WO2018012497A1

WO2018012497A1 - Disease model animal and disease therapeutic agent

Info

Publication number: WO2018012497A1
Application number: PCT/JP2017/025273
Authority: WO
Inventors: 元祖父江; 診祐石垣; 聡横井
Original assignee: 国立大学法人名古屋大学
Priority date: 2016-07-14
Filing date: 2017-07-11
Publication date: 2018-01-18
Also published as: JPWO2018012497A1

Abstract

The present invention mainly addresses the problem of providing a novel and specific knockout model animal that presents a symptom of a disease accompanied by a synapse abnormality in the brain, a method for producing same, and a therapeutic agent for said disease. The present invention provides a model animal that presents a symptom of a disease (e.g., frontotemporal lobar degeneration (FTLD)) accompanied by a synapse abnormality, which animal is characterized by being produced by specifically knocking out the FUS gene in the brain, for example, by using the Cre-loxP system and CamK2 promotor, for example and a method for producing same, or a screening method for the therapeutic agent for said disease, in which said model animal is used or the like.

Description

Disease model animals and disease treatment agents

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.

In recent years, life expectancy has been greatly extended, especially in developed countries, due to advances in medicine and science and technology. However, along with this, various functional declines, diseases and pathologies caused by aging of the brain, which has not been a problem in the past, have become major social problems. Among them, 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.

認知 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. However, 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.

In order to further advance the detailed study of FTLD that exhibits very complicated pathological conditions, it is essential to have a uniform model animal system showing the pathological conditions. Such a model animal system is preferably a genetically modified model animal such as a knockout mouse. Conventionally, 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. However, there is a criticism that 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.

In order to proceed more precisely with the FUS gene whose expression suppression (loss of function) was suggested as one of the causes of FTLD in the above-mentioned experiment, it is necessary to prepare a FUS gene knockout mouse. However, since FUS gene knockout mice are usually embryonic lethal, although there have been reports on generation of cross-breed mice so far (see, for example, Non-Patent Document 2 and Non-Patent Document 3), other reports are found. Absent. In addition, the creation of a knockout mouse in an inbred mouse having almost the same gene background is even more difficult and has not been reported so far. Since FTLD type dementia has a very complicated pathology, there is a limit in a hybrid system with large individual differences in order to obtain reliable experimental results.

On the other hand, SynGAP (Synthetic Ras GTPase Activating Protein) is a protein present in the postsynaptic thick part (PSD), and is known to negatively regulate small molecule G protein signaling downstream of glutamate receptor activation. Yes. 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 (α1, α2, β, γ, 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).

International Publication No. 2013/180214

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. On the other hand, there is no known analysis example using a FUS gene knockout mouse unified in the background of inbred lines.
Although 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.

As a result of intensive studies, 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 SynGAPα2 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.
[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.

[7] 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.
[8] 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.
[9] 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.
[10] The production method according to any one of [7] to [9] above, wherein the animal is a mouse.
[11] The production method according to any one of [7] to [10] above, wherein the animal is an inbred strain.

[12] 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.
[14] 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 SynGAPα2 due to application of a target drug.
[15] The screening method according to [14] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.

[16] Treatment of a disease accompanied by synaptic abnormalities characterized by containing, as an active ingredient, a gene encoding SynGAPα2 protein or a SynGAPα2 protein, or a drug that promotes the expression of the SynGAPα2 gene in the brain or a drug that can increase the SynGAPα2 protein Agent.
[17] The therapeutic agent according to [16] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
[18] A method for treating a disease accompanied by synaptic abnormalities, comprising a step of supplementing or increasing SynGAPα2 in the brain.
[19] The method according to [18] above, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.

According to the present invention, it is possible to research and develop diseases and conditions associated with synaptic abnormalities such as frontotemporal lobar degeneration (FTLD) and motor neuron diseases, or to screen for therapeutic agents for the diseases. Can do. In addition, a drug containing a therapeutic drug found from such screening can be used for the treatment of the disease.

