WO2022138943A1 - Procédé de production d'un modèle d'animal non humain souffrant d'un trouble mental ou d'un trouble neurologique, ledit modèle d'animal non humain et procédé de criblage d'un agent prophylactique ou thérapeutique pour ledit trouble mental ou trouble neurologique - Google Patents

Procédé de production d'un modèle d'animal non humain souffrant d'un trouble mental ou d'un trouble neurologique, ledit modèle d'animal non humain et procédé de criblage d'un agent prophylactique ou thérapeutique pour ledit trouble mental ou trouble neurologique Download PDF

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WO2022138943A1
WO2022138943A1 PCT/JP2021/048329 JP2021048329W WO2022138943A1 WO 2022138943 A1 WO2022138943 A1 WO 2022138943A1 JP 2021048329 W JP2021048329 W JP 2021048329W WO 2022138943 A1 WO2022138943 A1 WO 2022138943A1
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ampa receptor
disorder
expression
animal
receptor gene
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PCT/JP2021/048329
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Japanese (ja)
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琢哉 高橋
智之 宮崎
亨 實木
航 太田
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公立大学法人横浜市立大学
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    • 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
    • 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
    • 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
    • A61P25/18—Antipsychotics, i.e. neuroleptics; Drugs for mania or schizophrenia
    • 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
    • A61P25/24—Antidepressants
    • 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
    • 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

Definitions

  • the present invention relates to a method for producing a model non-human animal for a psychiatric disorder or a neurological disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor.
  • the present invention relates to a non-human animal model of a psychiatric disorder or a neurological disorder, and a screening method for discovering a prophylactic or therapeutic agent for the psychiatric disorder or a neurological disorder using the model non-human animal.
  • AMPA ⁇ -amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid
  • AMPA receptor glutamate receptor
  • Non-Patent Documents 1-8 it has been reported that AMPA receptors are involved in many psychiatric and neurological disorders such as bipolar disorder, schizophrenia, drug dependence, autism, epilepsy, and ALS, mainly in the field of postmortem brain research. Has been done. Preclinical and clinical development of AMPA receptor agonists is being actively carried out for such diseases.
  • Patent Document 1 a patent for a novel compound that specifically binds to an AMPA receptor
  • Patent Document 9 in animals and humans (healthy subjects, epilepsy patients), the above compounds are used. Shows effectiveness.
  • Patent Document 2 a patent application has been filed for an imaging method of AMPA receptors in the brain of a primate organism (see Patent Document 2).
  • human psychiatric disorders or neurological disorders have increased or decreased expression of AMPA receptor gene in a predetermined region in the brain, or expression or function of AMPA receptor.
  • it correlates with an increase or decrease.
  • the above correlation for example, the following (1) to (5) are found.
  • AMPA receptor density In depression based on the HAM-D rating scale of human bipolar disorder patients, there is an increase in AMPA receptor density in the parietal lobe and occipital lobe, that is, there is a positive correlation. (3) In human schizophrenia patients, AMPA receptor density is relatively low in the cingulate gyrus. (4) In human autism patients, AMPA receptor density increases in the middle frontal gyrus orbit, and AMPA receptor density decreases in the occipital lobe and cerebellum. (5) In human depression patients, AMPA receptor density decreases in the frontal lobe and cerebellum.
  • the present invention has been made in view of the above findings, and is a psychiatric disorder or neurological disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor.
  • the present inventors knock down the expression of the AMPA receptor gene in a predetermined region in the brain of a mouse or rat, or increase the function of the AMPA receptor, or the above-mentioned predetermined region and others. It has been further found that the mouse or rat exhibits a corresponding mental or neurological disorder by suppressing or activating a neural pathway connecting the region (region other than the predetermined region). More specifically, for example, the following (6) to (11) have been further found. (6) By knocking down the AMPA receptor gene in the cerebellum of a mouse, the mouse significantly exhibits a manic state similar to that of a patient with bipolar disorder.
  • the mouse By knocking down the AMPA receptor gene during the anterior cingulate gyrus of the mouse, the mouse significantly exhibits a schizophrenic state.
  • the mouse By increasing the AMPA receptor in the middle frontal gyrus orbit of a mouse, the mouse significantly exhibits an autistic state.
  • the mouse In a mouse, by suppressing the claustrum having a neural connection with the anterior cingulate cortex and the neural connection with the anterior cingulate cortex, the mouse significantly exhibits a schizophrenic state.
  • the rat By knocking down the AMPA receptor gene in the medial prefrontal cortex of a rat, the rat significantly exhibits a depressive state.
  • the first aspect of the present invention is a model non-human animal of a mental disease or a neurological disease caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. It is a manufacturing method of A step of reducing or increasing (improving) at least one selected from the group consisting of the expression of an AMPA receptor gene or AMPA receptor in the predetermined region of a non-human animal and the function of the AMPA receptor as compared with the wild type. , Or a method comprising a step of suppressing or activating a neural pathway (nerve transmission pathway) connecting the predetermined region and another region (brain region other than the predetermined region).
  • a second aspect of the invention is a model non-human animal for mental or neurological disorders resulting from increased or decreased expression of the AMPA receptor gene in a predetermined region of the brain or increased or decreased expression or function of the AMPA receptor. And At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor is decreased or increased (improved) as compared with the wild type, or the predetermined region.
  • a third aspect of the present invention is a method for screening a prophylactic or therapeutic agent for a psychiatric disorder or a neurological disease, wherein the test substance is administered to the animal according to the second aspect, and the psychiatric disorder or the neurological disease in the animal is concerned. It is a method of screening a prophylactic or therapeutic agent for the psychiatric disorder or a neurological disorder by using the prevention, suppression or cure of the above as an index.
  • the model non-human animal of the psychiatric disorder or the neurological disorder according to the second aspect is a psychiatric disorder caused by an increase or decrease in the expression of the AMPA receptor gene in a predetermined region in the brain or an increase or decrease in the expression or function of the AMPA receptor. It is useful as a model animal for a disease or neurological disorder (eg, at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism).
