Model animal of neurodevelopmental disorder

Abstract

To provide a model animal in which the symptom of an autistic spectrum disease is more severe than that of a conventional model mouse of the autistic spectrum disease, or a neurodevelopmental disorder model animal showing other symptoms of the autistic spectrum disease, and to, by using the model animal, contribute to elucidation of the pathology of the neurodevelopmental disorder, search for a new improving agent for the neurodevelopmental disorder, or the like.SOLUTION: Provided is a model animal of neurodevelopmental disorders caused by genetic abnormality having four syntenic regions in other animals to a human chromosomal 15q11-13 region.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an animal model for neurodevelopmental disorders. More specifically, the present invention relates to an animal model for neurodevelopmental disorders caused by genetic abnormalities, which has four syntenic regions in other animals that are related to the human chromosome 15q11-13 region, and a method for producing the same. [Background technology]

[0002] Neurodevelopmental disorders are a group of diseases caused by underdevelopment of the nervous system. Neurodevelopmental syndromes become evident early in development, often before school age, and patients who develop the disorder have difficulty fulfilling expected personal, social, academic or professional functions. Genetic factors are cited as one of the causes of neurodevelopmental disorders, and in particular, the relationship between neurodevelopmental disorders and partial duplication or deletion of chromosomes has been attracting attention.

[0003] Autism spectrum disorder (ASD), also known as autism spectrum disorder, is a diagnostic name classified as a group of neurodevelopmental disorders. ASD is a diagnostic name that encompasses various conditions as a continuum (spectrum), such as symptoms related to communication and language in patients with ASD and stereotyped behaviors in patients with ASD. Criteria for diagnosing ASD include the Diagnostic and Statistical Manual of Mental Disorders (DSM) created by the American Psychiatric Association and the International Statistical Classification of Diseases and Related Health Problems (ICD) created by the World Health Organization (WHO).

[0004] For more accurate diagnosis of neurodevelopmental disorders in clinical practice, elucidation of the pathology of neurodevelopmental disorders is required, and for example, candidate causative genes of neurodevelopmental disorders are being examined using model animals of neurodevelopmental disorders. As such model animals of neurodevelopmental disorders, ASD model mice produced by drug administration such as a method of administering valproic acid, a histone deacetylase inhibitor, to pregnant mice, a method of inactivating the immunity of pregnant mice by administering double-stranded RNA, lipopolysaccharide, or leukemia inhibitory factor (LIF) (Patent Document 1), and a method of administering a GABAA receptor enhancer or inhibitor to the animal during the neurogenesis period of the animal's fetus (Patent Document 2) are known. However, these methods of producing model mice by drug administration have problems in that offspring mice exhibiting autism-like behavior cannot be stably obtained, and the disorders caused in animals by the administered drugs are non-specific, making it difficult to produce model animals with disorders specific to the target disease.

[0005] In addition, the present inventor previously developed an ASD model animal and focused on the duplication of the human chromosome 15q11-13 region, which is one of the causes of human ASD. Then, the inventor succeeded in producing an ASD model mouse (hereinafter also referred to as a "chromosome duplication ASD model mouse") in which a syntenic region (i.e., mouse chromosome 7c region) with respect to the human chromosome 15q11-13 region is duplicated (i.e., having three syntenic regions in chromosome 7) (Non-Patent Document 1, Patent Document 3). The chromosome duplication ASD model mouse exhibits symptoms similar to those of human autism spectrum disorder (ASD-like), and is therefore useful for elucidating the pathology of ASD and screening for drugs to improve ASD. However, since the chromosome duplication ASD model mouse exhibits ASD-like symptoms slowly, a model animal with more severe ASD-like symptoms or a model animal showing other symptoms of ASD was required. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent Publication No. 2004-180655 [Patent Document 2] Patent Publication No. 2016-027792 [Patent Document 3] JP2007-166966A [Non-patent literature]

[0007] [Non-Patent Document 1] Nakatani J. et al., Cell. 137(7):1235-1246 (2009) Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present invention aims to provide a model animal for neurodevelopmental disorders in which ASD-like symptoms are more severe than those of the chromosomal duplication ASD model mouse, or a model animal for neurodevelopmental disorders that exhibits other ASD-like symptoms, and to use the model animal to contribute to elucidating the pathology of neurodevelopmental disorders and discovering new drugs to improve neurodevelopmental disorders. [Means for solving the problem]

