JPWO2018012497A1 - Disease model animal and disease treatment agent - Google Patents

Abstract

An object of the present invention is to provide a novel specific knockout model animal exhibiting symptoms of a disease accompanied by synaptic abnormalities in the brain, a method for producing the same, or a therapeutic agent for the disease.
In the present invention, for example, a disease associated with synapse abnormality (for example, the frontal side characterized by being produced by specifically knocking out the FUS gene in the brain using, for example, the Cre-loxP system and the CamK2 promoter) A model animal exhibiting symptoms of temporal lobe degeneration (FTLD), a method for producing the same, or a method for screening a therapeutic drug for the disease using the model animal can be mentioned.

Description

  The present invention belongs to the technical field of genetically modified laboratory animals and therapeutic agents for diseases. The present invention is effective for a FUS gene-specific knockout model animal in the brain, a method for producing the same, or the disease using the model animal, which exhibits symptoms of a disease accompanied with synaptic abnormality (for example, frontotemporal lobe degeneration). The present invention relates to a screening method for a therapeutic agent, or a therapeutic agent for the disease.

  Recent advances in medicine and technology have significantly extended the life expectancy, especially in developed countries. However, along with that, various functional declines, diseases and pathologies caused by aging of the brain, which have not been a problem in the past, have become major social problems. Among them, dementia significantly reduces the quality of life of the patient, and the burden on the caring family is also very large. Dementia-related symptoms range from memory impairment, visual impairment, speech disorder, behavioral problems, sleep disorder, depression symptoms, etc. Known degenerative diseases of the brain that cause such dementia are Alzheimer's disease, Lewy body dementia, cerebrovascular dementia, frontotemporal lobar degeneration (FTLD) and the like.

  Dementia due to FTLD, especially in the West, is the second most frequent progressive dementia following Alzheimer's disease. The main feature of FTLD is that the frontal lobe and the temporal lobe are degeneration sites, and the histopathological findings are three of frontal lobe degeneration type, Pick's disease type, and motor neuron type disease. Symptoms include behavioral abnormalities attributed to higher brain dysfunction such as personality disorder and social behavior abnormalities. However, the pathophysiology of FTLD is so complicated that research on diagnostic methods and treatments has not been sufficiently advanced, and a fundamental treatment has not yet been established.

  In order to further advance detailed studies of FTLD exhibiting a very complicated pathological condition, the existence of a homogeneous model animal system showing the pathological condition is essential. Such a model animal system is preferably a genetically modified model animal such as a knockout mouse. Conventionally, FTLD is said to cause neurodegeneration in nerve cells by abnormal aggregation / accumulation or abnormal localization of FUS or TDP-43 protein. Therefore, dementia model animals such as FTLD have been produced by overexpression of dementia related genes, for example, by gene knock-in. However, it can not be denied that the toxicity caused by the expression of a large amount of the particular gene may cause neurodegeneration, and there has been a criticism that the model itself is due to abnormal findings.

  Patent Document 1 discloses a mouse model of amyotrophic lateral sclerosis and / or frontotemporal lobar degeneration. The model mouse is a knock-in mouse in which mutant TDP-43 is substituted for at least one allele of endogenous TDP-43. The model mouse exhibits the same pathological condition as amyotrophic lateral sclerosis and frontotemporal lobar degeneration, but since it is a knock-in mouse, it is difficult to determine whether the model itself is an abnormal finding.

  The inventors of the present group have studied the above-mentioned pathology of FTLD using mice, and as a result, mice in which FUS (Fused in sarcoma) gene was knocked down in the mouse brain using shRNA expression lentivirus And FTLD-like pathological conditions (see Non-Patent Document 1). Furthermore, in cultured cells in which the FUS gene has been knocked down, the amount of glutamate receptor GluA1 protein present at synapses is significantly reduced, and FUS has a function to specifically bind to and stabilize GluA1 mRNA, It was shown that the FTLD-like pathological condition of the above-mentioned FUS-knocked-down mice is restored by gene transfer of GluA1.

  In order to further study the FUS gene whose expression suppression (loss of function) is suggested as one of the causes of FTLD in the above-mentioned experiment, it is necessary to create a FUS gene knockout mouse. However, since FUS gene knockout mice are usually embryonic lethal, although there have been reports on preparation of mice of crossbring lines so far (see, for example, Non-patent document 2 and Non-patent document 3), other reports were found. Absent. Also, the generation of knockout mice in inbred mice with approximately the same gene background is even more difficult and has not been reported so far. Because FTLD-type dementia has a very complicated disease state, there are limitations in hybridization systems with large individual differences in order to obtain reliable experimental results.

On the other hand, SynGAP (Synaptic Ras GTPase activating Protein) is a protein present in the postsynaptic thick region (PSD) and known to negatively regulate small molecule G protein signaling downstream of glutamate receptor activation. There is. SynGAP proteins have been found to be present from nematodes to higher mammals. In addition, SynGAP protein has a variety of splices consisting of combinations of multiple types of C-terminal domains (α1, α2, β, γ, etc.) and multiple types of N-terminal domains (A, B, C, etc.) to date The presence of the variant is shown, suggesting its diverse functions (see eg non-patent documents 4 and 5).

International Publication No. 2013/180214

Udagawa T, Fujioka Y, Tanaka M, Honda D, Yokoi S, Riku Y, Ibi D, Nagai T, Yamada K, Watanabe H, Katsuno M, Inada T, Ohno K, Sokabe M, Okado H, Ishigaki S, Sobue G ., Nat Commun., (2015), May 13; 6: 7098. doi: 10.1038 / ncomms8098. Kino et al. Acta Neuropathologica Communications 2015; 3:24 Kuroda M, Sok J, Webb L, Baechtold H, Urano F, Yin Y, Chung P, de Rooij DG, Akhmedov A, Ashley T, Ron D. EMBO J. 2000 Feb 1; 19 (3): 453-62. AC McMahon, MW Barnett, TS O'Leary, PN Stoney, MO Collins, S. Papadia, J. Choudhary, NH Komiyama, SGN Grant, GE Hardingham, DJ Wyllie & PC Kind, Nat communi., (2012), June 12; 3 : 900. doi: 10.1038 / ncomms 1900. Jeyabalan N, Clement JP., Front Cell Neurosci. (2016), Feb 15; 10: 32. doi: 10.3389 / fncel.

The model in which the FUS gene is injected and knocked down with, for example, adeno-associated virus has limited effect only on the hippocampus, and it is not easy to learn the procedure. All reported FUS gene knockout mice are hybrid lines, and their behavior analysis may differ depending on the strain. On the other hand, no analysis example using FUS gene knockout mice standardized in the background of inbred strains is known.
In addition, techniques for tissue-specific knockout using the loxP-Cre system are known, but since the phenotypes differ depending on the promoter selection, the Cre expression method, etc., the optimized pathological model animal (pathological model mouse The preparation of) is not always easy.

  An object of the present invention is to provide a novel model animal exhibiting symptoms of a disease accompanied with synaptic abnormalities (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, and a therapeutic agent and a treatment method for the disease using the model animal.

  As a result of intensive studies, the present inventors have found that the above-mentioned problems can be solved by conditional knockout of FUS gene in the brain, and came to complete the present invention. The present inventors also found that increasing the protein SynGAPα2 present in postsynaptic thickness is effective for the treatment of diseases associated with synaptic abnormalities.