  • the production method according to the first aspect can produce a model animal for the above-mentioned mental disease or neurological disease.
  • the screening method according to the third aspect can screen a prophylactic or therapeutic agent for a psychiatric disorder or a neurological disorder.
  • FIG. 6 is a brain atlas showing the coordinates of introduction of shRNA into the cerebellum of mice for AMPA receptors. It is a figure which shows the result of the posalt forced swimming test. It is a figure which shows the tail suspension test result. It is a synthetic figure of the brain atlas which shows the introduction coordinates of the shRNA for the AMPA receptor into the anterior cingulate gyrus of the mouse, and the brain section GFP fluorescence imaging result for confirmation of the introduction position. It is a figure which shows the prepulse inhibition test result. It is a figure which shows the open field test result.
  • (A) is a diagram showing the results of a three-chamber social interaction (social preference) test
  • (b) is a diagram showing a test device.
  • FIG. 1 It is a composite diagram of the brain atlas showing the introduction coordinates to the middle frontal gyrus orbit of a mouse of CPTX, and the brain section Alexa594 fluorescence imaging result for confirming the introduction position. It is a figure which shows the 3 chamber social interaction (social preference) test result.
  • (A) is a diagram showing the results of a three-chamber social interaction (social recognition) test
  • (b) is a diagram showing a test device. It is a figure which shows the prepulse inhibition test result. It is a figure which shows the forced swimming test result. It is a figure which shows the forced swimming test result.
  • the AMPA receptor has four subunits, GluA1, GluA2, GluA3 and GluA4, which form homo or heterotetramers.
  • the coding regions (CDS) of mouse GluA1, mouse GluA2, mouse GluA3 and mouse GluA4 have the sequences represented by SEQ ID NOs: 1 to 4 in the sequence listing below.
  • the CDSs of human GluA1, human GluA2, human GluA3 and human GluA4 have the sequences represented by SEQ ID NOs: 5-8 below.
  • the method for obtaining the AMPA receptor gene is not particularly limited. Appropriate probes and primers were prepared based on the information on the base sequence and amino acid sequence of the AMPA receptor, and were prepared using them in a human cDNA library (appropriate cells expressing the AMPA receptor gene according to a conventional method).
  • the AMPA receptor gene can be isolated by selecting the desired clone from the above.
  • the method for producing a model non-human animal for a psychiatric disorder or a neurological disorder according to the first aspect is a method.
  • the predetermined areas include the cerebellum, the frontal cortex (eg, anterior cingulate gyrus, middle frontal gyrus, etc.), and other cerebral cortex (eg, motor area, somatic sensory area, associative area, thalamus, auditory area, broker). Field, Wernicke field, cingulate gyrus, etc.), striatum, hippocampus, thalamus, thalamus, pineapple, pituitary gland, mesencephalon, posterior brain and cingulate gyrus. At least one selected from the group consisting of cerebellum, frontal lobe, parietal lobe, occipital lobe, and cingulate gyrus is preferred.
  • psychiatric disorders or neurological disorders include (a) depression, major depression, bipolar depression, dysthymia, emotional disorders, recurrent depression, postpartum depression, winter depression (seasonal emotional disorder), etc.
  • Stress disorder, depressive symptoms manic disorder, anxiety, general anxiety disorder, anxiety syndrome, panic disorder, fear, social fear, social anxiety disorder, compulsive disorder, post-traumatic stress syndrome, post-traumatic stress disorder , Tauret Syndrome, Autism, Vulnerable X Syndrome, Let Syndrome, Adaptation Disorder, Bipolar Disorder, Neurology, schizophrenia, Chronic Fatigue Syndrome, Anxiety, Obstructive Disorder, Depressive Disorder, Depression, Psychiatric disorders such as divine hypersensitivity, attention deficit hyperactivity disorder, learning disorder, psychotic major depression, intractable major depression, treatment-resistant depression, drug addiction, alcohol dependence, gambling disorder, etc.
  • C Age-related cognitive and memory disorders such as age-related memory disorders and senile dementia.
  • the above mental or neurological disorders are selected from the group consisting of schizophrenia, autism, epilepsy, depression, cerebral ischemia, Parkinson's disease, Alzheimer's disease, attention hyperactivity disorder (ADHD) and multiple sclerosis. At least one psychiatric or neurological illness is preferred, and more preferably at least one psychiatric disorder selected from the group consisting of bipolar disorder, depression, schizophrenia and autism.
  • the above-mentioned increase is a statistically significant increase (for example, excess) with respect to the expression level or activity value when the subject was previously healthy, or a known range known as a normal value of the expression level or activity value.
  • 1.2 with respect to the expression level or activity value when the subject was healthy, or the known range known as the normal value of the expression level or activity value.
  • It is preferably fold or more, 1.4 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more, and particularly preferably 3 times or more. It is preferably 5 times or more, and most preferably 5 times or more.
  • the upper limit of the increase is not particularly limited, but may be 20 times or less, 10 times or less, 8 times or less, and the like.
  • the above-mentioned decrease refers to the expression level or activity value when the subject (for example, patient, non-human animal, etc.) was previously healthy, or the known range known as the normal value of the expression level or activity value.
  • the expression level or activity value when the subject was healthy before, or the normal value of the expression level or activity value.
  • It is preferably 3/4 or less, more preferably 1/2 or less, further preferably 1/4 or less, and particularly preferably 1/10 or less, based on the known range. Most preferably, there is no expression or activity.
  • the non-human animal subjected to the step of suppressing or activating the neural pathway connecting the predetermined region and the other region is not particularly limited as long as it is a non-human animal generally used as an experimental animal. It is preferably a non-human vertebrate, more preferably a non-human mammal, and even more preferably a rodent.