[0009] The present inventors came up with the idea that it would be possible to create an ASD model mouse with more severe symptoms by adding one more syntenic region to a conventional chromosome duplication ASD model mouse having three syntenic regions with the human chromosome 15q11-13 region. However, the above syntenic region in the mouse chromosome (i.e., the 7c region) is a very long region with a total length of about 6.3 Mbp, containing multiple genes (a comparison diagram of the human chromosome 15q11-13 region and the mouse chromosome 7c region is shown in Figure 1). Therefore, it was speculated that adding one more syntenic region to the chromosome of a conventional chromosome duplication ASD model mouse would cause embryonic lethality in the mouse. Furthermore, no method is known that can genotype whether the number of syntenic regions with the human chromosome 15q11-13 region is three or four. Due to these circumstances, there have been no reports on mammals having four syntenic regions with the human chromosome 15q11-13 region.

[0010] The present inventors came up with the idea that some mouse fetuses having four syntenic regions with respect to the human chromosome 15q11-13 region may by chance avoid embryonic lethality. Therefore, the present inventors searched for a method capable of genotyping whether the syntenic regions are three or four, and succeeded in developing such a method. As a result of further research, the present inventors actually succeeded in producing mice having four syntenic regions with respect to the human chromosome 15q11-13 region (hereinafter, 4-copy 7c region mice) (a schematic diagram of the chromosome is shown in FIG. 2). It was found that the number of mice having 4 copies of the 7c region obtained was very small compared to Mendel's law, which strongly suggested that many of the mice having 4 copies of the chromosome 7c region were embryonic lethal. The genotype pattern of the resulting mice is shown in FIG. 3. Furthermore, it was found that such mice, unlike conventional chromosome duplication ASD model mice, unexpectedly showed symptoms similar to those of attention deficit hyperactivity disorder (ADHD). The present inventors have conducted further research based on these findings and have completed the present invention.

[0011] That is, the present invention is as follows. [1] This animal model has four syntenic regions in other animals that are syntenic to the human chromosome 15q11-13 region, and is a model animal for a group of neurodevelopmental disorders caused by genetic abnormalities. [2] The animal described in [1], wherein the neurodevelopmental disorder is at least one selected from the group consisting of autism spectrum disorder, intellectual development disorder, attention deficit hyperactivity disorder, and stereotypic movement disorder. [3] The animal described in [1] or [2], wherein at least one of the neurodevelopmental disorders is an autism spectrum disorder. [4] An animal described in [3] that exhibits symptoms similar to attention deficit hyperactivity disorder. [5] The animal according to any one of [1] to [4], wherein the animal is a mammal. [6] The animal described in [5], wherein the mammal is a rodent. [7] (1) genotyping the offspring obtained by mating animals having three syntenic regions in another animal with respect to the human chromosome 15q11-13 region; and (2) selecting an offspring confirmed to have four syntenic regions in step (1) as an animal model for neurodevelopmental disorders caused by genetic abnormalities; The present invention relates to a method for producing a model animal for a neurodevelopmental disorder, the method comprising the steps of: [8] The method according to [7], wherein the neurodevelopmental disorder is selected from the group consisting of autism spectrum disorder, intellectual development disorder, attention deficit hyperactivity disorder, and stereotypic movement disorder. [9] The method according to [7] or [8], wherein the neurodevelopmental disorder is an autism spectrum disorder.

[10] The method according to any one of [7] to [9], wherein the animal is a mammal.

[11] The method according to

[10] , wherein the mammal is a rodent.

[12] The method according to any one of [7] to

[11] , wherein the genotyping is carried out by comparing the intensities of bands obtained by Southern blotting.