As the present invention, for example, the following can be mentioned.
[1] A model animal exhibiting symptoms of a disease accompanied by synaptic abnormality, which is produced by specific knockout of FUS gene in 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 the above-mentioned [1] or [2], wherein the disease accompanied by synapse abnormality is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.
[4] The model animal according to any one of the above [1] to [3], wherein the animal is a mouse.
[5] The model animal according to any one of [1] to [4] above, wherein the animal is inbred.
[6] A model neuron or a 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 symptoms 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 the above-mentioned [7] or [8], wherein the disease accompanied with synapse abnormality is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.
[10] The production method according to any one of the above-mentioned [7] to [9], wherein the animal is a mouse.
[11] The production method according to any one of the above-mentioned [7] to [10], wherein the animal is inbred.

[12] A synapse comprising: 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 agent for a disease accompanied by an abnormality.
[13] The screening method of the above-mentioned [12], wherein the disease accompanied with synaptic abnormalities is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.
[14] A screening method for searching for a therapeutic agent for a disease associated with synapse abnormality, comprising the step of measuring increase or decrease of SynGAPα2 by application of a target drug.
[15] The screening method of the above-mentioned [14], wherein the disease accompanied by synapse abnormality is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.

[16] A treatment for a disease associated with synapse abnormality, which comprises as an active ingredient a gene encoding SynGAPα2 or a SynGAPα2 protein, or a drug capable of enhancing SynGAPα2 gene expression in the brain or SynGAPα2 protein Agent.
[17] The therapeutic agent according to the above-mentioned [16], wherein the disease accompanied by synapse abnormality is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.
[18] A method for treating a disease associated with synaptic abnormality, comprising the step of supplementing or increasing SynGAPα2 to the brain.
[19] The method according to the above-mentioned [18], wherein the disease accompanied by synaptic abnormality is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.

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

It is a schematic diagram of the vector gene inserted in 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 the 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 the wild type gene (WT), the middle figure is a schematic diagram of the recombinant gene (KO) into which the vector has been inserted, and the lower figure is a conditional knockout completed by removing the Neo cassette by crossing with the FLP mouse. It is a schematic diagram of a mouse gene (Neo Deletion). In creating a conditional knockout mouse, the region including the exons 1 to 6 in the wild-type gene sequence of the FUS gene targeted for genetic manipulation is represented. The continuation of the gene sequence of FIG. 4A. Among gene sequences inserted into conditional knockout mice, a region including exons 1 to 6 of the FUS gene is shown. The continuation of the gene sequence of FIG. 5A. FIG. 10 shows the gene sequence of an AAV vector into which the gene sequence of SynGAP Aα2 has been inserted. The continuation of the gene arrangement | sequence of FIG. 6A. Figure (A) shows the immunostaining image of FUS protein in the hippocampus CA1 region for the control mouse (Cre-) and the FUS gene conditional knockout mouse (FUS-cKO mouse (Cre +)) expressing Cre, Figure (B) The respectively represents the result of the western blot of the hippocampus CA1 area | region sample. The Golgi stained images of brain tissue sections in the hippocampus CA1 region are shown for control mice (Cre−) and FUS-cKO mice (Cre +). It represents the total number of spines in the brain tissue section. The vertical axis shows the total number of spines per 100 μm of neurites. It represents the percentage of mature spines in the brain tissue section. The vertical axis shows the percentage (%) of mature spines in the total number of spines. The results of open field tests in control mice (Cre-) and FUS-cKO mice (Cre +) are shown. The vertical axis indicates the movement distance (mm) of the mouse in the arena. Fig. (A) shows the results of "the path with wall" or "the path without wall" in the elevated plus maze test in control mice (Cre-) and FUS-cKO mice (Cre +) . The vertical axis indicates the staying time (seconds). Figure (B) represents the results for the "wallless road". The vertical axis shows the number of intrusions (times). For control mice (Cre-) and FUS-cKO mice (Cre +), Figure (A) shows the results of the novel object recognition test. The vertical axis shows search preference (%). Figure (B) represents the results of the fear conditioning test. The vertical axis shows the freezing reaction time (seconds). Figure (A) shows the fluorescence immunostaining image in the hippocampus CA1 region and Figure (B) shows the result of the western blot of the hippocampus CA1 region sample for control mice (Cre-) and FUS-cKO mice (Cre +). Represent. 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 spines in the brain tissue section. The vertical axis shows the percentage (%) of mature spines in the total number of spines. The results of the open field test are shown for FUS-cKO mice (Cre +), FUS-cKO mice (Cre +), and control mice (Cre-) supplemented with SynGAP Aα2. The vertical axis indicates 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 on a road without walls.

1 Model Animal According to the Present Invention A model animal according to the present invention (hereinafter referred to as "the present invention model animal") is characterized by being produced by specific knockout of FUS gene in the brain, Present with symptoms.
Here, the "brain" in the present invention is also present in lower organisms, but in particular in higher organisms, the cerebrum or its corresponding part can be mentioned mainly, especially frontal lobe, temporal lobe, hippocampus and the like.

1.1 FUS gene The FUS gene is a nematode, a fish such as zebrafish, an amphibian such as a frog, a mouse, a rat, a rodent such as a guinea pig, a rabbit, a ferret, a dog, a cat, a pig, a sheep, a goat, a cow, It is a gene widely present in higher mammals such as horses and monkeys and humans. A common structure of the FUS protein encoded by the FUS gene has a transcription activation domain at the N-terminal side and an RNA binding motif and a zinc finger domain at the C-terminal side. The nucleotide sequence information of the FUS gene in each animal can be obtained, for example, from Genbank, which is a gene information database.
In the preparation of the model animal of the present invention, the FUS gene of the target animal is genetically manipulated using data from a gene information database etc. 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 brain.
As a method for performing specific knockout, usually, conditional knockout method, which is a method for knocking out a target gene of a target model animal in time and space can be mentioned. As a system used in such a conditional knockout method, for example, a system of a recombinant enzyme Cre (Cre recombinase) derived from bacteriophage P1 and its target sequence loxP sequence (Cre-loxP system), budding yeast (Saccharomyces cerevisiae) Of recombinant enzyme FLP and its target sequence FRT sequence, system of recombinant enzyme R from soy sauce yeast (Zygosaccharomyces rouxii) and system of RS sequence as its target sequence, or recombinant enzyme Gin and its target from bacteriophage Mu A system of Gix sequence which is a sequence, a tetracycline ON-OFF system can be mentioned. Among these, the Cre-loxP system is preferred.

  The Cre-loxP system is a system using Cre recombinase, which is a recombinant enzyme, and loxP sequence, which is its recognition sequence. The system usually links Cre recombinase to a promoter that functions in a time-specific and / or site-specific manner in a genetically modified model animal, with the target gene flanked by loxP sequences, and the time-specific, site-specific Expressed Cre recombinase causes deletion of the target gene flanked by loxP sequences. When this method is used, the FUS gene can be made to function normally without knocking out at the stage of the 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 adult growth. . Usually, a gene of interest is manipulated in the fertilized egg or early stages of development of the model animal, eg, 2-cell stage to blastocyst stage, blastocyst stage, and the FUS gene conditional knockout which is the model animal of the present invention A model animal (FUS-cKO model animal) is produced.