It is a schematic diagram of the vector gene inserted into the ES cell. 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, and 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, and the lower figure is a conditional knockout completed by removing the Neo cassette by crossing with FLP mice. It is a schematic diagram of a mouse gene (Neo Selection). In creating a conditional knockout mouse, a region containing exons 1 to 6 in the wild-type gene sequence of the FUS gene targeted for genetic manipulation is represented. Continuation of the gene sequence of FIG. 4A. Of the gene sequence inserted into the conditional knockout mouse, this represents the region containing exons 1 to 6 of the FUS gene. Continuation of the gene sequence of FIG. 5A. Represents the gene sequence of the AAV vector into which the SynGAP Aα2 gene sequence has been inserted. Continuation of the gene sequence of FIG. 6A. For the control mouse (Cre−) and the FUS gene conditional knockout mouse expressing Cre (FUS-cKO mouse (Cre +)), FIG. (A) shows the immunostained image of FUS protein in the hippocampal CA1 region. Represents the results of Western blotting of hippocampal CA1 region samples, respectively. 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 μm 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. For the control mouse (Cre−) and the FUS-cKO mouse (Cre +), FIG. (A) shows the fluorescence immunostained image in the hippocampal CA1 region, and FIG. (B) shows the Western blot result of the hippocampal CA1 region sample. To express. 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 Aα2. 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 Aα2. 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 Aα2. The vertical axis shows the number of times of intrusion (times) on a road without a wall.

1. 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.
Here, 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.

1.1 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. As a common structure of the FUS protein encoded by the FUS gene, it has a transcription activation domain on the N-terminal side and an RNA-binding motif and a zinc finger domain on the C-terminal side. The base sequence information of the FUS gene in each animal can be obtained from, for example, Genbank, which is a gene information database.
In the production of the model animal of the present invention, the FUS gene of the target animal is genetically manipulated using data such as a gene information database and specifically knocked out.

1.2 Specific knockout of FUS gene in brain The model animal of the present invention is produced by specifically knocking out the FUS gene of the animal in the brain.
As a method for performing specific knockout, 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. As a system used in such a conditional knockout method, for example, a recombinant enzyme Cre (Cre recombinase) derived from bacteriophage P1 and its target sequence, a loxP sequence system (Cre-loxP system), budding yeast (Saccharomyces cerevisiae) Recombinant enzyme FLP and its target sequence FRT sequence system, soy sauce yeast (Zygosaccharomyces rouxii) recombinant enzyme R and its target sequence RS sequence system, or 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. When this method is used, 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. . Usually, 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.

1.3 Diseases with Synaptic Abnormalities Examples of the diseases with synaptic abnormalities exhibited by the model animals of the present invention 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). In neurodegenerative diseases, it is suggested that 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. In the model animal of the present invention, 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 SynGAPα2.

The disease accompanied by synaptic abnormality exhibited by the model animal of the present invention may show higher brain dysfunction as a result. Examples of 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. These higher brain dysfunctions can be evaluated by behavioral tests used in the field of brain science. Examples of behavior tests for abnormal behavioral quantities include an open field test and a home cage activity analysis test. Examples of the 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. These behavioral tests can be performed alone or in a plurality depending on the symptoms and the type of higher brain function to be evaluated, and can be evaluated for higher brain dysfunction.
When the above behavioral test is performed on the model animal of the present invention exhibiting symptoms of FTLD or frontotemporal dementia (FTD), a significant increase in momentum and lack of anxiety are observed, while memory impairment is It shows a tendency not to be seen.

1.4 Animal species 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. For example, 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. Among them, preferred are 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.
One feature of the present invention is that an inbred model animal (especially a mouse) suitable for experiments is also provided.

1.5 Model Neuron and Model Neuron Cell Line According to the Present Invention 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.
Here, 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. By using the model neuronal cell line of the present invention, a reproducible and stable experiment can be performed.

1.6 Production Method 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”. As 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.
In the knockout operation, it is preferable to use the Cre-loxP system and the CamK2 promoter.