[13] (1) A step of administering a test substance to an animal according to any one of [1] to [6], and (2) A step of selecting the test substance as a candidate substance for improving neurodevelopmental disorders when the symptoms of neurodevelopmental disorders caused by genetic abnormalities are improved compared to a control group not administered the test substance or the animals before administration of the test substance. A method for screening for a drug for improving a neurodevelopmental disorder, comprising:

[14] (1) A step of administering a test substance to an animal according to any one of [1] to [6], and (2) selecting the test substance as a candidate for a drug for improving autism spectrum disorder or attention deficit hyperactivity disorder when the autism spectrum disorder-like symptoms or attention deficit hyperactivity disorder-like symptoms are improved compared to a control group not administered the test substance or the mammal before administration of the test substance; A method for screening for a drug for improving autism spectrum disorder or attention deficit hyperactivity disorder, comprising:

[15] The method according to

[14] , wherein the autism spectrum disorder-like symptoms or attention deficit hyperactivity disorder-like symptoms are at least any one of impaired social interaction, increased anxiety levels, tendencies towards fixation, tendencies towards inattention, and increased hyperactivity / impulsivity compared to wild-type animals. Effect of the Invention

[0012] According to the present invention, a model animal for neurodevelopmental disorders is provided, and by using this model animal it is possible to confirm the effects of existing drugs for improving neurodevelopmental disorders such as ASD, and also to aid in the development of drugs and appropriate drug interventions that improve the symptoms of neurodevelopmental disorders such as ASD. [Brief description of the drawings]

[0013] [Figure 1] Human chromosome 15 region 11-13 (left) and its conserved region, mouse chromosome 7 region c (right). [Diagram 2] Schematic diagram of copy number increase. Two-copy mouse (wild type: left), three-copy mouse (previously reported ASD model mouse: middle), four-copy mouse (newly invented ASD model mouse: right). [Diagram 3] How to create a four-copy ASD model mouse. When a three-copy male and female mouse are crossed, two-copy, three-copy, and four-copy mice are born simultaneously as littermates. [Figure 4] The first row shows a photograph of the actual Southern blot (first row), and the quantification results of the bands for each genotype (second row: wild-type control (Ctrl), third row: 3-copy mouse (Dp), fourth row: 4-copy mouse (DpDp)). [Diagram 5]MRI of the chest immediately after birth. Comparison of a 2-copy mouse (left) and a 4-copy mouse (right). The 2-copy mouse has air in its lungs (black spots), and breathing has begun at birth, but this has not started in the 4-copy mouse. [Figure 6] The results of survival rate for mice of each genotype are shown (dotted line: wild-type mice, thin line: 3-copy mice, thick line: 4-copy mice). From the left, the survival rate at 3 weeks, the change in survival rate when 3 weeks is set as 1, the weight change of males from 3 to 10 weeks, the weight change of males from 10 weeks to 15 months, the weight change of females from 3 to 10 weeks, and the weight change of females from 10 weeks to 15 months are shown. [Figure 7] The top left shows the results of a three-chamber social behavior test, showing the time that two mice (one of which is the model and the other is wild type) engaged in social behavior. Immediately after the two mice met, the wild type (dotted line) was hesitant to the other mouse's presence and its social behavior was reduced, but the 4-copy mouse (shown by the thick line) was observed to approach the other mouse without hesitation. The top middle shows the results of placing a mouse in a square chamber measuring 42.5 cm on each side and tracing its behavior for 10 minutes. The vertical axis shows the percentage of mice that stayed in the center. The top right shows a prepulse inhibition test, which examines how much the startle level of the sound decreases when a sound is played after a sound of 70 or 74 decibels is played. The bottom row shows the results of a fear conditioning test, where the left shows the increase in startle level when sound and electrical stimulation are given, the middle shows the startle level (without electrical or sound stimulation) when the mouse is placed in the same chamber 24 hours later, and the right shows the results of placing the mouse in a chamber of a different shape and observing the startle level. In the case of the right, the sound is played from 4 minutes. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] 1. Disease models and their preparation methods As shown in the examples below, animals with four syntenic regions (hereinafter, sometimes referred to as "15q11-13 syntenic regions") in other animals with respect to the human chromosome 15q11-13 region, unlike animals with three of the regions (i.e., copy number variation (CNV)), have a high mortality rate and show not only symptoms similar to autistic spectrum disorder (ASD) (ASD-like) (also referred to as "symptoms of ASD"), but also symptoms similar to attention deficit hyperactivity disorder (ADHD) (ADHD-like) (also referred to as "symptoms of ADHD"). Thus, animals with four 15q11-13 syntenic regions can be used more preferably as models of neurodevelopmental disorders (preferably ASD model animals) than conventional model animals. Thus, the present invention provides an animal model for neurodevelopmental disorders (hereinafter sometimes referred to as "the animal model of the present invention") having four 15q11-13 synteny regions.