  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. Among these, the CamK2 promoter is preferred. The mouse CamK2 promoter is a promoter in which significant gene expression is observed in the brains of mice after 2 months of age and can specifically knock out the FUS gene in adult brains.

1.3 Diseases Associated with Synapse Abnormalities Examples of diseases associated with synapse abnormalities exhibited by the model animal of the present invention include, for example, frontotemporal lobe degeneration (FTLD) and motor neuron diseases. The model animal of the present invention mainly causes degeneration in the frontal lobe and temporal lobe, and exhibits histopathological findings of frontal lobe degeneration type and motor neuron disease type. Symptoms include, for example, significant atrophy of the frontal and temporal lobes, or behavioral abnormalities resulting from higher brain dysfunction.

  Examples of synaptic abnormalities exhibited by the model animal of the present invention include abnormalities in the spines of dendrites present in cell bodies of neurons. The morphology of spines present in the postsynaptic state is said to change to a mushroom shape when the synapse formation matures, and to increase the surface area in contact with the presynaptic synapse, resulting in synaptic potentiation and affecting behavioral physiology (Hippocampus 2000; 10: 501, JCB 2010; 189: 619). In neurodegenerative diseases, it has been suggested that changes in the postsynaptic dendrite spine may be involved in the pathological condition (Annu Rev Pathol 2016; 11: 221, Neurosci Biobehav Rev 2015; 59: 208). Examples of spine abnormalities exhibited by the model animal of the present invention include, for example, a reduction in the number of spines, a reduction in the number of mature spines, and an abnormality in spine morphology. In the model animal of the present invention, a synapse abnormality causes an abnormality in a protein present in the synapse. Examples of abnormalities in proteins present in synapses include localization abnormalities of PSD-95 and reduction of SynGAPα2.

  Diseases with synaptic abnormalities exhibited by the model animal of the present invention may show higher brain dysfunction as a result. Examples of the above-mentioned higher brain dysfunction include hyperactivity (abnormality of movement amount), anxiety behavior, memory disorder, depression-like behavior, personality disorder, disorder of social behavior, and disorder of pain sensitivity. These higher brain dysfunctions can be evaluated by behavioral tests used in the field of brain science. Examples of the behavioral test for behavioral abnormality include an open field test and a home cage activity test. Examples of behavioral tests for anxiety include, for example, Elevated plus maze test and Light / dark transition test. Behavioral tests for memory impairment include, for example, Novel object recognition test, Fear conditioning test, and Morris water maze test.

The behavioral test of depression-like behavior includes, for example, Porsolt forced swim test, Tail suspension test. Behavioral tests for social behavior abnormalities include the Social Interaction Test and the 24 hour Homecage Social Interaction Test. Examples of pain sensitivity test include hot plate test and formalin test. These behavioral tests can be performed alone or in combination depending on the symptoms and types of higher brain functions to be evaluated, and can be used to evaluate higher brain dysfunction.
When the behavioral test as described above is performed on the animal model of the present invention exhibiting symptoms of FTLD or frontotemporal dementia (FTD), significant increase in exercise amount and lack of anxiety are observed, while memory impairment is It shows the tendency not to be seen.

1.4 Animal species The animal species used for producing the model animal of the present invention is not particularly limited as long as it is generally used as an experimental animal, and includes, 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, cattle and horses. Among them, preferably rodents such as mice, rats and guinea pigs, or primates such as monkeys and marmosets, more preferably mice. Many of these animal strains are readily available from laboratory animal vendors.

The animal line may be a cross line or an inbred line, and is not particularly limited, but in order to minimize individual differences in results in animal experiments, an inbred line having a uniform gene background is preferable. In the case of mice, inbred strains of experimental animals are usually obtained by inbreding for more than 20 generations, so each has approximately 0.01% or less of the heterozygous gene, so each is approximately genetic. It can be regarded as the same individual. Examples of general inbred experimental animals include the Wister strain in rats, the DBA / 2 strain in mice, the C57BL / 6 strain, and the BALB / c strain. These inbred experimental animals can be easily obtained from the laboratory animal supplier. It can also be established using inbred animal production services such as the MAX-BAX program provided by experimental animal vendors such as Charles River et al.
One feature of the present invention is to provide inbred model animals (in particular, mice) suitable for experiments.

1.5 Model nerve cell according to the present invention, and model nerve cell line The present invention relates to a model nerve cell obtained from the model animal of the present invention (hereinafter referred to as "model nerve cell according to the present invention") , “The model neural cell line of the present invention”.
The model neuron of the present invention is a primary culture 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 nerve cells and the like. The model neuron of the present invention is usually a neuron of the brain or nervous system of the model animal of the present invention. Further, the model nerve cell line of the present invention is a cell line (cell line established) established by repeating subculture from the model nerve cell of the present invention. The model nerve cell of the present invention and the model nerve cell line of the present invention have the advantage of being easily handled as compared to the model animal of the present invention, and are cryopreserved in liquid nitrogen etc. It can also be done. Methods known in the art of cell culture can be used for culturing, subculturing, cryopreservation, thawing, etc. of the model neuron etc. of the present invention. For research, experiments and tests using the model neuron of the present invention, methods known in the cell culture art can also be used.
Here, the "cell line" refers to a cell which has been maintained by subculture in vitro for a long period of time and has reached a certain stable property. By using the model neural cell line of the present invention, reproducible and stable experiments can be performed.

1.6 Production method The present invention is characterized by comprising an operation step of specifically knocking out the FUS gene in the brain, a method for producing a model animal exhibiting symptoms of a disease accompanied with synaptic abnormality (hereinafter referred to as “production according to the present invention Method).
“FUS gene”, “disease with synaptic abnormality”, “animal species” and the like are as defined above.

Examples of the operation steps for specifically knocking out the FUS gene in the brain according to the production method of the present invention include the conditional knockout method mentioned in the section "1.2 Specific knockout of FUS gene in brain" above. As a system used by this conditional knockout method, although the same system as what was shown by the paragraph can be mentioned, for example, Cre-loxP system is preferred among them.
In addition, it is preferable to use the Cre-loxP system and the CamK2 promoter in the knockout operation.

In the method of the present invention, a specific gene is genetically engineered at the developmental stage of the target animal. Examples of such genetic engineering methods include microinjection, in which DNA is directly injected into pronuclei of fertilized eggs, a method using a retrovirus vector, a method using ES cells, and a method using iPS cells. be able to. The gene injected into a fertilized egg or the like is, for example, integrated into the chromosome by homologous recombination. In the production method of the present invention, when the Cre-loxP system is used, two genes of the FUS gene sandwiched between loxP sequences and a sequence encoding the full-length Cre recombinase gene (hereinafter referred to as "Cre gene") are genetically manipulated. And be incorporated into fertilized eggs etc. In the production method of the present invention, although it is possible to integrate the Cre gene and the FUS gene between the loxP sequences into one individual at one time, the Cre gene and the above FUS gene are integrated into separate individuals, By mating, individuals having both genes integrated can also be obtained.
Hereinafter, as a specific example of the production method of the present invention, the case of microinjection using a mouse will be described.