  • mice specifically examples thereof include mice, rats, guinea pigs, hamsters, rabbits, cows, sheep, goats, pigs, horses, dogs, chickens, monkeys, etc., preferably mice, rats, guinea pigs, or hamsters, and more preferably.
  • mice a mouse or rat, particularly preferably a mouse.
  • the strain of the mouse may be C57BL / 6 or BALB / c strain, but it is preferable that the gene background is closer to the pure strain by backcrossing or the like, and it is preferable that the strain is the same as that of the wild-type mouse to be compared.
  • a method for reducing or increasing the amount as compared with the wild type specifically, at least one substance selected from the group consisting of the following (1) to (4) is placed in the above-mentioned predetermined region in the brain stereotactically in the brain. Examples thereof include a method of internal injection (preferably intraventricular injection).
  • AMPA receptor gene or a substance that reduces the expression of AMPA receptor (2) Substances that reduce the function of AMPA receptors, (3) A substance that increases the expression of the AMPA receptor gene or AMPA receptor and (4) a substance that increases the function of the AMPA receptor
  • the dose varies depending on the weight and age of the animal, the administration method, etc. A trader can appropriately select an appropriate dose.
  • the above-mentioned neural pathway is introduced into the above-mentioned neural pathway, for example, the above-mentioned neural pathway is introduced into the above-mentioned neural pathway.
  • the predetermined region is the anterior cingulate cortex (ACC)
  • the other regions include other brain regions having a neural connection with the ACC, and more specifically, the claustrum, thalamus, and insular cortex. And so on.
  • examples of the other region include other brain regions having a neural connection with the medial prefrontal cortex, and more specifically, the amygdala basolateral nucleus and the like.
  • examples of the active or inhibitory DREADD or RASSL include hM4Gi, hM3Gq, hM4Di, mAChR and the like.
  • Examples of the method of introduction include introduction using a viral vector (for example, an adeno-associated virus vector), and examples thereof include a method of administering the viral vector to the other region (for example, stereotactic injection administration in the brain).
  • the introduction amount varies depending on the body weight and age of the animal, the administration method, and the like, but those skilled in the art can appropriately select an appropriate introduction amount.
  • the active or inhibitory DREADD or RASSL can be activated by any agonist, eg, by administration of any agonist to the other regions mentioned above (eg, stereotactic injection in the brain). be able to.
  • the agonist include clozapine-N-oxide (CNO) and the like.
  • CNO clozapine-N-oxide
  • the dose varies depending on the body weight and age of the animal, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.
  • the above-mentioned suppression is known as the expression level or activity value related to neural connection when the subject (for example, patient, non-human animal, etc.) was healthy, or the normal value of the expression level or activity value.
  • the expression level or activity value related to neural connection when the subject (for example, patient, non-human animal, etc.) was healthy, or the normal value of the expression level or activity value.
  • the above activation is statistically significant with respect to the expression level or activity value related to neural connection when the subject was previously healthy, or the known range known as the normal value of the expression level or activity value.
  • the range it is preferably 1.2 times or more, more preferably 1.4 times or more, further preferably 1.5 times or more, particularly preferably 2 times or more, 3 It is particularly preferable that the amount is double or more, and most preferably 5 times or more.
  • the upper limit of the activation is not particularly limited, and examples thereof include 20 times or less, 10 times or less, 8 times or less, and the like.
  • the above-mentioned step of reducing the amount as compared with the wild-type is a step of knocking down the above-mentioned AMPA receptor gene.
  • knockdown of the AMPA receptor gene means an operation of reducing the transcription or expression of the AMPA receptor gene
  • knockout of the AMPA receptor gene means disruption of the AMPA receptor gene.
  • AMPA receptor gene or substance that reduces the expression of AMPA receptor examples include nucleic acids having RNAi action (for example, siRNA, shRNA), antisense oligonucleotides, artificial nucleases, and the like, which are described below.
  • RNAi action for example, siRNA, shRNA
  • antisense oligonucleotides for example, antisense oligonucleotides
  • artificial nucleases and the like.
  • (2) a substance that lowers the function of the AMPA receptor (3) a substance that increases the expression of the AMPA receptor gene or the AMPA receptor, and (4) a substance that increases the function of the AMPA receptor. It will be described later.
  • Examples of the above-mentioned (1) AMPA receptor gene or a substance that reduces the expression of the AMPA receptor include a nucleic acid having an RNAi action capable of suppressing the expression of the AMPA receptor gene.
  • the nucleic acid having RNAi action preferably contains a continuous partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene or a sequence complementary thereto.
  • RNAi RNA interference
  • Examples of the nucleic acid having an RNAi action include siRNA (small interfering RNA), shRNA (small hairpin RNA) and the like.
  • siRNA and shRNA will be described.
  • siRNA examples include double-stranded RNA capable of suppressing the expression of the AMPA receptor gene by the action of RNAi or DNA encoding the above-mentioned double-stranded RNA, and suppressing the expression of the AMPA receptor gene by the action of RNAi.
  • a double-stranded RNA having a continuous partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene or a DNA encoding the double-stranded RNA is preferable.
  • the siRNA may have a sequence complementary to the number of consecutive sequences required to suppress the expression of the AMPA receptor gene in the base sequence of RNA transcribed from the AMPA receptor gene.
  • double-stranded RNA containing at least 17 consecutive nucleotides in the base sequence of RNA transcribed from the AMPA receptor gene or DNA encoding the double-stranded RNA is preferred and transcribed from the AMPA receptor gene. It is more preferably a double-stranded RNA containing at least 19 consecutive nucleotides in the base sequence of the RNA to be obtained, or a DNA encoding the above double-stranded RNA, and more preferably in the base sequence of the RNA transcribed from the AMPA receptor gene. It is more preferably a double-stranded RNA containing at least 21 contiguous nucleotides or a DNA encoding the double-stranded RNA.
  • the siRNA is preferably a double-stranded RNA containing 30 or less consecutive nucleotides in the base sequence of RNA transcribed from the AMPA receptor gene, or a DNA encoding the double-stranded RNA.