[0015] As used herein, the term "model animal for neurodevelopmental disorders" refers to an animal that exhibits a phenotype similar to that of a human neurodevelopmental disorder due to genetic abnormality. The neurodevelopmental disorders that serve as models in the model animal of the present invention are not particularly limited, and examples thereof include intellectual developmental disorders, developmental speech or language disorders, ASD, developmental learning disorders, developmental coordination disorder, ADHD, stereotypic movement disorders, and other clearly defined neurodevelopmental disorders.

[0016] Intellectual development disorders include, for example, X-linked mental retardation, X-linked mental retardation syndromes, autosomal recessive intellectual developmental disorders, autosomal dominant intellectual developmental disorders, FRA12A mental retardation, autosomal recessive mental retardation syndromes, syndromic intellectual developmental disorders, etc. Also included in the group of neurodevelopmental disorders are conditions with intellectual developmental disorders as an associated clinical feature. Conditions with intellectual developmental disability as an associated clinical feature include, for example, Angelman syndrome, Prader-Willi syndrome, Rett syndrome, Schneider-Robinson syndrome, hypergamy, Cohen syndrome, ATR-X syndrome, ZTTK syndrome, Rempenning syndrome, Christianson syndrome, Stocco dos Santos X-linked mental retardation syndrome, Partington syndrome, childhood hypotonia with psychomotor retardation and characteristic facies, Laurence-Moon syndrome, PEHO syndrome, Schaaf-Yang syndrome, Skraban-Deardorff syndrome, PEHO-like syndrome, intellectual developmental disability with short stature, hypergamy with epilepsy, Ververi-Brady syndrome, Helsmoortel-van der Aa syndrome, hypotonia-ataxia and growth retardation syndrome, growth retardation, intellectual developmental disability, hypotonia and liver damage, Hao-Fountain syndrome, etc. Among them, the model neurodevelopmental disorder is preferably a disease caused by duplication, deletion and / or insertion of a part of a chromosome, and more preferably a disease in which the part of the chromosome (i.e., the region of genetic abnormality) is the human chromosome 15q11-13 region or its syntenic region. Unless otherwise specified, the definition, classification, name, etc. of the disease shall follow the provisions of ICD-11.

[0017] ASD is a continuum of pervasive developmental disorders characterized by (A) impaired interpersonal communication, (B) impaired interpersonal interaction, and (C) restricted, repetitive, and stereotyped patterns of behavior, interests, and activities. ASD includes, but is not limited to, (so-called traditional) autistic disorder, Asperger syndrome, Rett syndrome, childhood disintegrative disorder, and pervasive developmental disorder not otherwise specified. ASD is typically diagnosed according to the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V) published by the American Psychiatric Association. When the model neurodevelopmental disorder group is ASD (i.e., when it is an ASD model animal), the model animal may have a CNV in its chromosome and thus exhibit a phenotype similar to ASD in humans, such as impaired social interaction, elevated anxiety, and / or a tendency to fixation, as compared with wild-type animals.

[0018] In the case of humans, there are many cases in which several neurodevelopmental disorders are present at the same time. For example, cases are known in which a patient has ADHD characteristics, but also has ASD and / or specific learning disorder, and in almost all patients with chromosomal abnormalities in the 15q11-13 region, retardation of intellectual development is observed. Therefore, the neurodevelopmental disorder model animal of the present invention may also have the neurodevelopmental disorder at the same time (in other words, a plurality of neurodevelopmental disorders may be present). In addition, stereotypic behavior is observed in animals with three 15q11-13 synteny regions (Non-Patent Document 1). Therefore, the model animal of the present invention may be a model animal of at least one disease selected from the group consisting of ASD, intellectual developmental disorder, ADHD, and stereotypic movement disorder. In addition, the neurodevelopmental disorder model animal of the present invention is preferably a model having ASD-like symptoms, and may also have ADHD-like symptoms.