  In the microinjection method, a fertilized egg is first collected from the oviduct of a female mouse whose mating is confirmed, and after culturing, the desired nucleus is injected into the pronucleus. As a DNA construct, one containing a Cre gene is used. As a DNA construct to be used, a CamK2 promoter, which is a promoter sequence capable of performing stage-specific expression of a transgene, can be used. In addition, the FUS gene with loxP sequences is inserted into the chromosome by homologous recombination. The fertilized egg which has finished the injection operation is transplanted into the oviduct of a pseudopregnant mouse, and the transplanted mouse is reared for a predetermined period to obtain a pup mouse (F0).

In order to confirm that the FUS gene having Cre gene and / or loxP sequence is properly integrated into the chromosome of the offspring mouse, DNA is extracted from the tail of the offspring mouse, etc. and a primer specific for the transgene is used The PCR method, the dot hybridization method using a probe specific for the transgene, etc. are performed, and a suitable recombinant mouse is selected.
In adulthood of the model animal of the present invention, the evaluation of whether or not conditional knockout was properly performed can be confirmed by examining the target tissue, ie brain tissue. As a test method, for a test for FUS protein, for example, immunostaining of tissue sections, Western blot of extracted protein, for a test for FUS mRNA, for example, quantitative PCR, Northern hybridization, in In situ hybridization is included.

2 Screening method according to the present invention The present invention searches for a therapeutic drug for a disease associated with synapse abnormality characterized by having a step using the model animal of the present invention, the model neuron of the present invention or the model neuronal cell line of the present invention. Screening method (hereinafter referred to as "the screening method of the present invention").
The screening method of the present invention, when using the animal model of the present invention, is usually directed to the animal model of the present invention exhibiting symptoms such as frontotemporal lobe degeneration (FTLD), motor neuron disease and the like with synaptic abnormalities. It is carried out by administering the drug and evaluating whether or not its characteristic symptoms, pathological findings, or higher brain dysfunction are alleviated or treated. As a result of administering the subject drug to the model animal of the present invention, if the characteristic symptoms, pathological findings or higher brain dysfunction are 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 neuron of the present invention or the model nerve cell of the present invention, for example, the target drug is administered to the model animal of the present invention as a step of evaluating the efficacy of the target drug. After that, it is possible to cite the step of measuring the variation (changes before and after administration) of the amount of protein or mRNA, particularly in the brain, of the gene associated with the disease accompanied by synaptic abnormality as a result. Such genes associated with diseases associated with synaptic abnormalities include, for example, SynGAPα2, Tau and GluA1. Among these, it is preferable to measure increase or decrease of SynGAPα2 protein or mRNA.

  When the target drug is administered to the model animal of the present invention and the amount of SynGAPα2 protein or mRNA in the diseased part is increased compared to before administration or in the control group, the drug may treat or alleviate the disease. It 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 mRNA measurement methods include quantitative RT-PCR and northern hybridization. Methods of measuring protein include, for example, Western blot and immunostaining. Usually, the evaluation of efficacy by the above-mentioned measurement is performed using, for example, an extract of tissue at a disease site or a tissue section.

  In addition, a disease associated with synapse abnormality (eg, frontotemporal lobar degeneration (eg, frontal temporal lobe degeneration) by having a step of measuring increase or decrease of SynGAPα2 by application of the target drug in vivo or in vitro without using the model animal etc. Therapeutic agents for FTLD), motor neuron diseases, etc.) can also be screened. Such screening methods are also included in the present invention (hereinafter, also including the screening methods, also referred to as "the screening methods of the present invention").

  The increase or decrease of SynGAPα2 can be measured by a conventional method. Then, if application of the target drug results in an increase in SynGAPα2 before application or in comparison to the control group, it can be evaluated that the drug may treat or alleviate the disease. In the said in vivo said screening method, the increase / decrease in SynGAP (alpha) 2 in the brain of the animal used is measured normally. On the other hand, in the screening method in the above in vitro, usually, increase and decrease of SynGAPα2 in cells used and the like are measured.

The animal species which can be used in the screening method in vivo described above is not particularly limited as long as it is a non-human animal generally used as an experimental animal, and examples include primates (monkeys, marmosets), rodents (for example) Mouse, rat, guinea pig etc.), rabbit, dog, cat, pig, cow, sheep, horse.
In the said in vitro said screening method, the culture | cultivated cell etc. of the brain of the human and the non-human animal used as an experimental animal which were mentioned above, etc., etc. are used. Examples of human cultured cells include NSC-34 which is a cultured cell derived from motor nerves. As cultured cells of non-human animals, for example, mouse primary cultured neurons, Neuro2A can be mentioned. In addition, iPS cells produced from human or non-human animal cells with or without a disease exhibiting the above synaptic abnormalities can also be used.

The target drug (therapeutic agent candidate) applicable to the screening method of the present invention is not particularly limited as long as it is generally used as a drug, and, for example, low molecular weight organic compounds, inorganic compounds, peptides, proteins, glycoproteins, Lipids, glycolipids, sugars, nucleic acids can be mentioned. 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, as a method of applying the target drug to the model animal of the present invention (administration method), a usual administration method to experimental animals can be mentioned. Specifically, for example, oral administration, intravascular Injection, administration using a catheter, application to the epidermis, subcutaneous administration, intraperitoneal administration, intrathecal administration, intracerebroventricular administration, brain parenchymal injection, etc., but since the target site is a diseased part of the brain, oral Administration, intravascular injection, administration to a disease site using a catheter, intrathecal administration, intracerebroventricular administration, brain parenchymal injection is preferred.

3 Therapeutic agent according to the present invention The present invention is characterized by containing a gene encoding SynGAPα2 or a SynGAPα2 protein, or a drug capable of increasing the expression of SynGAPα2 gene in brain or a drug capable of increasing SynGAPα2 protein as an active ingredient. And a therapeutic agent for diseases associated with synaptic abnormalities (hereinafter referred to as “the therapeutic agent of the present invention”).
Here, "treatment" not only directly or indirectly ameliorates, alleviates or cures the target disease, symptoms or their associated symptoms, but also includes, for example, prevention of the disease, etc. It is a term.
Here, SynGAPα2 in the present invention has an α2 domain at the C-terminal side among the splicing variants of SynGAP, and the domain at the N-terminal side may be any of A, B, C and the like.

While examining the involvement of FUS in the pathophysiology of the present invention using the animal model of the present invention, the present inventors confirmed that mature spines in brain neurons are significantly reduced and, at the same time, SynGAP2α in brain neurons. It was also confirmed that the expression of the gene was reduced, that selective knockdown of SynGAP2α had the same phenotype as that of the model animal of the present invention, and that the mature spine was restored by supplementation of the SynGAP2α gene.
Thus, agents that can increase SynGAPα2 in the brain may be effective for treating diseases with synaptic abnormalities such as FTLD. As such an agent, for example, an agent containing a gene encoding SynGAPα2 or SynGAPα2 protein, or a drug that promotes expression of SynGAPα2 gene in the brain or a drug capable of increasing SynGAPα2 protein as an active ingredient can be mentioned.