  • Examples of the DNA encoding the double-stranded RNA include DNA having an inverted repeat sequence of a partial sequence of the AMPA receptor gene.
  • the inverted repeat sequence of a partial sequence of the AMPA receptor gene can be expressed in the cell, thereby receiving AMPA by RNAi action.
  • the expression of body genes can be suppressed.
  • An inverted repeat sequence is a sequence in which a partial sequence of an AMPA receptor gene and a complementary reverse sequence thereof are arranged in parallel via an appropriate sequence. Specifically, when the partial sequence of the AMPA receptor gene has a double strand consisting of the n base sequences shown below, 5'-X 1 X 2 . .. . ..
  • the inverted repeat has the following sequence. 5'-Y n Y n-1 . .. . . . . Y 2 Y 1-3 ' 3'-X n X n-1 . .. . . . . X 2 X 1-5 ' (Here, among the bases represented by X and the bases represented by Y, those having the same subscript number are complementary bases to each other).
  • the inverted repeat sequence is a sequence in which the above two types of sequences are mediated by an appropriate sequence. There are two cases of inverted repeats, one is when the partial sequence of the target gene is upstream of the complementary sequence of the target gene, and the other is when the inverted sequence is upstream of the partial sequence of the complementary target gene. Can be considered.
  • the inverted repeat sequence used in the present invention may be any of the above, but the inverted repeat is preferably located upstream of the partial sequence of the target gene complementary thereto.
  • shRNA an inverted repeat sequence in which a partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene and an inverted sequence complementary thereto are paralleled via a sequence capable of forming a hairpin loop. Examples thereof include single-stranded RNA having or DNA encoding the above RNA.
  • the shRNA is suitable for a method of introducing a vector or virus expressing shRNA into a cell, and can function in the cell in the same manner as the siRNA described above.
  • the sequence may be complementary to the contiguous sequence of, and typically a partial sequence containing at least 17 consecutive nucleotides in the base sequence of the RNA transcribed from the AMPA receptor gene is preferred. It is more preferably a partial sequence containing at least 19 consecutive nucleotides in the base sequence of the RNA to be produced, and it is a partial sequence containing at least 21 consecutive nucleotides in the base sequence of the RNA transcribed from the AMPA receptor gene. Is more preferable.
  • the partial sequence in the base sequence of RNA transcribed from the AMPA receptor gene is preferably a partial sequence containing 30 consecutive nucleotides or less in the base sequence of RNA transcribed from the AMPA receptor gene.
  • the length of the sequence capable of forming the hairpin loop is not particularly limited as long as the hairpin loop can be formed, but is preferably 0 to 300 bp, more preferably 1 to 100 bp, still more preferably 2 to 75 bp, and particularly preferably 3 to 50 bp. Is. Restriction enzyme sites may be present in this sequence.
  • the inverted repeat sequence of the target gene By incorporating the inverted repeat sequence of the target gene downstream of the promoter sequence that can be actuated in the mammal, the inverted repeat sequence of the target gene can be expressed in the intracellular cells of the mammal.
  • the promoter sequence is not particularly limited as long as it can be operated in mammals.
  • the nucleic acid having RNAi action described above may be functionally bound to an appropriate terminator such as TPI1 terminator or ADH3 terminator for a human growth hormone terminator or a fungal host, if necessary.
  • the recombinant vector also has elements such as polyadenylation signals (eg, from the SV40 or adenovirus 5E1b region), transcription enhancer sequences (eg, SV40 enhancers) and translation enhancer sequences (eg, encoding adenovirus VARNA). You may be doing it.
  • the recombinant vector or virus may further comprise a DNA sequence that allows the vector or virus to replicate within the packaging cell, an example of which is the SV40 origin of replication.
  • the recombinant vector or virus may further contain a selectable marker.
  • Selectable markers include genes lacking its complement in packaging cells, such as, for example, dihydrofolate reductase (DHFR) or the sidosaccharomyces ponbe TPI gene, or, for example, ampicillin, kanamycin, tetracycline, chloramphenicol. , Neomycin or drug resistance genes such as dihydromycin.
  • DHFR dihydrofolate reductase
  • sidosaccharomyces ponbe TPI gene or, for example, ampicillin, kanamycin, tetracycline, chloramphenicol.
  • Neomycin or drug resistance genes such as dihydromycin.
  • Examples of the packaging cell for preparing the vector or virus by introducing the nucleic acid having the RNAi action described above or the vector or virus containing the nucleic acid thereof include higher eukaryotic cells, bacteria, yeasts, fungi and the like, but in mammalian cells. It is preferable to have.
  • a method for transforming a mammalian cell to express the gene introduced into the cell is also known, and for example, a lipofection method, an electroporation method, a calcium phosphate method and the like can be used.
  • the nucleic acid having RNAi action described above can be administered alone or by injection or the like together with a transfection agent or a carrier for lipofection used to assist uptake into cells.
  • a transfection agent or a carrier for lipofection used to assist uptake into cells.
  • the carrier for lipofection include carriers having a high affinity for cell membranes (for example, liposomes, cholesterol, etc.), and lipofectamine or lipofectin is preferable, and lipofectamine is more preferable.
  • AMPA receptor gene or a substance that reduces the expression of the AMPA receptor examples include antisense oligonucleotides having a sequence complementary to a continuous sequence in the AMPA receptor gene.
  • a strand-specific nuclease eg, RNase H
  • RNase H can degrade the mRNA of an AMPA receptor containing a nucleotide chain and suppress transcription, translation, etc. of the AMPA receptor gene.
  • the antisense oligonucleotide may be DNA or RNA, but is preferably DNA from the viewpoint that mRNA is cleaved by the specific nuclease.
  • the antisense oligonucleotide may have a sequence complementary to the number of consecutive sequences required for suppressing the expression of the AMPA receptor gene in the base sequence of the AMPA receptor gene, and is typically used.