[0019] In addition, as shown in the examples below, the ASD model animal of the present invention has four 15q11-13 synteny regions (i.e., has excess CNVs) compared to conventional ASD model animals, and therefore may exhibit a high (90% or more) mortality rate. In addition, clear low body weight may be observed in surviving individuals. The high mortality rate may also be an important point in the study of neurodevelopmental disorders such as ASD. In recent years, the proportion of children diagnosed with neurodevelopmental disorders has been increasing. The exact reason is unknown, and the elderly childbirth age and the increased awareness of neurodevelopmental disorders have also been pointed out. However, when neurodevelopmental disorders are caused by genetic abnormalities, the patient is thought to have some abnormality not only in the central nervous system but also in the circulatory system and respiratory system, so that the prognosis of life may be poor in the first place before the onset of neurodevelopmental disorders. With the improvement of modern medical technology, even if abnormalities occur in the circulatory system or respiratory system, the chances of life being saved by neonatal emergency care are high, and it is speculated that this will contribute to an increase in the number of patients with neurodevelopmental disorders. In addition, abnormalities in the circulatory and respiratory systems can lead to poor growth in newborns, and this may be related to the high rate of low birth weight infants who are later diagnosed with neurodevelopmental disorders. In other words, the model animals of the present invention may exhibit characteristics closer to the pathological conditions of actual human neurodevelopmental disorders.

[0020] The animal species from which the model animal of the present invention is derived is not particularly limited as long as it is an animal other than a human, and includes mammals, birds, reptiles, amphibians, fish, etc., but is preferably a mammal. Examples of such mammals include rodents (e.g., mice, rats, guinea pigs, gerbils, hamsters, etc.), primates (e.g., rhesus monkeys, cynomolgus monkeys, Japanese monkeys, chimpanzees, etc.), laboratory animals such as ferrets, rabbits, dogs, and minipigs, pet animals such as dogs and cats, and livestock such as cows, horses, pigs, and sheep. Among these, rodents are preferred because of the established strains (for example, pure mouse strains include the C57BL / 6 strain, the BALB / c strain, and the DBA2 strain; hybrid mouse strains include the B6C3F1 strain, the BDF1 strain, the B6D2F1 strain, and the ICR strain; and rat strains include Wistar and SD) and ease of handling. Additionally, non-human primates are also preferred because they are genetically closer to humans.

[0021] The human chromosome 15q11-13 region is an imprinting region, and the gene expression patterns of paternal and maternal chromosomes are determined by the presence or absence of DNA methylation at the imprinting center. Therefore, the gene expression levels of paternally derived duplications and maternally derived duplications differ due to imprinting. Representative paternally expressed genes contained in the human chromosome 15q11-13 region include MKRN3, MAGEL2, NDN, and SNRPN, maternally expressed genes include UBE3A and ATP10A, and bisexually expressed genes include GABRB3, GABRA5, GABRG3, OCA2, and HERC2. Therefore, in this specification, the "syntenic region in other animals with respect to the human chromosome 15q11-13 region" may be a region of the chromosome of another animal that contains at least orthologs of all of the above genes. Examples of such synteny regions include the chromosome 7c region in mice, the chromosome 17 region in rabbits, the chromosome 6 region in zebrafish, and the chromosome 7 region in macaques (e.g., rhesus monkeys, cynomolgus monkeys, Japanese macaques, etc.), but are not limited to these animal species.