  The nucleotide sequence of human SynGAPα2 gene and the amino acid sequence of the protein encoded by the gene are shown in 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 shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

3.1 Gene Encoding SynGAPα2 The DNA of SynGAPα2 contained in the therapeutic agent of the present invention is hybridized under stringent conditions with a DNA having a base sequence complementary to the base sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3. DNA to be soybeanized, the nucleotide sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3 and BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) For example, it is preferably at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably, when calculated using default or default parameters). Or one or more or a few (1 to 10, preferably 1 to 5, preferably 1 to 5), more preferably 97% or more, of the DNA having a homology of 97% or more or the amino acid sequence of a protein encoded by the DNA Is a DNA encoding a protein consisting of an amino acid sequence in which one or two amino acids are substituted, deleted and / or added, and so on, as long as it encodes a protein having an activity to promote formation of mature spines And the therapeutic agent of the present invention. Here, the “stringent conditions” are, for example, conditions of “1 × SSC, 0.1% SDS, 37 ° C.”, and more severe conditions “0.5 × SSC, 0.1% SDS, 42 ° C.” The more severe condition is the condition of “0.2 × SSC, 0.1% SDS, 65 ° C.”. Thus, the more stringent the hybridization conditions are, the more isolation of DNA having high homology with the probe sequence can be expected. However, the combination of SSC, SDS and temperature conditions described above is an example, and necessary stringency can be realized by appropriately combining the probe concentration, the length of the probe, the reaction time of hybridization, etc. 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. Also, 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 comprises a vector containing the SynGAPα2 gene. By introducing the vector into a subject, SynGAPα2 protein can be expressed in the subject and exert a mature spine formation promoting effect. Such introduction of the gene of interest into the subject upon administration of the therapeutic agent of the present invention can be carried out by known methods. Methods for introducing genes into subjects include methods using viral vectors and methods using non-viral vectors, and various methods are known (Separate volume experimental medicine, basic technology for gene therapy, Yodosha, 1996; separate volume Experimental medicine, gene transfer and expression analysis experimental method, Yodosha, 1997; edited by Japan Society for Gene Therapy, Handbook of Gene therapy development research, NTS, 1999).

  Examples of viral vectors used for the therapeutic agent of the present invention include adenovirus, adeno-associated virus (AAV), virus vectors such as retrovirus, detoxified retrovirus, herpes virus, vaccinia virus, pox virus, polio virus, syn virus DNA virus or RNA virus such as bis virus, Sendai virus, SV40, immunodeficiency virus (HIV) and the like. Among them, adenovirus and adeno-associated virus (AAV) are preferable. It is possible to introduce a gene into cells by introducing the target gene into the above-mentioned viral vector and infecting cells with a recombinant virus.

  Adeno-associated virus vectors (AAV vectors) are preferably used for the therapeutic agent of the present invention. The AAV vector may contain regulatory elements for efficiently expressing the DNA of interest, such as a promoter, an enhancer, a transcription terminator, etc., and as necessary, a translation initiation codon, a translation termination codon, etc. may be inserted. It is also good.

  AAV vectors can be used for gene transfer to any growth / non-proliferation cells, and in non-dividing cells, in particular, long-term target gene expression is possible. It is less immunogenic than adenoviral and retroviral vectors and is also suitable for gene transfer to animals. In addition, since it is a nonpathogenic virus, it can be handled even at P1 level facilities, and is widely used as a safe and easy-to-handle virus vector for research. In addition, there are over 100 serotypes in AAV, and it is known that different serotypes have different host range and virus characteristics. Serotype 2 (AAV2) is one of the long-studied serotypes 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 tropism, and AAV1 includes muscle, liver, airway, central nervous system, etc., AAV5 The central nervous system, liver, retina, etc., AAV6 is said to have a high gene transfer efficiency to the heart, muscle, liver, etc., and can be used properly depending on the target tissue. Serotype 9 (AAV9) was used in the test examples described below. In Non-Patent Document 1, there is a history of using a virus generated from a plasmid of the same backbone as AAV9 used in this experiment.

  AAV vectors for use in the therapeutics of the invention can be prepared by standard methods well known in the art. For example, US Patent No. 5,858, 351 and the references cited therein describe various recombinant AAVs suitable for use in gene therapy, as well as methods of making and propagating these vectors ( For example, Kotin (1994) Human Gene Therapy 5: 793 to 801, or Berns "Parvoviridae and their Replication" Fundamental Virology, Second Edition, edited by Fields & Knipe, etc.).

  According to a preferred method for producing an AAV vector, a plasmid is first produced by leaving ITRs at both ends of wild-type AAV and inserting a gene of interest therebetween (AAV vector plasmid). On the other hand, a plasmid that expresses the Rep gene (a gene encoding a replication protein) and the Cap gene (a gene encoding a viral head shell protein), and a plasmid that expresses the adenovirus genes E2A, E4, and VA, respectively. Prepare. Then, packaging cells expressing the E1 gene, such as HEK 293 cells, are cotransfected with these three plasmids, and the cells are cultured. This makes it possible to produce AAV vector particles having high infectivity to mammalian cells. In the test example to be described later, the virus was produced by the same method as Non-Patent Document 1.

  The non-viral vector used for the therapeutic agent of the present invention is not particularly limited as long as it is a vector capable of expressing a target gene in vivo, and examples thereof include pCAGGS (Gene 108, 193-200 (1991)) and pBK -Expression vectors such as CMV, pcDNA3, 1, pZeoSV (Invitrogen, Stratagene), pVAX1 and the like can be mentioned. When using a nonviral vector, it is preferable to use a known method for efficiently incorporating a gene into a target cell or tissue. As the method, for example, lipofection method, phosphate-calcium coprecipitation method, DEAE-dextran method, direct injection of DNA using a micro glass tube, gene transfer method by internal liposome, electrostatic liposome Gene transfer method (electorostatic type liposome), HVJ-liposome method, modified 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 to a cell together with a carrier (metal particle), a method of direct introduction of naked-DNA, a method of introduction using various polymers, and the like.

The vector containing the SynGAPα2 gene may optionally contain a promoter or enhancer for transcribing the gene, a poly A signal, a marker gene for labeling and / or selection of cells into which the gene has been introduced, and the like. A well-known promoter can be used as a promoter in this case.
In order to introduce the therapeutic agent of the present invention containing the SynGAPα2 gene into a subject, an in vivo method of directly introducing the therapeutic agent of the present invention into the body, and removing certain cells from human and ex vivo the therapeutic agent of the present invention Either ex vivo method or the like can be used which is introduced into cells and the cells are put back into the body (Nikkei Science, April 1994, pp. 20-45; Monthly Journal, 36 (1), 23- 48 (1994); Experimental Medicine Supplement, 12 (15), (1994); Japan Gene Therapy Association, edited by Gene Therapy Development Research Handbook, NTS (1999).

3.2 SynGAPα2 Protein The protein encoded by SynGAPα2 gene contained in the therapeutic agent of the present invention has an amino acid sequence substantially identical to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4 and induces formation of mature spines Proteins having activity can be used. Here, as substantially the same amino acid sequence, one or more or several (1 to 10, preferably 1 to 5, more preferably 1 or 2) amino acids relative to the amino acid sequence are used. A substituted, deleted and / or added amino acid sequence or the amino acid sequence, BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) Have a homology of at least 85% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 97% or more when calculated using default or default parameters). It is something that can be cited.