In the production method of the present invention, a specific gene is genetically manipulated at the developmental stage of the target animal. Examples of 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. When the Cre-loxP system is used in the production method of the present invention, two genes, a FUS gene sandwiched between loxP sequences and a sequence encoding the full length of the Cre recombinase gene (hereinafter referred to as “Cre gene”), are genetically manipulated. And incorporated into fertilized eggs. In the production method of the present invention, the integration of the Cre gene and the FUS gene sandwiched between the loxP sequences can be integrated into one individual at a time, but the Cre gene and the FUS gene are integrated into separate individuals, Individuals in which both genes are integrated can also be obtained by mating.
Below, the case by the microinjection method using a mouse | mouth is demonstrated as a specific example of this invention manufacturing method.

In the microinjection method, 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. For the DNA construct to be used, the CamK2 promoter, which is a promoter sequence that enables time-specific expression of the transgene, can be used. In addition, 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).

In order to confirm that 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.
In the adult stage of the model animal of the present invention, 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. As a 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.

2. Screening Method According to the Present Invention 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”).
When the model animal of the present invention is used, 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. As a result of administering 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.

In the screening method of the present invention 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, as a step of evaluating the effectiveness of the target drug, for example, the target drug is administered to the model animal of the present invention. Thereafter, 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. Examples of genes associated with diseases associated with such synaptic abnormalities include SynGAPα2, Tau, and GluA1. Among these, it is preferable to measure increase / decrease in SynGAPα2 protein or mRNA.

As a result of administering the target drug to the model animal of the present invention, when the amount of SynGAPα2 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 SynGAPα2 can be confirmed by measuring the amount of mRNA encoding SynGAPα2 or the amount of SynGAPα2 protein by a conventional method. Examples of the mRNA measurement method include quantitative RT-PCR and Northern hybridization. Examples of protein measurement methods include Western blotting and immunostaining. Usually, 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.

In addition, by using a step of measuring the increase or decrease of SynGAPα2 due to application of the target drug in vivo or in vitro without using the model animal of the present invention, a disease accompanied by synaptic abnormality (eg, frontotemporal lobar degeneration ( FTLD), motor neuron diseases, etc.) can also be screened. 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 SynGAPα2 can be measured by a conventional method. As a result of application of the target drug, when SynGAPα2 increases before application or compared to the control group, it can be evaluated that the drug may treat or alleviate the disease. In the above in vivo screening method, the increase or decrease of SynGAPα2 in the brain of the animal used is usually measured. On the other hand, in the in vitro screening method, the increase or decrease of SynGAPα2 in cells or the like to be used is usually measured.

In the above in vivo screening method, 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. For example, 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. 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.
In the screening method of the present invention, 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. Specifically, for example, 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.

3. Therapeutic Agents According to the Present Invention The present invention is characterized by containing as an active ingredient a gene encoding SynGAPα2 or a SynGAPα2 protein, or a drug that promotes the expression of the SynGAPα2 gene in the brain or a drug that can increase the SynGAPα2 protein. And a therapeutic agent for diseases associated with synaptic abnormalities (hereinafter referred to as “the therapeutic agent of the present invention”).
Here, “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.
Here, SynGAPα2 in the present invention has an α2 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, synGAP2α in brain neurons. It has also been confirmed that the expression of this gene is decreased, that the selective phenotype of SynGAP2α 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 SynGAP2α gene.
Therefore, a drug capable of increasing SynGAPα2 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 SynGAPα2 or a SynGAPα2 protein, a drug that promotes the expression of the SynGAPα2 gene in the brain, or a drug that can increase the SynGAPα2 protein as an active ingredient.

The nucleotide sequence of the human SynGAPα2 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 SynGAPα2 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.