[0022] In the model animal of the present invention, the 15q11-13 synteny region has two exogenous 15q11-13 synteny regions (hereinafter, sometimes referred to as "exogenous 15q11-13 synteny regions") in addition to the two 15q11-13 synteny regions that wild-type animals naturally have (hereinafter, sometimes referred to as "endogenous 15q11-13 synteny regions"). Each exogenous 15q11-13 synteny region may be present in any region of any chromosome (i.e., it may be a chromosome other than the chromosome in which the endogenous 15q11-13 synteny region exists). That is, each exogenous 15q11-13 synteny region may be present on a chromosome other than the chromosome where the endogenous 15q11-13 synteny region is present, and when it is present on the same chromosome as the endogenous 15q11-13 synteny region, it may be present adjacent to the endogenous 15q11-13 synteny region or may be present at a distant position. In addition, both exogenous 15q11-13 synteny regions may be present on the same chromosome (paternal homologous chromosome or maternal homologous chromosome). Preferably, at least one exogenous 15q11-13 synteny region is present on the same chromosome as the endogenous 15q11-13 synteny region, and more preferably adjacent to the endogenous 15q11-13 synteny region. In particular, both exogenous 15q11-13 synteny regions are present on the same chromosome as each endogenous 15q11-13 synteny region, and more preferably, both exogenous 15q11-13 synteny regions are adjacent to each endogenous 15q11-13 synteny region (i.e., two 15q11-13 synteny regions are present adjacent (in tandem) on the paternal and maternal homologous chromosomes, respectively).

[0023] The model animal of the present invention can be obtained, for example, by mating animals having three conventional 15q11-13 synteny regions (hereinafter, sometimes referred to as "conventional model animals for neurodevelopmental disorders"), although the survival rate is low. Therefore, in another embodiment of the present invention, (1) Genotyping the offspring obtained by mating conventional neurodevelopmental disorder model animals; and (2) selecting an offspring confirmed to have four syntenic regions in step (1) as an animal model for neurodevelopmental disorders caused by genetic abnormalities; Also provided is a method for producing a model animal for a neurodevelopmental disorder (hereinafter sometimes referred to as a "method for producing the model animal of the present invention") comprising the steps of:

[0024] The conventional model animal of neurodevelopmental disorder used in step (1) of the method for producing the model animal of the present invention may be an existing one, or may be produced by the method described in Non-Patent Document 1 or Patent Document 3. Specifically, for example, it can be produced by the following procedure. (i) A targeting vector is constructed in which 5'Hprt-loxP and 3'Hprt-loxP are placed in the outer introns of the Mkrn3 and Herc2 genes, respectively, present in the 15q11-13 syntenic region. (ii) The constructed targeting vector containing 5'Hprt-loxP is introduced into pluripotent stem cells (e.g., induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells)) by electroporation or other methods, and the introduced cells are selected based on their drug resistance. Insertion into the desired position by homologous recombination may be confirmed by Southern blotting (Southern method). (iii) The constructed targeting vector containing 3'Hprt-loxP is introduced into the pluripotent stem cells selected in step (ii), and 3'Hprt-loxP is similarly confirmed by drug resistance and Southern blotting. (iv) The pluripotent stem cells selected in step (iii) are treated with Cre recombinase to induce duplication of the 15q11-13 synteny region, and pluripotent stem cells with a duplicated target region are identified by Southern blotting. (v) The pluripotent stem cells for which duplication has been confirmed are introduced into mammalian blastocysts, which are then transplanted into recipients (foster parents). After a certain period of time, offspring are obtained by Caesarean section. The introduction of the chromosomal duplication into the germline cells of the offspring is confirmed (Southern blotting, RT-PCR, FISH, BAC-CGH).

[0025] Alternatively, it can be prepared by genome editing using ZFN, TALEN, CRISPR / Cas9 system, etc. For such a method, see, for example, Tamada K. et al., Nat Commun. 12(1):4056 (2021).

[0026] The offspring used in step (1) of the method for producing the model animal of the present invention may be offspring obtained by new mating, or offspring that already exists. Thus, the method for producing the model animal of the present invention may include step (0) of obtaining offspring obtained by mating conventional model animals for neurodevelopmental disorder with each other, or a step of preparing the offspring. Steps (0) to (2) of the method for producing the model animal of the present invention may all be performed by the same person, or some steps may be performed by different people. The offspring obtained or prepared in step (0) has a genotype of either one of two 15q11-13 synteny regions (i.e., wild-type animals), three 15q11-13 synteny regions (i.e., conventional model animals for neurodevelopmental disorder), or four 15q11-13 synteny regions (i.e., the model animal of the present invention). Therefore, the model animal of the present invention can be obtained by genotyping the above-mentioned offspring and selecting an animal confirmed to have four 15q11-13 synteny regions as a model for neurodevelopmental disorder.