  The uptake of SynGAPα2 protein into a nerve cell may be carried out, for example, by binding to a peptide capable of passing SynGAPα2 protein through the cell membrane and administering it to a brain tissue site. Various peptides are known as peptides capable of passing through the cell membrane, and known peptides thereof can be used, for example, the protein transduction domain of HIV-1 · TAT, Drosophila homeobox protein Antennapedia cell membrane domain, C-terminal (267-300) peptide of VP22, HIV-1 / Rev (34-50) peptide, FHV / coat (35-49) peptide, N-terminal (7-22 of K-FGF) And the hydrophobic region of Alternatively, they may be administered in combination with a compound capable of specifically binding to nerve cells. In this case, it may be locally administered to a brain tissue site, or may be administered orally or the like to deliver SynGAPα2 protein to nerve cells. Such compounds capable of specifically binding to nerve cells include homing signal peptides that bind to receptors expressed on the surface of nerve cells. In order to bind the above-mentioned cell membrane transmembrane peptide or signal peptide to SynGAPα2 protein, for example, a DNA encoding SynGAPα2 protein and a DNA encoding the above-mentioned cell membrane transmembrane peptide or homing signal peptide are linked in frame, and known genetic engineering It may be produced as a fusion protein by techniques.

  Furthermore, the SynGAPα2 protein or 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, and a peptide having a mature spine formation promoting activity, and the nucleotide sequence of the DNA A fragment nucleotide consisting of a partial base sequence, which is a nucleotide encoding a peptide having a mature spine formation promoting activity is also included. Such fragmented peptides or nucleotide fragments can be easily obtained by cleaving full-length protein or full-length DNA at an appropriate site, and determining whether it has a mature spine formation promoting activity.

3.3 Preparation The therapeutic agent of the present invention is a fusion protein of SynGAPα2 protein or SynGAPα2 protein and a cell membrane transmembrane peptide or homing signal peptide, or a carrier that is pharmacologically acceptable in addition to SynGAPα2 gene DNA or a vector containing the DNA. A diluent or excipient may be included.
The therapeutic agent of the present invention can be administered in various forms, and orally administered by tablets, capsules, granules, powders, syrups, etc., or injections, drips, suppositories, sprays, eye drops, transnasal Parenteral administration by administration agent, patch, etc. can be mentioned.

  The therapeutic agent of the present invention comprises carriers, diluents and excipients commonly used in the pharmaceutical field. For example, as a carrier for tablets and excipients, lactose, magnesium stearate and the like are used. As the aqueous solution for injection, physiological saline, isotonic solution containing glucose and other adjuvants, etc. are used, and suitable solubilizers such as alcohol, polyalcohol such as propylene glycol, nonionic surfactant etc. You may use it together with As an oily liquid, sesame oil, soybean oil, etc. are used, and as a solubilizer, benzyl benzoate, benzyl alcohol etc. may be used in combination.

3.4 Administration Target, Administration Method, Dosage The therapeutic agent of the present invention is usually administered in an animal (in vivo). The subject animal to which it can be administered is not particularly limited, and examples include human or non-human animals. The non-human animals are not particularly limited, but non-human mammals, specifically, for example, primates (monkeys, marmosets), rodents (mice, rats, guinea pigs etc.), rabbits, dogs, cats, pigs, cattle , Sheep, 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 according to the treatment method and the like. The administration method may be either systemic administration or local administration. Administration of the therapeutic agent of the present invention can be carried out by injection (needle type, needleless type), administration using a catheter, oral administration, or external use. As for the administration route, it is preferable to select an effective route for treatment and the like. For example, in the case of systemic administration, in addition to oral administration, it is administered by parenteral administration such as intravenous administration, intraarterial administration, subcutaneous administration, intramuscular administration, and interstitial 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, mucosa, 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 the diseased part of the brain, oral administration, intravascular injection, administration using a catheter to the disease site, intrathecal administration, intracerebroventricular Administration, brain parenchymal injection is preferred.

  The dose of the therapeutic agent of the present invention can be appropriately selected according to the type of active ingredient, type of disease, condition of patient, age, body 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 adult, preferably in the range of 1 ng to 500 mg, and more preferably in the range of 10 ng to 200 mg. Sometimes you need more than this, or sometimes less than this. In addition, such a dose can be divided into one dose once or multiple times a day.

  When the active ingredient is SynGAPα2 protein, about 0.001 mg to 100 mg per day is preferable for oral administration, and it is preferable to administer once or several times. In parenteral administration, it is preferable to administer 0.001 mg to 100 mg at a time by subcutaneous injection, intramuscular injection or intravenous injection. In addition, when the active ingredient is SynGAPα2 gene DNA inserted into an expression vector or the like which is translated in a subject, 0.001 mg to 100 mg is subcutaneously injected or intramuscularly injected once every several days, several weeks or several months. Preferably, administration is by intravenous injection.

3.5 Drug The therapeutic agent of the present invention can contain, as an active ingredient, a drug that promotes expression of SynGAPα2 gene in the brain or a drug that can increase SynGAPα2 protein, in addition to the gene encoding SynGAPα2 and SynGAPα2 protein. As the drug, for example, low molecular weight organic compounds, inorganic compounds, peptides, proteins, glycoproteins, lipids, glycolipids, sugars, nucleic acids, plants, as long as they have the efficacy found by the screening method of the present invention. No particular limitation is imposed on the extract of culture products such as microorganisms and cells, culture supernatants and the like.

  In addition to the drug, a pharmaceutically acceptable carrier and 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, and examples include edible carbohydrates such as starch and mannitol. The amount of these carriers etc. in the preparation is suitably in the range of 0.5 to 99.999%, preferably in the range of 10 to 99.99% by weight.

  In addition to the above carriers, pharmaceutically acceptable additives, excipients and the like can be blended with the therapeutic agent of the present invention containing the drug as an active ingredient, as long as the effects of the present invention are not impaired. As such additives or excipients, for example, an emulsifying auxiliary (for example, a fatty acid having 6 to 22 carbon atoms or a pharmaceutically acceptable salt thereof, albumin, dextran), a stabilizer (for example, cholesterol, phosphatidic acid) , Tonicity 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) It can be mentioned. One or more of these can be used. 90 weight% or less is suitable for content of the said additives etc. in this invention therapeutic agent, 70 weight% or less is preferable, and 50 weight% or less is more preferable.

The dosage form of the therapeutic agent of the present invention containing the drug as an active ingredient is not particularly limited and may be appropriately selected depending on the administration method etc., and examples of preparations for parenteral administration include injections, ointments, Gels, creams, patches, liniments, suppositories, sprays, inhalants, sprays, eye drops, nasal drops are included. Among these, injections are preferred. Examples of preparations for oral administration include powders, granules, tablets, capsules, syrups and solutions. In the treatment method of the present invention comprising the drug as an active ingredient, the administration subject, administration subject, dose and the like are the same as described above.