3.1 Gene Encoding SynGAPα2 The SynGAPα2 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. Or one or more or several (1 to 10, preferably 1 to 5, more preferably) DNA having a homology of 97% or more, or the amino acid sequence of the protein encoded by the DNA. Is one that encodes a protein having activity of promoting the formation of mature spine among DNAs encoding a protein comprising an amino acid sequence in which one or two amino acids are substituted, deleted, and / or added. Are included in the therapeutic agent of the present invention. Here, “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.”. Thus, isolation of DNA having high homology with the probe sequence can be expected as the hybridization conditions become more severe. However, 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 SynGAPα2 gene can be obtained from human cells, human tissues and the like based on the sequence information of SEQ ID NO: 1. Further, the mouse SynGAPα2 gene can be obtained from mouse cells, mouse tissues and the like based on the sequence information of SEQ ID NO: 3.
Furthermore, the therapeutic agent of the present invention also includes a vector containing the SynGAPα2 gene. By introducing the vector into a subject, the SynGAPα2 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. As a method for introducing a gene into a subject, there are a method using a viral vector and a method using a non-viral vector, and various methods are known (separate volume experimental medicine, basic technology of gene therapy, Yodosha, 1996; separate volume). Experimental Medicine, Gene Transfer & Expression Analysis Experimental Method, Yodosha, 1997; edited by Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTS, 1999).

Examples of 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.

For the therapeutic agent of the present invention, 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. In a test example described later, 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. For example, 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).

According to a preferred method for producing an AAV vector, first, 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). On the other hand, a 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 Prepare. These three plasmids are then simultaneously transfected into packaging cells that express the E1 gene, eg, HEK293 cells, and the cells are cultured. As a result, AAV vector particles having a high infectivity for mammalian cells can be produced. In a test example to be described later, a virus was prepared in the same manner as in Non-Patent Document 1.

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. For example, pCAGGS (Gene 108, 193-200 (1991)), pBK -Expression vectors such as CMV, pcDNA3, 1, pZeoSV (Invitrogen, Stratagene), pVAX1 and the like. When a non-viral vector is used, it is preferable to use a known technique for efficiently incorporating a gene into a target cell or tissue. 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 SynGAPα2 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. As the promoter in this case, a known promoter can be used.
In order to introduce the therapeutic agent of the present invention containing a SynGAPα2 gene into a subject, an in vivo method in which the therapeutic agent of the present invention is directly introduced into the body, and certain cells from a human are taken out and the therapeutic agent of the present invention is introduced outside the body. Either ex vivo method such as introducing into cells and returning the cells to the body can be used (Nikkei Science, April 1994, pages 20-45; Monthly Pharmaceutical Affairs, 36 (1), 23- 48 (1994); experimental medicine extra number, 12 (15), (1994); edited by Japanese Society of Gene Therapy, Gene Therapy Development Research Handbook, NTS, 1999).

3.2 SynGAPα2 protein The protein encoded by the SynGAPα2 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. Here, as 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) (for example, a basic local alignment search tool of the National Center for Biological Information) (Using default or default parameters) and having a homology of at least 85%, preferably 90%, more preferably 95%, particularly preferably 97% or more. It is something that can be cited.

Incorporation of the SynGAPα2 protein into the nerve cell may be performed, for example, by combining the SynGAPα2 protein with a peptide capable of passing through the cell membrane and administering it to the brain tissue site. Various peptides are known as peptides that can pass through the cell membrane, and these known peptides can be used. For example, 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. Moreover, you may couple | bond and administer with the compound which can couple | bond with a nerve cell specifically. In this case, it may be administered locally to the brain tissue site, or by oral administration or the like, and the SynGAPα2 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. In order to bind the above-mentioned cell membrane-passing peptide or signal peptide to the SynGAPα2 protein, for example, a DNA encoding the SynGAPα2 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.

Furthermore, the SynGAPα2 protein or the SynGAPα2 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.

3.3 Formulation The therapeutic agent of the present invention is a pharmacologically acceptable carrier in addition to a SynGAPα2 protein or a fusion protein of a SynGAPα2 protein and a cell membrane-passing peptide or a homing signal peptide, or a SynGAPα2 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. For example, lactose and magnesium stearate are used as carriers and excipients for tablets. As an 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.

3.4 Administration Subject, Administration Method, Dose 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. For example, in the case of systemic administration, it is administered by parenteral administration such as intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, and tissue administration in addition to oral administration. In the case of local administration, for example, it is administered to the ventricle, medullary cavity, brain parenchyma, skin, mucous membrane, lung, bronchi, nasal cavity, nasal mucosa, eye and the like. Among these administration methods, since 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. Specifically, for example, 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.