  • Is preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 10 consecutive nucleotides, preferably an antisense oligonucleotide having a sequence complementary to an oligonucleotide containing at least 13 nucleotides. More preferred.
  • the antisense oligonucleotide has a sequence complementary to a continuous oligonucleotide of 20 nucleotides or less in the base sequence of the AMPA receptor gene. It is more preferably an antisense oligonucleotide having a sequence complementary to a contiguous 17-nucleotide or less oligonucleotide.
  • the antisense oligonucleotide is preferably an antisense oligonucleotide containing at least one nucleotide having at least one structure selected from the group consisting of a phosphorothioate structure, a crosslinked structure and an alkoxy structure.
  • the antisense oligonucleotide it is preferable that at least one of the nucleotides at one end (preferably 1 to 3 bases from the end) has a crosslinked structure or an alkoxy structure.
  • the antisense oligonucleotide can be produced by a conventional method using a DNA synthesizer and a known organic synthesis technique.
  • the intracellular (preferably intranuclear) uptake of the antisense oligonucleotide may be free uptake.
  • a carrier for lipofection may or may not be used.
  • the vector or virus is prepared after being inserted into a suitable vector or virus and further introduced into a suitable packaging cell to prepare the vector or virus. It may be an embodiment in which the virus infects the target cancer cells.
  • the type of the above-mentioned suitable vector or virus is not particularly limited, and may be, for example, an autonomously replicating vector or virus, but a chromosome integrated into the genome of the packaging cell when introduced into the packaging cell. It is preferable that it is duplicated with.
  • Examples of the packaging cell for preparing the vector or virus by introducing the antisense oligonucleotide or the vector or virus containing the same include higher eukaryotic cells, bacteria, yeasts, fungi and the like, but the cells are mammalian cells. Is preferable.
  • AMPA receptor gene or a substance that reduces the expression of the AMPA receptor include artificial nucleases for genome editing such as CRISPR (Crust Generally Interspaced Short Palindromic Repeats) / Cas nuclease, and TranscrictionActivator-Li It may be an artificial restriction enzyme (artificial nuclease) using Nuclease (TALEN) and a zinc finger nuclease (ZFN).
  • CRISPR Crust Generally Interspaced Short Palindromic Repeats
  • Cas nuclease Cas nuclease
  • TranscrictionActivator-Li TranscrictionActivator-Li It may be an artificial restriction enzyme (artificial nuclease) using Nuclease (TALEN) and a zinc finger nuclease (ZFN).
  • TALEN Nuclease
  • ZFN zinc finger nuclease
  • the CRISPR / Casnuclease contains a guide RNA and a Casnuclease (preferably Cas9). At least a partial sequence in the target AMPA receptor gene includes oligonucleotides contained in the AMPA receptor gene.
  • a nucleic acid or Cas nuclease encoding a Cas nuclease into eukaryotic cells or eukaryotes containing the AMPA receptor gene.
  • Nucleic acids or Casnucleases encoding Casnucleases and DNAs encoding guide RNAs or guide RNAs can be obtained from a variety of methods known in the art such as microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticles.
  • nucleic acid encoding the Cas nuclease or the Cas nuclease and guide RNA can be transferred into the organism by various methods known in the art for administering genes or proteins such as infusions.
  • the nucleic acid or Cas protein encoding the Cas nuclease can be transferred into the cell in the form of a complex with a guide RNA or separately. Cas nuclease fused to a protein transduction domain such as Tat can also be efficiently delivered intracellularly.
  • eukaryotic cells or eukaryotes are co-transfected or serially transfected with Cas9 nuclease and guide RNA.
  • Serial transfection can be performed first by transfection with a nucleic acid encoding Casnuclease, followed by a second transfection with a naked guide RNA.
  • the second transfection is after 3, 6, 12, 18, 24 hours, but is not limited to them.
  • AMPA receptor gene or a substance that reduces the expression of the AMPA receptor is as described above.
  • the substance (2) that lowers the function of the AMPA receptor include an AMPA receptor antagonist, a negative allosteric modulator of the AMPA receptor, a nucleic acid such as an aptamer, and an antibody.
  • the negative allosteric modulator of the AMPA receptor any compound known as the negative allosteric modulator of the AMPA receptor can be used.
  • AMPA receptor antagonist examples include CNQX, DNQX, NBQX, perampanel and the like.
  • An aptamer is a nucleic acid that is composed of single-stranded RNA or DNA and binds to a target protein by its three-dimensional structure to inhibit its function. Aptamers have high binding and specificity to target proteins, low immunogenicity, can be produced by chemical synthesis, and have high storage stability. Aptamers that selectively bind to AMPA receptors can be obtained by the SELEX (Systematic Evolution of Ligands by EXPonential evolution) method.
  • the antibody that selectively binds to the AMPA receptor may be either a polyclonal antibody or a monoclonal antibody as long as it can selectively bind to the AMPA receptor.
  • Examples of the above-mentioned (3) AMPA receptor gene or a substance that increases the expression of the AMPA receptor include a nucleic acid that knocks in the AMPA receptor gene, an artificial nuclease, and the like.
  • Examples of the substance (4) that increases the function of the AMPA receptor include an AMPA receptor agonist, a positive allosteric modulator of the AMPA receptor, a nucleic acid such as an aptamer, a synapse organizer, and an antibody.
  • Examples of the AMPA receptor agonist include glutamic acid, bromo-willardine, ibotenic acid, ACPA, kainic acid, domoic acid and the like.
  • Positive allosteric modulators of AMPA receptors include CX-516, CX-614, aniracetam, CTZ, IDR-21, diazoxide and the like.
  • Synapse organizers which may be natural or artificial (synthetic), include chimeric molecules that link the Neurexin binding domain of Celeberin 1 (Cbln1) with the AMPA receptor binding domain of Neuronal Pentraxin 1. Cerebello-Pentraxin (CPTX) is preferred.