4 Therapeutic method according to the present invention The present invention includes a method for treating a disease associated with synapse abnormality (hereinafter referred to as “the therapeutic method according to the present invention”) comprising a step of supplementing or increasing SynGAPα2 to the brain. .
Examples of diseases associated with synapse abnormalities targeted by the treatment method of the present invention include frontotemporal lobar degeneration (FTLD) and motor neuron diseases. In the treatment method of the present invention, in order to supplement or increase SynGAPα2 to the brain, for example, administering a gene encoding SynGAPα2 or SynGAPα2 protein, a drug that promotes expression of SynGAPα2 gene in the brain or a drug that can increase SynGAPα2 protein Can be achieved directly or indirectly by administering

As a drug that promotes the expression of SynGAPα2 gene in the brain or a drug that can increase SynGAPα2 protein, for example, low molecular weight organic compounds, inorganic compounds, peptides, and the like having the corresponding efficacy found by the screening method of the present invention There are no particular limitations on the extract, culture supernatant, etc. of proteins, glycoproteins, lipids, glycolipids, sugars, nucleic acids, plants, microorganisms, cells, etc.
In the treatment method of the present invention, the preparation for administering the active ingredient, the subject of administration, the dose and the like are the same as described above.

  EXAMPLES The present invention will next be described in more detail by way of examples and test examples, which should not be construed as limiting the invention in any manner.

Example 1 Preparation of Model Animal of the Present Invention (FUS-cKO Mouse (Cre +)) The model animal of the present invention was prepared using the Cre-loxP system. A vector having a gene sequence in which a loxP sequence having the same orientation was inserted into the 5 'UTR and intron 3 of the FUS gene together with a neomycin cassette having the Frt sequence was designed (see FIG. 1) to produce a vector having the sequence. Schematic representation of the region of exons 1 to 6 of wild-type (WT) gene (FIGS. 4A and 4B, SEQ ID NO: 5), vector, and gene into which the vector was inserted (KO gene, FIGS. 5A and 5B, SEQ ID NO: 6) A diagram is shown in FIG. In order to confirm the insertion of the target sequence, the DNA was treated with restriction enzymes with BamHI and HindIII, and Southern blotting was performed to confirm the target gene transfer. Next, iTL BA1 ES cells (C57BL / 6J × 129 / SvEv) into which the vector was inserted were prepared. The gene-introduced ES cells were confirmed by amplifying the target sequence of the DNA by PCR. The ES cells having the insertion of the prepared vector were inserted by microinjection into blastocysts of C57BL / 6J strain mice. The born offspring were mated with C57BL / 6J FLP mice to remove the neomycin cassette (the above process was commissioned to inGenious Targeting laboratory). A schematic diagram of the WT gene, the KO gene, and the gene (Neo deletion) from which the neomycin cassette has been removed is shown in FIG. Mouse DNA was confirmed by PCR for detection of mice with Neo deletion containing LoxP sequences. Since the WT is 286 bp and the sequence of Neo deletion is 451 bp, homo mice in which only 451 bp was confirmed were used in the experiment. Thereafter, the mice were backcrossed to C57BL / 6J (purchased from Chubu Chemicals Co., Ltd.). Then, using the MAX-BAX program (Charles River), a Cre-loxP sequence-introduced mouse with a completely identical gene background was generated.

Next, Camk2a-Cre mice (Cell 1996; 87: 1917) were mated to the above-mentioned mice with the gene background completely matched. Of the born offspring, FUS ^{fl / fl} / Cre + mice are used as FUS-cKO mice (Cre +) (model animal of the present invention), and FUS ^{fl / +} and FUS ^{fl / fl} / mouse (Cre-) as control mice. used.

In the following test examples, males of 3 to 4 months old were mainly used. In the case of brain parenchymal injection of the AAV vector of Test Example 7, injection was performed at 6 weeks of age, and analysis was performed at 3 to 4 months of age.

Test Example 1 Evaluation of FUS-cKO Mouse (Cre +) of Example 1 The FUS-cKO mouse (Cre +) prepared in Example 1 was 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 and paraffin-embedded samples were prepared, and anti-FUS antibody immunostaining was performed with DAB color in its hippocampal tissue section went. Next, the hippocampus CA1 region was cut off from the section, and the amount of protein expression was confirmed by Western blotting using a sample disrupted by ultrasound.

In a model animal of the present invention (FUS-cKO mouse (Cre +)) expressing Cre and brain-specifically knocking out the FUS gene as compared to a control mouse not expressing Cre (Cre-), in brain tissue, Immunostaining with an anti-FUS antibody resulted in a decrease in the stainability of the FUS protein (see FIG. 7 (A)). The decrease in the staining ability of FUS protein was particularly remarkable in hippocampus CA1 area, dentate gyrus neurons. In addition, as a result of experiments by Western blotting using an anti-FUS antibody, a decrease in the expression level of FUS protein was observed in the hippocampus CA1 region of FUS-cKO mice (Cre +) (see FIG. 7 (B)).
As mentioned above, in the FUS-cKO mouse | mouth (Cre +) produced in Example 1, it confirmed that the knockout of FUS gene was materialized.

Test Example 2 Analysis of Phenotype of FUS-cKO Mouse (Cre +) Using the brain tissue section of 3- to 4-month-old FUS-cKO mouse (Cre +) prepared in Example 1, FD Rapid Golgi Stain kit (FD) Golgi staining was performed by Neuro Technologies, Inc.) to evaluate spine morphology in the postsynaptic region. In addition, the total number of spines per 100 μm of neurites in the tissue section and the percentage of mature spines of mushroom type were evaluated.

As compared with control mice, FUS-cKO mice (Cre +) mice had reduced mushroom-type mature spines indicated by arrows (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 percentage of mature spines in the total number of spines was significantly reduced in FUS-cKO mice (Cre +) (Figure 8C: N = 8 from 4 mice each).
From the above, in the FUS-cKO mice (Cre +) prepared in Example 1, it was revealed that there is a spine maturation disorder, ie, a postsynaptic maturation disorder, which contributes to the reduction in neural activity. Suggested a possibility.

[Test Example 3] Behavioral test (action amount)
Higher-order brain dysfunction in FUS-cKO mice generated in Example 1 was evaluated by a behavioral test used in the study of brain science. The mice used for behavioral tests are backcrossed to the C57BL / 6J strain, and mice with a 99.5% or higher concordance rate by genetic testing (MAX-BAX, Charles River) are bred, 3-4 Evaluation was performed at the age of the month. For the experiment, 10 control mice (Cre-) group and 11 FUS-cKO mice (Cre +) group were used. An open field test for evaluating the amount of activity was conducted. As a test method, a subject mouse was placed in an arena of 60 cm in diameter, and the amount of activity of the mouse for 5 minutes was measured with EthoVision (automatic tracking program, manufactured by Brainscience Idea).

The total migration distance was significantly increased in the FUS-cKO mouse (Cre +) group as compared to the control mouse (Cre-) group (see FIG. 9). When the arena was divided into a circle with a diameter of 40 cm and divided into a central part and an outer periphery, the movement distance of the outer periphery in particular increased.
As described above, the FUS-cKO mouse (Cre +) prepared in Example 1 exhibited a "hyperactivity" phenotype.

[Test Example 4] Behavioral test (anxiety behavior)
Anxiety behavior was evaluated by the elevated plus maze method. The test mice were used as test mice. As a method, one side is a “walled road” with fences on both sides, and the other is a cross path made “open wallless road” at a height of 50 cm above the floor. Body mice were placed and evaluated by evaluating the behavior pattern for 5 minutes.