[0027] The genotyping in the above step (1) can be performed, for example, by comparing the density (intensity) of the band obtained by Southern blotting. More specifically, for example, it can be performed by the following method. Genomic DNA is extracted from the tail of an animal, cleaved with a restriction enzyme (e.g., SacI, etc.), and electrophoresed on an agarose gel. Transferred to a nylon membrane and hybridized with a probe capable of distinguishing the exogenous 15q11-13 synteny region from the endogenous 15q11-13 synteny region by band length (in the examples described below, a probe was used that detects a band of a 8.9 kb DNA fragment in the case of a wild-type allele, and a band of 8.9 kb and 32 kb DNA fragments in the case of an allele having two synteny regions (Dp allele)). After washing, the intensity of the obtained Southern blotting band is quantified using an imaging plate. Specifically, when the probe and restriction enzyme used in the examples described later are used, a band of a 8.9 kb DNA fragment is detected in the case of a mouse having only a wild-type allele, and a band of 8.9 kb and a band of 32 kb DNA fragment are detected in the case of a mouse having a wild-type allele and a Dp allele (Dp mouse) and a mouse having only a Dp allele (DpDp mouse). In the case of a DpDp mouse, the amount of a 32 kb DNA fragment obtained is about twice that of a Dp mouse, so that it is possible to distinguish between a Dp mouse and a DpDp mouse by comparing the density of the bands.

[0028] Animals that have been confirmed by genotyping to have four 15q11-13 synteny regions will, in theory, show symptoms similar to those of neurodevelopmental disorders such as ASD (hereinafter referred to as "neurodevelopmental disorder-like symptoms" or alternatively "neurodevelopmental disorder symptoms"). However, whether or not they actually show neurodevelopmental disorder-like symptoms in humans can be evaluated using open field tests, social behavior tests, prepulse tests (tests to observe startle responses to sound), fear conditioning tests (tests to observe fear learning by administering electric shocks, etc.), etc.

[0029] Furthermore, since the model animal of the present invention has such chromosomal abnormalities introduced into the germline cells, a model of a neurodevelopmental disorder can be obtained by mating the models of the present invention with each other, but in this case, genotyping is no longer necessary. Therefore, in yet another aspect of the present invention, a method for producing a model animal of a neurodevelopmental disorder caused by genetic abnormalities is also provided, which includes a step of mating animals having four 15q11-13 synteny regions with each other to obtain an offspring animal. The above-mentioned methods for producing a model animal are illustrative and are not limited to these methods.

[0030] 2. Uses of disease models Since the model animal of the present invention can show symptoms similar to those of human neurodevelopmental disorders, the model animal of the present invention can be used to screen for drugs that improve neurodevelopmental disorders, and in this respect, the model animal of the present invention is also useful. Furthermore, when the model animal of the present invention is an ASD model animal, the behavioral pattern of the animal can also show a tendency toward hyperactivity, i.e., an ADHD-like tendency, and therefore the model animal of the present invention can also be used, for example, to screen for drugs that improve ASD or ADHD. Furthermore, the model animal of the present invention can also be used to elucidate the onset mechanism and pathology of neurodevelopmental disorders such as ASD or ADHD. Thus, in yet another aspect, the present invention provides (1) administering a test substance to the model animal of the present invention; and (2) There is also provided a screening method for a drug for improving neurodevelopmental disorders (hereinafter sometimes referred to as the "screening method of the present invention"), which includes a step of selecting the test substance as a candidate drug for improving neurodevelopmental disorders when neurodevelopmental disorder-like symptoms improve compared to a control group not administered the test substance or the animals before administration of the test substance.

[0031] In addition, in one embodiment of the screening method of the present invention, (1') administering a test substance to the model animal of the present invention; and (2') A step of selecting the test substance as a candidate for an ASD or ADHD improving drug (including both ASD and ADHD improving drugs) when the ASD-like symptoms or ADHD-like symptoms have improved (including when both ASD and ADHD-like symptoms have improved) compared to a control group not administered the test substance or the mammal before administration of the test substance. The present invention provides a method for screening for a drug for improving ASD or ADHD, comprising:

[0032] In the screening method of the present invention, the test substance can be administered to animals orally or parenterally (e.g., subcutaneous injection, intramuscular injection, local injection (e.g., intraventricular administration), intraperitoneal administration, etc.), but parenteral administration is preferable. The test substance may be mixed with a medicamentically acceptable carrier in a conventional manner to prepare a parenteral preparation such as an injection, suspension, or drip infusion. Examples of medicamentically acceptable carriers that can be contained in the parenteral preparation include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.).