  • the degree of the decrease is not particularly limited as long as it is a statistically significant decrease, but the AMPA receptor gene or AMPA is compared with the wild type non-human animal.
  • the expression of the receptor or the function of the AMPA receptor is preferably 3/4 or less, more preferably 1/2 or less, further preferably 1/4 or less, and 1/10 or less. Is particularly preferable, and it is most preferable that the expression or function is lost.
  • the degree of the increase is not particularly limited as long as it is a statistically significant increase, but the expression of the AMPA receptor gene or the AMPA receptor or the function of the AMPA receptor is higher than that of the wild type non-human animal. It is preferably 1.2 times or more, more preferably 1.4 times or more, further preferably 1.5 times or more, particularly preferably 2 times or more, and 3 times or more. It is particularly preferable, and it is most preferable that it is 5 times or more.
  • the upper limit of the increase is not particularly limited, but may be 20 times or less, 10 times or less, 8 times or less, and the like.
  • the expression of the AMPA receptor gene in various animal tissues can be detected even in Incilico.
  • the expression of the AMPA receptor gene in various animal tissues can be detected.
  • the expression of the AMPA receptor gene can be detected by a conventional method such as RT-PCR, Northern blot, Southern blot and the like.
  • the expression level of the AMPA receptor gene at the mRNA level can also be measured by a conventional method such as RT-PCR, Northern blot, Southern blot or the like.
  • the primer is not particularly limited as long as it can specifically amplify only the AMPA receptor gene, and can be appropriately set based on the sequence information of the AMPA receptor gene.
  • an oligonucleotide containing at least 10 consecutive nucleotides in the base sequence of the AMPA receptor gene, and an antisense oligonucleotide having a sequence complementary to the oligonucleotide can be used as a probe or primer. More specifically, an oligonucleotide having a continuous 10 to 60 residue, preferably 10 to 40 residue base sequence in the base sequence of the AMPA receptor gene, and an anti having a sequence complementary to the oligonucleotide. Sense oligonucleotides can be used.
  • the above-mentioned oligonucleotide and antisense oligonucleotide can be produced by a conventional method using a DNA synthesizer.
  • a sense primer corresponding to the base sequence on the 5'end side for example, in a part of the base sequence of the mRNA to be detected, a sense primer corresponding to the base sequence on the 5'end side, an antisense primer corresponding to the base sequence on the 3'end side, and the like.
  • the sense primer and the antisense primer are oligonucleotides whose melting temperature (Tm) and number of bases do not change drastically, and examples thereof include those having about 10 to 60 bases and those having about 10 to 40 bases. Is preferable.
  • the above-mentioned derivative of the oligonucleotide can be used, and for example, a methyl form or a phosphorothioate form of the oligonucleotide can be used.
  • the expression level of the AMPA receptor protein can be measured by Western blotting using an antibody described later or by ordinary immunoassay such as ELISA. Specifically, it can be carried out by a conventional method known to those skilled in the art described in Molecular Cloning 2nd Edition or Current Protocols in Molecular Biology and the like.
  • model non-human animal for a mental or neurological disorder according to the second aspect (hereinafter, also simply referred to as “model animal according to the second aspect”) has increased expression of the AMPA receptor gene in a predetermined region in the brain.
  • a non-human animal model of a psychiatric or neurological disorder resulting from a decrease or an increase or decrease in the expression or function of the AMPA receptor At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor is decreased or increased (improved) as compared with the wild type, or the above.
  • the neural pathways that connect a predetermined region to another region are suppressed or activated.
  • Examples of the predetermined region include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
  • Examples of the above-mentioned psychiatric disorder or neurological disorder include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
  • Examples of the non-human animal used for producing the model animal according to the second aspect include the same as the above-mentioned specific examples and preferable examples for the production method according to the first aspect.
  • the first aspect is as a method for reducing or increasing at least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor in the predetermined region and the function of the AMPA receptor as compared with the wild type.
  • at least one substance selected from the group consisting of the above (1) to (4) is stereotactically injected into the predetermined region of the brain (preferably the brain).
  • the method of indoor injection) and the like can be mentioned.
  • a substance that reduces the expression of the AMPA receptor gene or AMPA receptor (2) a substance that reduces the function of the AMPA receptor, and (3) a substance that increases the expression of the AMPA receptor gene or the AMPA receptor.
  • Specific examples and preferable examples of the substance and the substance that increases the function of the above-mentioned (4) AMPA receptor include the same as the above-mentioned specific example and preferable example for the production method according to the first aspect.
  • an active or inhibitory DREADD or RASSL is used as a method for suppressing or activating a neural pathway connecting the predetermined region and another region. Examples thereof include a method of introducing into the above-mentioned other region where a nerve pathway exists, and specific examples and preferable examples include the same as the above-mentioned specific examples and preferable examples of the production method according to the first aspect.
  • the degree of the decrease is not particularly limited as long as it is a statistically significant decrease, and examples of the production method according to the first aspect are the same as those of the above-mentioned specific examples and preferable examples.
  • the degree of the increase is not particularly limited as long as it is a statistically significant improvement, and examples of the production method according to the first aspect are the same as those of the above-mentioned specific examples and preferable examples.
  • At least one selected from the group consisting of the expression of the AMPA receptor gene or the AMPA receptor and the function of the AMPA receptor in the frontal lobe, the occipital lobe, the cerebellum or the zonal gyrus is selected. It is reduced or reduced compared to the wild type of the non-human animal.
  • At least one selected from the group consisting of AMPA receptor gene or AMPA receptor expression and AMPA receptor function in the parietal lobe, occipital lobe, or middle frontal gyrus fossa is a wild-type non-human animal. It is preferably a model non-human animal that has been increased (improved) as compared to the model.