Compared to the control mice (Cre-) group, the FUS-cKO mice (Cre +) group significantly increased the staying time of the "no-wall way" and decreased significantly in the "walled way" (FIG. 10) (A)). The number of penetrations into the "wallless path" in 5 minutes was also significantly increased in the FUS-cKO mouse (Cre +) group (see FIG. 10 (B)).
As described above, the FUS-cKO mouse (Cre +) prepared in Example 1 exhibited the phenotype of "anxiety".

[Test Example 5] Behavioral test (memory disorder)
Memory impairment was evaluated in the novel object recognition test and the fear conditioning test. The test mice were used as test mice.
First, in the new object recognition test, place two objects in a 30 cm square box and let the subject mouse freely explore for 5 minutes there, and then change only one of them on the next day, and change the new object on the next day. The procedure of the "holding trial" step to assess the cognition of
In addition, the fear conditioning test puts the subject mouse in a 30 cm square box (context conditioning), makes a sound of 85 dB for 15 seconds (sound conditioning), and gives an electric shock of 0.8 mA for the last 5 seconds. Performed times and conditioned by context and sound. Then, put them in the same box on the next day, and without subjecting them to electric shock, observe the subject mouse for "shrinkage response" or observe for 2 minutes and let the fear context conditioning test hear the same sound as conditioning for 4 hours later, sound stimulation A fear sound conditioning test was performed to observe the “stagnation response” for 1 minute each during back and forth and during sound stimulation.

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

  As a summary of the behavioral test, the FUS-cKO mouse (Cre +) group exhibited behavioral abnormalities such as "hyperactivity" and "lack of anxiety", but "memory disorder" was not recognized. This feature is similar to that of early clinical symptoms in FTLD patients (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 LC / MS analysis of a protein extracted from a FUS expression suppression model in mouse primary culture neurons yielded SynGAPα2 as a therapeutic target. Therefore, the amount of SynGAPα2 protein in the brain tissue of FUS-cKO mice was subjected to fluorescence immunostaining using anti-SynGAPα2 antibody on paraffin sections of brain hippocampus CA1 region prepared in the same manner as in Test Example 1. In addition, Western blotting using an anti-SynGAPα2 antibody was performed using a sample obtained by sonicating hippocampus CA1 of the tissue section.

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

[Test Example 7] Supplementation Experiment of SynGAP Aα2 The cDNA sequence of mouse SynGAP A α2 with FLAG attached at the N-terminus was incorporated to create an AAV vector expressing under the CAG promoter, and AAV loaded with this vector was created (FIG. 6A and See Figure 6B, 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 replenished by expressing SynGAP Aα2 to conduct a phenotypic improvement test.
The experiment was performed using 1.5 × 10 13 AAV in the bilateral hippocampus of the 6-week-old FUS-cKO mouse (Cre +) prepared in Example 1 using the stereotaxic device (Angel Two, Leica). This was done by stereotactic injection of 1 μL at a concentration of raised VG / mL. For evaluation, as in Test Example 2, Golgi staining is performed with FD Rapid Golgi Stain kit (manufactured by FD Neuro Technologies) using brain tissue sections of FUS-cKO mice, and the spine morphology in the postsynaptic area is evaluated to achieve maturation. It was done by evaluating the percentage of spines. The experiment was performed using three FUS-cKO mice (Cre +). As a negative control group of the experiment, two FUS-cKO mice (Cre +) and three control mice (Cre-) injected with AAV loaded with an AAV vector incorporating a cDNA sequence of GFP were prepared.

  As a result of observation of Golgi staining images, in FUS-cKO mice (Cre +) supplemented with SynGAP Aα2, a decrease in the percentage of mature spines of mushroom type in the total number of spines observed in FUS-cKO mice (Cre +) It significantly improved (see FIGS. 13A and 13B).

[Test Example 8] Behavioral test (amount of behavior, anxiety behavior)
For the FUS-cKO mouse (Cre +) group supplemented with SynGAP Aα2 in Test Example 7, recovery of higher brain dysfunction was evaluated by a behavioral test. The amount of behavior was evaluated by the open field test in the same manner as in Test Example 3, and the anxiety behavior was evaluated by the elevated plus maze method in the same manner as in Test Example 4. In the same manner as in Test Example 7, FUS-cKO mice (Cre +) and control mice (Cre-) were injected with AAV vector loaded with AAV vector into which the cDNA sequence of GFP was inserted as negative control group of experiment. .

In FUS-cKO mice supplemented with SynGAP Aα2, “hyperactivity”, which is the behavioral abnormality observed in Test Examples 3 to 4, was improved compared to FUS-cKO mice supplemented with GFP as a control. (See Figure 14A. From the left column, N = 14, 11, 11). As for “anxiety”, FUS-cKO mice supplemented with SynGAP Aα2 were significantly improved as 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 it is effective to increase or decrease SynGAP Aα2 by application of a drug, or to improve the phenotype of this mouse as an index for the search for therapeutic agents such as FTLD.
As described above, the FUS-cKO mouse of the present invention was shown to be useful as a model animal for the study of treatments such as FTLD.

The model animal of the present invention can be used as an industry in researching and developing the pathophysiology and treatment of diseases accompanied 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 industrially in searching for a therapeutic agent for diseases associated with synaptic abnormalities. Furthermore, since the therapeutic agent of the present invention is effective for the treatment of a disease accompanied by synaptic abnormality, it can be used for production, sales and the like.

SEQ ID NO: 5: This is the base sequence of the region of exons 1 to 6 of the mouse FUS gene.
SEQ ID NO: 6: The nucleotide sequence of the region of exons 1 to 6 of the mouse FUS gene after insertion of the vector.
SEQ ID NO: 7: A base sequence of an AAV vector into which cDNA of SynGAP A2α is incorporated.

Claims (19)

A model animal exhibiting symptoms of a disease accompanied by a synaptic abnormality, which is produced by specific knockout of 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 with synaptic abnormalities is frontotemporal lobe 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. A method for producing a model animal exhibiting symptoms of a disease accompanied by a synaptic abnormality, comprising: an operation step of specifically knocking out a FUS gene in the brain. The production method according to 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 method according to claim 7 or 8, wherein the disease accompanied with synaptic abnormalities is frontotemporal lobe degeneration (FTLD) or a motor neuron disease. The method according to any one of claims 7 to 9, wherein the animal is a mouse. The method according to any one of claims 7 to 10, wherein the animal is inbred. A therapeutic agent for a disease associated with synapse abnormality, comprising: 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 with synaptic abnormalities is frontotemporal lobe 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 the step of measuring increase or decrease of SynGAPα2 by application of a target drug. The screening method according to claim 14, wherein the disease accompanied with synaptic abnormalities is frontotemporal lobe degeneration (FTLD) or a motor neuron disease. A therapeutic agent for a disease associated with a synaptic abnormality, which comprises as an active ingredient a gene encoding SynGAPα2 or a SynGAPα2 protein, or a drug capable of increasing the expression of SynGAPα2 gene in brain or a drug capable of increasing SynGAPα2 protein. The therapeutic agent according to claim 16, wherein the disease accompanied with synaptic abnormalities is frontotemporal lobe degeneration (FTLD) or a motor neuron disease. A method for treating a disease associated with synaptic abnormality, comprising the step of supplementing or increasing SynGAPα2 to the brain. 19. The method according to claim 18, wherein the disease accompanied by synaptic abnormalities is frontotemporal lobe degeneration (FTLD) or a motor neuron disease.