When the active ingredient is SynGAPα2 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. For parenteral administration, 0.001 mg to 100 mg per administration is preferably administered by subcutaneous injection, intramuscular injection, or intravenous injection. In addition, when the active ingredient is SynGAPα2 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.

3.5 Drug In addition to the gene encoding SynGAPα2 and the SynGAPα2 protein, the therapeutic agent of the present invention may contain a drug that promotes the expression of the SynGAPα2 gene in the brain or a drug that can increase the SynGAPα2 protein as an active ingredient. Examples of 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.

In addition to the drug, a pharmaceutically acceptable carrier or the like can be included as long as the effects of the present invention are not impaired. Examples of 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.

In addition, 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. Examples of such 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) Can be mentioned. One or more of these can be used. 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. As 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. Among these, an injection is preferable. Examples of preparations for oral administration include powders, granules, tablets, capsules, syrups, and liquids. In the treatment method of the present invention using the drug as an active ingredient, the administration subject, administration subject, dosage and the like are the same as described above.

4. Treatment Method According to the Present Invention 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 SynGAPα2 in the brain. .
Examples of the disease accompanied by synaptic abnormality targeted by the treatment method of the present invention include frontotemporal lobar degeneration (FTLD) and motor neuron disease. In the treatment method of the present invention, in order to supplement or increase SynGAPα2 in the brain, for example, a gene encoding SynGAPα2 or a SynGAPα2 protein, a drug that promotes the expression of SynGAPα2 gene in the brain, or a drug that can increase the SynGAPα2 protein Can be achieved directly or indirectly.

Examples of the drug that promotes the expression of the SynGAPα2 gene in the brain or the drug that can increase the SynGAPα2 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.
In the treatment method of the present invention, the preparation for administering the active ingredient, the administration subject, the dosage, etc. are the same as described above.

Next, the present invention will be described in more detail with reference to examples and test examples, but the present invention is not limited to these examples and test examples.

[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 (FIGS. 4A and 4B, SEQ ID NO: 5), vector, and gene into which the vector has been inserted (KO gene, FIGS. 5A and 5B, SEQ ID NO: 6) The figure is shown in FIG. In order to confirm the insertion of the target sequence, 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. Next, iTL BA1 ES cells (C57BL / 6J × 129 / SvEv) into which the vector was inserted were prepared. 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.

Next, Camk2a-Cre mice (Cell 1996; 87: 1917) were mated to the above-mentioned mice whose gene backgrounds were completely matched. Among the offspring born, 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.

In the following test examples, males aged 3-4 months were mainly used. When 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.

[Test 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.

In the model animal of the present invention (FUS-cKO mouse (Cre +)) in which Cre was expressed and the FUS gene was knocked out specifically in the brain, compared with the control mouse (Cre−) in which Cre was not expressed, According to immunostaining using an anti-FUS antibody, the staining ability of the FUS protein was reduced (see FIG. 7A). The decrease in FUS protein staining was particularly remarkable in the hippocampal CA1 region and neurons in the dentate gyrus. In addition, as a result of Western blotting using an anti-FUS antibody, a decrease in the expression level of FUS protein was observed in the hippocampal CA1 region of FUS-cKO mice (Cre +) (see FIG. 7B).
From the above, it was confirmed that the FUS gene knockout was established in the FUS-cKO mouse (Cre +) prepared in Example 1.

[Test Example 2] Phenotypic analysis of FUS-cKO mice (Cre +) Using the brain tissue sections of 3-4 month old FUS-cKO mice (Cre +) prepared in Example 1, FD Rapid GolgiStain kit (FD) Golgi staining was performed by Neuro Technologies), and the spine morphology at the post-synaptic portion was evaluated. In addition, the total number of spines per 100 μm of neurites in the tissue section and the ratio of mushroom-type mature spines were evaluated.