  • the psychiatric or neurological disorder is at least one psychiatric disorder selected from the group consisting of bipolar disorder and schizophrenia
  • the AMPA receptor gene or AMPA receptor in the frontal lobe, occipital lobe, cerebellum or zonal gyrus It is preferred that at least one selected from the group consisting of expression and function of the AMPA receptor is reduced as compared to the wild type.
  • the above mental or neurological disorder is autism, select from the group consisting of the expression of AMPA receptor gene or AMPA receptor and the function of AMPA receptor in the parietal lobe, occipital lobe, or mid-frontal gyrus. It is preferable that at least one of these is increased (improved) as compared with the wild type.
  • the reduced animal is an animal in which the AMPA receptor gene has been knocked down.
  • a test substance is administered to the non-human animal, and a preventive or therapeutic agent for the mental disease or the neurological disease is used as an index for the prevention, suppression or cure of the mental disease or the neurological disease in the non-human animal.
  • a preventive or therapeutic agent for the mental disease or the neurological disease is used as an index for the prevention, suppression or cure of the mental disease or the neurological disease in the non-human animal.
  • the screening method according to the third aspect the screening means to at least narrow down the population of the test substance using the above as an index.
  • the screening method according to the third aspect is to determine whether or not the lesion occurs after the above-mentioned administration as compared with the absence of the test substance (for example, a system before administration of the test substance or a negative control system).
  • a screening step of selecting the preventive or therapeutic agent candidate by judging the action of the test substance by observation using whether or not the lesion is healed or relieved as an index.
  • it is possible to judge the action of the test substance by observing whether or not the lesion occurs, or whether or not the lesion is healed or relieved, for example, in the Posalt forced swimming test. It can be performed by evaluating the tail suspension test, the open field test, the rotarod test, the prepulse inhibition test, the 3-chamber social interaction test, the life span, the weight, and the like.
  • the test substance can be administered by a method known to those skilled in the art such as intracerebral injection (preferably intracerebroventricular injection), oral administration, intraarterial injection, intravenous injection, intraperitoneal injection, and subcutaneous injection. ..
  • intracerebral injection preferably intracerebroventricular injection
  • oral administration intraarterial injection
  • intravenous injection intraperitoneal injection
  • subcutaneous injection ..
  • the above-mentioned predetermined regions in the brain for example, cerebellum, prefrontal cortex, other cerebral cortex, striatum, hippocampus, thorax, hypothalamus, pineapple, pituitary gland, middle brain, posterior brain, choroidal flora, etc.
  • intracerebral injection preferably intraventricular injection
  • the non-human animal when the non-human animal is a bipolar disorder model animal, it is preferable to inject it into the cerebellum in a stereotactic manner.
  • the above-mentioned non-human animal is a schizophrenia model animal, it is preferable to perform intracerebral injection stereotactically in the anterior cingulate gyrus.
  • the non-human animal is an autism model animal, it is preferable to inject it into the middle frontal gyrus orbit in a stereotactic manner.
  • the non-human animal is a depression model animal, it is preferable to inject it into the medial prefrontal cortex stereotactically in the brain as the other region.
  • test substance used in the screening method according to the third aspect.
  • the type of the test substance is not particularly limited, and may be a nucleic acid molecule, an antibody, a synthetic peptide (oligo or polypeptide), an individual small molecule synthetic compound, or is present in a natural product extract. It may be a compound or the above-mentioned artificial nuclease for genome editing.
  • the test compound may also be a synthetic peptide (oligo or polypeptide) library (eg, a random peptide library with 10 or less amino acid residues), a compound library, a phage display library or a combinatorial library.
  • the test substance is preferably a random peptide library with 10 or less amino acid residues, a low molecular weight compound (eg, a compound library), a nucleic acid molecule, an artificial nuclease or antibody for genome editing, and an AMPA receptor gene or AMPA receptor.
  • a nucleic acid molecule or antibody is more preferable, and an aptamer or antibody that selectively binds to the nucleic acid molecule or AMPA receptor having a sequence complementary to the oligonucleotide contained in the AMPA receptor gene. Is more preferable.
  • FIG. 1 is a brain atlas showing the coordinates of introduction of shRNA into the cerebellum of mice for AMPA receptors.
  • Lentivirus expressing shRNA capable of selectively suppressing the expression of AMPA receptors (GluA1-4) or scrambled RNA (non-functional shRNA as a control) is shown in FIG. 1 using a glass micropipette.
  • the AP coordinates represent the anterior distance from the bregma suture in the brain map.
  • the ML coordinates represent the distances from the median to the left and right sides of the brain map.
  • the H coordinate represents the depth from the bregma suture in the brain map.
  • the obtained knockdown mice were subjected to a posalt forced swimming test, a tail suspension test, an open field test, and a rotarod test, and evaluated as bipolar disorder model mice.
  • the Posalt forced swimming test and the tail suspension test are known as despair models of depression, and the onset time of immobile behavior can be evaluated as depression-like behavior.
  • the obtained knockdown mouse is suitable as a bipolar disorder model mouse.
  • FIG. 4 is a synthetic diagram of a brain map showing the coordinates of introduction of shRNA into the anterior cingulate gyrus of AMPA receptors and the results of GFP fluorescence imaging of brain sections for confirming the introduction position.
  • Lentivirus expressing shRNA capable of selectively suppressing the expression of AMPA receptors (GluA1-4) or scrambled RNA (non-functional shRNA as a control) is shown in FIG. 4 using a glass micropipette.
  • nl 300 nl (0.3 ⁇ l) was introduced stereotactically into the brain coordinates of the following (1) and (2), which are the anterior cingulate cortex of the mouse.
  • (1) AP coordinates 1.2 mm, ML coordinates ⁇ 0.5 mm, and H coordinates 1.6 mm.
  • (2) AP coordinates 0 mm, ML coordinates ⁇ 0.5 mm, and H coordinates 1.2 mm.
  • the obtained knockdown mice were evaluated as schizophrenia model mice by performing a prepulse inhibition (hereinafter, also simply referred to as “PPI”) test, an open field test, and a three-chamber social interaction test.
