homozygous mice in which only 451 bp are confirmed were used in the experiment. Thereafter, the mice were backcrossed to C57BL / 6J (purchased from Chubu Chemical Materials Co., Ltd.) and mated. Then, using the MAX-BAX program (Charles River), a Cre-loxP sequence-introduced mouse having a completely matched genetic background was prepared.
  • Camk2a-Cre mice (Cell 1996; 87: 1917) were mated to the above-mentioned mice whose gene backgrounds were completely matched.
  • FUS fl / fl / Cre + mice were used as FUS-cKO mice (Cre +) (model animal of the present invention), and FUS fl / + , FUS fl / fl / mouse (Cre ⁇ ) were used as control mice. used.
  • males aged 3-4 months were mainly used.
  • brain parenchymal injection of the AAV vector of Test Example 7 was performed, the injection was performed at 6 weeks of age, and the analysis was performed at 3 to 4 months of age.
  • Example 1 Evaluation of FUS-cKO mice (Cre +) of Example 1
  • the FUS-cKO mice (Cre +) prepared in Example 1 were evaluated for the expression level of FUS protein in the hippocampus of the brain by immunostaining.
  • the brains of 3-month-old FUS-cKO mice (Cre +) and control mice (Cre ⁇ ) were fixed with paraformaldehyde to prepare paraffin-embedded samples, and anti-FUS antibody immunostaining was performed with DAB coloring in the hippocampal tissue sections. went. Next, the hippocampal CA1 region was separated from the section, and the protein expression level was confirmed by Western blotting using a sample crushed by ultrasonic waves.
  • the total distance of movement was significantly increased in the FUS-cKO mouse (Cre +) group (see FIG. 9).
  • the FUS-cKO mouse (Cre +) prepared in Example 1 exhibited a “hyperactivity” phenotype.
  • Test Example 4 Behavior test (anxiety behavior) Anxiety behavior was evaluated by the elevated plus maze method. The test mouse used in Test Example 3 was used. As a method, a cross passage with one side set as a “walled road” with fences on both sides and the other opened as a “wallless road” is placed at a height of 50 cm above the floor and tested there. This was done by placing a body mouse and evaluating the behavior pattern for 5 minutes.
  • the FUS-cKO mouse (Cre +) group had a significant increase in residence time of the “wallless road” and significantly decreased in the “walled road” group (FIG. 10). (See (A)). The number of intrusions into the “wallless road” in 5 minutes was also significantly increased in the FUS-cKO mouse (Cre +) group (see FIG. 10B). As described above, the FUS-cKO mouse (Cre +) prepared in Example 1 exhibited a “lack of anxiety” phenotype.
  • Test Example 5 Behavioral test (memory impairment) Memory impairment was assessed with a novel object recognition test and a fear conditioning test.
  • the test mouse used in Test Example 3 was used.
  • the fear conditioning test the subject mouse was placed in a 30 cm square box (context conditioning), allowed to hear 85 dB of sound for 15 seconds (sound conditioning), and subjected to an electric shock of 0.8 mA for the last 5 seconds. Turned and conditioned by context and sound. Then, put it in the same box the next day and observe for 2 minutes whether the subject mouse caused a “shrinking reaction” without giving an electric shock.
  • a 2019 sound conditioning test was performed to observe the “freezing reaction” for 1 minute each before and after sound stimulation.
  • FUS-cKO mice (Cre +) group exhibited behavioral abnormalities such as “hyperactivity” and “lack of anxiety”, but “memory impairment” was not observed. This feature is similar to that of early clinical symptoms in patients with FTLD (Dement Geriatr Cogn Disord 2006; 21: 74). Therefore, it is clear that the FUS-cKO mouse (Cre +) prepared in Example 1 is appropriate as a model animal such as FTLD.
  • SynGAP A ⁇ 2 supplementation experiment A cDNA vector of mouse SynGAP A ⁇ 2 with FLAG attached to the N-terminus was inserted into an AAV vector that was expressed under the CAG promoter, and an AAV equipped with this vector was prepared (FIG. 6A and FIG. 6). FIG. 6B, see SEQ ID NO: 7).
  • a Flag peptide is inserted as a labeling sequence at the N-terminus of SynGAP. It was administered to 6-week-old mice, and SynGAP A ⁇ 2 protein was supplemented by expressing SynGAP A ⁇ 2 to conduct a phenotypic improvement test.
  • the experiment was carried out using the stereotaxic device (Angel Two, Leica) on the bilateral hippocampus of 6-week-old FUS-cKO mice (Cre +) prepared in Example 1, and AAV was 1.5 ⁇ 10 13 on the bilateral hippocampus. This was done by stereotaxic injection of 1 ⁇ L at a concentration of squared VG / mL. Evaluation was performed in the same manner as in Test Example 2, using a brain tissue section of a FUS-cKO mouse, Golgi staining was performed by FD Rapid GolgiStain kit (manufactured by FD Neuro Technologies), and the spine morphology at the post-synaptic part was evaluated, and matured This was done by evaluating the proportion of spine.
  • the experiment was performed using 3 FUS-cKO mice (Cre +).
  • Re + As a negative control group for the experiment, two FUS-cKO mice (Cre +) and three mice in which AAV loaded with an AAV vector incorporating the GFP cDNA sequence was injected into control mice (Cre ⁇ ) were prepared.
  • Test Example 8 Behavior test (action amount, anxiety behavior) For the FUS-cKO mice (Cre +) group supplemented with SynGAP A ⁇ 2 in Test Example 7, recovery of higher brain dysfunction was evaluated by behavioral tests. The amount of behavior was evaluated by the open field test as in Test Example 3, and the anxiety behavior was evaluated by the elevated plus maze method as in Test Example 4. As in Test Example 7, FUS-cKO mice (Cre +) and control mice (Cre ⁇ ) were injected with AAV loaded with an AAV vector incorporating the GFP cDNA sequence, as a negative control group for the experiment. .
  • the model animal of the present invention can be used as an industry for research and development of diseases associated with synaptic abnormalities such as frontotemporal lobar degeneration (FTLD) and motor neuron diseases.
  • FTLD frontotemporal lobar degeneration
  • the screening method of the present invention can be used as an industry in searching for therapeutic agents for diseases associated with synaptic abnormalities.
  • the therapeutic agent of the present invention is effective for the treatment of diseases accompanied by synaptic abnormalities, it can be used for production and sales.
  • SEQ ID NO: 5 is the nucleotide sequence of exon 1 to 6 of mouse FUS gene.
  • SEQ ID NO: 6 nucleotide sequence of exon 1-6 region of mouse FUS gene after insertion of vector.
  • SEQ ID NO: 7 This is the base sequence of an AAV vector incorporating SynGAP A2 ⁇ cDNA.