Compared to control mice, mushroom-type mature spines indicated by arrows were decreased in FUS-cKO mice (Cre +) mice (see FIG. 8A). In quantitative analysis, there was no change in the total number of spines per 100 μm of neurites (see FIG. 8B), but the proportion of mature spines in the total number of spines was significantly decreased in FUS-cKO mice (Cre +) (FIG. 8). 8C: N = 8 from 4 mice each).
From the above, the FUS-cKO mouse (Cre +) prepared in Example 1 was found to have a spine maturation disorder, that is, a post-synaptic maturation disorder, which contributes to a decrease in nerve activity. Suggested the possibility.

[Test Example 3] Behavior test (action amount)
Higher-order brain function abnormalities in the FUS-cKO mice prepared in Example 1 were evaluated by behavioral tests used in brain science research. The mice used in the behavioral test were backcrossed to the C57BL / 6J strain, and mice that had a coincidence rate of 99.5% or more by genetic testing (MAX-BAX, Charles River) were bred and 3-4 Evaluation was performed by age. In the experiment, 10 mice were used for the control mouse (Cre−) group, and 11 mice were used for the FUS-cKO mouse (Cre +) group. An open field test for evaluating the amount of behavior was performed, and as a test method, a subject mouse was placed in an arena having a diameter of 60 cm, and the amount of behavior of the mouse for 5 minutes was measured with EthoVision (automatic tracking program, manufactured by Brainscience Idea).

Compared with the control mouse (Cre−) group, the total distance of movement was significantly increased in the FUS-cKO mouse (Cre +) group (see FIG. 9). When the arena was divided into a circle having a diameter of 40 cm and divided into a central portion and an outer periphery, the movement distance of the outer periphery increased in particular.
As described above, 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.

Compared to the control mouse (Cre−) group, 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.
First, in the new object recognition test, two objects are placed in a 30 cm square box, and a “training trial” step in which the subject mouse freely searches for 5 minutes, and then only one of the next day is changed. The procedure of the “holding trial” step to evaluate cognition was performed.
In 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 terrifying sound conditioning test was performed to observe the “freezing reaction” for 1 minute each before and after sound stimulation.

As a result, there was no significant difference in the search time for two objects in the “training trial” of the new object recognition test, and the search time for the new object was significant in both groups compared to the familiar object (familiar) in the “hold trial” The difference in search preference was not seen in both groups (see FIG. 11A). Therefore, in this test, no “memory impairment” was observed in the FUS-cKO mouse (Cre +) group prepared in Example 1.
In the fear conditioning test, there was no significant difference in “shrinkage response” between the two groups in both the place and the sound stimulus (see FIG. 11B). Therefore, also in this test, “memory impairment” of the FUS-cKO mouse (Cre +) group prepared in Example 1 was not observed.

As a summary of behavioral tests, 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.

[Test Example 6] Search for therapeutic target such as FTLD SynGAPα2 was obtained as a therapeutic target by LC / MS analysis of the protein extracted from the FUS expression suppression model in mouse primary cultured neurons. Therefore, the amount of SynGAPα2 protein in the brain tissue of FUS-cKO mice was subjected to fluorescent immunostaining using an anti-SynGAPα2 antibody on paraffin sections of the brain hippocampal CA1 region prepared in the same manner as in Test Example 1. Further, Western blotting using an anti-SynGAPα2 antibody was performed using a sample obtained by ultrasonically disrupting the hippocampal CA1 of the tissue section.

As a result of Western blotting of proteins extracted from the hippocampal CA1 region (see FIG. 12B) and fluorescent immunostaining of paraffin sections of the hippocampal CA1 region (see FIG. 12A), SynGAPα2 in FUS-cKO mice (Cre +) It was confirmed that the amount of the protein decreased significantly.

[Test Example 7] SynGAP Aα2 supplementation experiment A cDNA vector of mouse SynGAP Aα2 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). In the protein expressed by the vector, 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 Aα2 protein was supplemented by expressing SynGAP Aα2 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 μL 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 +). 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.