  • PPI prepulse inhibition
  • the prepulse inhibition refers to a phenomenon in which a startle response is significantly suppressed by a weak stimulus (prepulse) preceding immediately before the startle stimulus (pulse), and is used in schizophrenia patients and schizophrenia. It can be used as an endophenotype for schizophrenia because it shows a decline in both animal models.
  • PPI test From 3 weeks to 6 days after the introduction of the wrench virus, acclimatization to the PPI test device and mouse handling were performed for 10 minutes each. The PPI test was performed on the 7th day from the start of acclimatization to the above PPI test apparatus, and the Inhibition ratio was calculated. The Inhibition ratio was calculated in "pleplese74dB pulse120dB", “plepulus78dB pulse120dB” and "plepulus84dB pulse120dB". As is clear from the results shown in FIG.
  • the 3-chamber social interaction (social preference) test was performed one week after the above PPI test. Specifically, after acclimatization to the test device (see FIG. 7B) for 5 minutes, The residence time of the test mice in the chamber without other individuals (empty chamber) and the chamber with other individuals (stranger chamber) was measured for 10 minutes. The results are shown in FIG. 7 (a). As is clear from the results shown in FIG. 7 (a), in the mouse group into which the control scrambled RNA was introduced, the residence time in the stranger chamber was significantly longer than that in the empty chamber, and the rate of approaching other individuals was high. It can be said that the sex is high. On the other hand, in the mouse group into which shRNA for AMPA receptor was introduced, there was no significant difference between the staying time in the empty chamber and the staying time in the stranger chamber, the rate of approaching other individuals decreased, and the sociality was low. I can say.
  • the brain was collected by perfusion fixation (under anesthesia) using PFA solution. Then, a frozen section was prepared and the fluorescence of GFP was confirmed (the above-mentioned lentivirus expresses GFP together with shRNA) to identify the virus introduction site. The results are shown in the composite diagram of FIG.
  • FIG. 8 is a composite diagram of a brain map showing the coordinates of introduction of CPTX into the middle frontal gyrus orbital part of a mouse and a brain section Alexa594 fluorescence imaging result for confirming the introduction position.
  • CPTX a synthetic synapse organizer, or vehicle (solvent) as a control, using a glass micropipette
  • 300 ln 300 ln (0.3 ⁇ l) was introduced per location in a stereotactic manner.
  • (3) AP coordinates 2.5 mm, ML coordinates ⁇ 1.3 mm, and H coordinates 1.9 mm.
  • mice to which CPTX was administered to the middle frontal gyrus orbit were evaluated as autism model mice by conducting a 3-chamber social interaction test and a PPI test.
  • mice to which CPTX was administered to the middle frontal gyrus orbit is suitable as an autism model mouse.
  • HM4Gi which suppresses neural function using adeno-associated virus
  • ACC anterior cingulate cortex
  • claustrum which is the brain region that has neural connections.
  • CNO clozapine-N-oxide
  • Example 5 Production of depression model rat A depression model rat was produced as follows. As the rat, a 6-week-old male Wistar rat (manufactured by Charles River) was used. Short hairpin (sh) RNA that specifically suppresses the expression of AMPA receptor subunits 1-3 (GluA1-3) using lentivirus in the bilateral medial frontal cortex of Wistar rats or GluA1-3 as a control. A scrambled RNA that does not suppress expression (non-functional shRNA as a control) was introduced. (Forced swimming test) One week after the introduction, a forced swimming test was conducted. The forced swimming test was carried out by injecting tap water having a water temperature of 26 ⁇ 0.5 ° C.
  • Example 6 Production of depression model rat A depression model rat was produced as follows. As the rat, a 4-week-old male Wistar rat (manufactured by Charles River) was used. HM4Gi, which suppresses neural function using adeno-associated virus, was introduced bilaterally into the basolateral amygdala nucleus, which is a brain region with neural connections to the medial prefrontal cortex, which is the causative region. Three weeks after the introduction of the adeno-associated virus, hM4Gi was activated by clozapine-N-oxide (CNO), a forced swimming test was performed in the same manner as in Example 5, and the immobility time was measured. The results are shown in FIG.
  • CNO clozapine-N-oxide

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Abstract

L'invention concerne : un procédé de production d'un modèle d'animal non humain présentant un trouble mental ou un trouble neurologique qui est attribué à une augmentation ou une diminution de l'expression d'un gène du récepteur AMPA ou à une augmentation ou une diminution de l'expression ou de la fonction d'un récepteur AMPA dans une région prédéterminée du cerveau ; un modèle d'animal non humain présentant le trouble mental ou le trouble neurologique ; et un procédé de criblage permettant de découvrir un agent prophylactique ou thérapeutique pour le trouble mental ou le trouble neurologique à l'aide du modèle d'animal non humain.　Ce procédé permet de produire un modèle d'animal non humain présentant un trouble mental ou un trouble neurologique qui est attribué à une augmentation ou une diminution de l'expression d'un gène du récepteur AMPA ou à une augmentation ou une diminution de l'expression ou de la fonction d'un récepteur AMPA dans une région prédéterminée du cerveau. Le procédé comprend : une étape de réduction ou d'augmentation d'au moins un élément choisi dans le groupe constitué par l'expression du gène du récepteur AMPA ou du récepteur AMPA, et la fonction du récepteur AMPA, dans la région prédéterminée de l'animal non humain, par rapport à un type sauvage, ou une étape d'inhibition ou d'activation d'une voie nerveuse reliant la région prédéterminée à une autre région.
PCT/JP2021/048329 2020-12-25 2021-12-24 Procédé de production d'un modèle d'animal non humain souffrant d'un trouble mental ou d'un trouble neurologique, ledit modèle d'animal non humain et procédé de criblage d'un agent prophylactique ou thérapeutique pour ledit trouble mental ou trouble neurologique WO2022138943A1 (fr)

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