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Abstract

La présente invention traite principalement du problème de la fourniture d'un nouveau modèle animal inactivé et spécifique qui présente un symptôme d'une maladie accompagnée d'une anomalie de la synapse dans le cerveau, d'un procédé de production associé et d'un agent thérapeutique pour ladite maladie. La présente invention concerne un modèle animal qui présente un symptôme d'une maladie (par exemple, une dégénérescence lobaire frontotemporale (DLFT)) accompagnée d'une anomalie de la synapse, lequel animal est caractérisé en ce qu'il est produit par l'inactivation spécifique du gène FUS dans le cerveau, par exemple, à l'aide du système Cre-loxP et du promoteur de CamK2, par exemple, et un procédé de production associé, ou un procédé de criblage de l'agent thérapeutique pour ladite maladie, dans lequel on utilise ledit modèle animal, ou similaire.
PCT/JP2017/025273 2016-07-14 2017-07-11 Modèle animal de maladie et agent thérapeutique de maladie Ceased WO2018012497A1 (fr)

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WO2026015039A1 (fr) * 2024-07-09 2026-01-15 Bial - Portela & Ca., S.A. Thérapie génique pour le traitement de l'encéphalopathie épileptique associée à la syngap1

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2026015039A1 (fr) * 2024-07-09 2026-01-15 Bial - Portela & Ca., S.A. Thérapie génique pour le traitement de l'encéphalopathie épileptique associée à la syngap1

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