As a result of observing the Golgi-stained image, for the FUS-cKO mice (Cre +) supplemented with SynGAP Aα2, there was a decrease in the proportion of mushroom-type mature spines in the total number of spines seen in FUS-cKO mice (Cre +) There was a significant improvement (see FIGS. 13A and 13B).

[Test Example 8] Behavior test (action amount, anxiety behavior)
For the FUS-cKO mice (Cre +) group supplemented with SynGAP Aα2 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. .

In FUS-cKO mice supplemented with SynGAP Aα2, the behavioral abnormality “hyperactivity” observed in Test Examples 3 to 4 was improved compared to FUS-cKO mice supplemented with GFP as a control. (See FIG. 14A. N = 14, 11, 11 from the left column). In addition, regarding “lack of anxiety”, FUS-cKO mice supplemented with SynGAP Aα2 were significantly improved compared to FUS-cKO mice supplemented with GFP as a control (see FIG. 14B, from the left column). N = 8, 7, 8).
From these results, it is clear that SynGAP Aα2 can be a therapeutic target such as FTLD. In addition, it is clear that, for searching for therapeutic agents such as FTLD, it is effective to use increase or decrease of SynGAP Aα2 by application of drugs or improvement of phenotype of this mouse as an index.
As described above, it was shown that the FUS-cKO mouse of the present invention is useful as a model animal for research of treatment such as FTLD.

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. In addition, the screening method of the present invention can be used as an industry in searching for therapeutic agents for diseases associated with synaptic abnormalities. Furthermore, since 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 A2α cDNA.

Claims

A model animal exhibiting a symptom of a disease accompanied by a synaptic abnormality, which is produced by specific knockout of the FUS gene in the brain.
The model animal according to claim 1, wherein the knockout is performed using a Cre-loxP system and a CamK2 promoter.
The model animal according to claim 1 or 2, wherein the disease accompanied by a synaptic abnormality is frontotemporal lobar degeneration (FTLD) or a motor neuron disease.
The model animal according to any one of claims 1 to 3, wherein the animal is a mouse.
The model animal according to any one of claims 1 to 4, wherein the animal is an inbred strain.
A model neuron or a model neuron cell line obtained from the model animal according to any one of claims 1 to 5.
The manufacturing method of the model animal which exhibits the symptom of the disease accompanying a synaptic abnormality characterized by having the operation process which knocks out the FUS gene specifically in a brain.
The production method according to claim 7, 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 claim 7 or 8, wherein the disease accompanied by a synaptic abnormality is frontotemporal lobar degeneration (FTLD) or a motor neuron disease.
The production method according to any one of claims 7 to 9, wherein the animal is a mouse.
The production method according to any one of claims 7 to 10, wherein the animal is an inbred strain.
A therapeutic agent for diseases associated with synaptic abnormalities, comprising the step of using the model animal according to any one of claims 1 to 5, or the model nerve cell or model nerve cell line according to claim 6. Screening method to search for.
The screening method according to claim 12, wherein the disease accompanied by a synaptic abnormality is frontotemporal lobar degeneration (FTLD) or a motor neuron disease.
A screening method for searching for a therapeutic agent for a disease associated with a synaptic abnormality, comprising a step of measuring increase or decrease of SynGAPα2 due to application of a target drug.
The screening method according to claim 14, wherein the disease accompanied by synaptic abnormality is frontotemporal lobar degeneration (FTLD) or motor neuron disease.
A therapeutic agent for diseases accompanied by synaptic abnormalities, comprising as an active ingredient a gene encoding SynGAPα2 protein or a SynGAPα2 protein, or a drug that promotes the expression of the SynGAPα2 gene in the brain or a drug that can increase the SynGAPα2 protein.
The therapeutic agent according to claim 16, wherein the disease accompanied by a synaptic abnormality is frontotemporal lobar degeneration (FTLD) or a motor neuron disease.
A method for treating a disease accompanied by synaptic abnormality, comprising a step of supplementing or increasing SynGAPα2 in the brain.
The treatment method according to claim 18, wherein the disease accompanied by a synaptic abnormality is frontotemporal lobar degeneration (FTLD) or a motor neuron disease.