[0033] Preparations suitable for parenteral administration include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, isotonicity agents, etc. Also include aqueous and non-aqueous sterile suspensions, which may contain suspending agents, solubilizers, thickeners, stabilizers, preservatives, etc. The preparations can be sealed in unit doses or multiple doses in containers such as ampoules or vials. Also, the active ingredient and a pharma- ceutical acceptable carrier can be freeze-dried and stored in a state that can be dissolved or suspended in a suitable sterile vehicle immediately before use.

[0034] In the screening method of the present invention, "not administering the test substance" includes, for example, administering a formulation containing the same components except for the test substance when the test substance is administered in the form of a formulation, administering a formulation containing a substance other than the test substance that is known to have no effect of improving neurodevelopmental disorders, or administering none of these agents. In addition, as a model animal to which the test substance is not administered and used as a control in step (2), for example, a separate model animal (e.g., a littermate) prepared by a similar method may be used. As a result of the comparison in step (2), a test substance that improves the symptoms of the neurodevelopmental disorder group can be selected as a candidate substance for an improving drug for the neurodevelopmental disorder group.

[0035] The symptoms of the neurodevelopmental disorder group, when the neurodevelopmental disorder group is ASD, include impaired social interaction, increased anxiety, tendency to fixate, tendency to be inattentive, increased hyperactivity and impulsivity, etc., compared to wild-type animals. If the test substance improves at least one of the symptoms of the neurodevelopmental disorder group, it will be a candidate for a drug for improving the neurodevelopmental disorder group. Whether the symptoms of the neurodevelopmental disorder group have improved can be evaluated by, for example, an open field test, a social behavior test, a prepulse test, a fear conditioning test, etc., and, for example, when the scores of these tests are significantly high or low, it can be evaluated that the symptoms of the neurodevelopmental disorder group have improved. When a promising drug is selected as a candidate drug, the degree of improvement of symptoms when administered to the model animal of the present invention at various doses and routes can be evaluated to determine the optimal dose and administration route of the drug. The optimal dose of this neurodevelopmental disorder group model animal in humans can be calculated by a conventionally known method.

[0036] Test substances used in the screening method of the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0037] The test substance can also be obtained using any of the many approaches in combinatorial library methods known in the art, including (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound" library methods, and (4) synthetic library methods using affinity chromatography selection. The biological library method using affinity chromatography selection is limited to peptide libraries, while the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be produced in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9) or phages (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6; Cwirla et al. (1999) Science 249:404-6). al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

[0038] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these in any way. EXAMPLES

[0039] Example 1: Creation of an excess CNV model To generate CNV model mice, chromosome recombination was performed on mouse ES cells using the Cre / loxP system, and mice were generated using the ES cells (Non-Patent Document 1). These three-copy mice were crossed with C57BL / 6-Tyr-cBrd / cBrd for at least 10 generations. Male and female three-copy mice were crossed, and genotyping was performed by quantifying the band intensity of Southern blot.

[0040] The results of Southern blot are shown in Figure 4. The first row in Figure 4 is a photograph of an actual Southern blot, with electrophoresis from top to bottom. In lane 1 (Ctrl), only an 8.9 kb band was detected in the control, but this is because the wild type does not show an 8.9 kb band when detected with the white square radioactive probe as shown in the left figure in the second row. On the other hand, in lane 5 (Dp), two bands of 8.9 bp and 32 bp were detected. As can be seen in the third row, in Dp, there is a recombined Dp allele, and there are two detection regions in a single allele, and the band lengths are designed to be different, so bands are detected at 8.9 bp and 32 bp. However, since the 8.9 bp region is wider, the band quantification is approximately 2:1 (graph on the right of the third row). In 4-copy mice, the number of 8.9 bp and 32 bp regions is equal (left figure in the fourth row), so although the number of bands is the same at two, the band intensities are different. Quantitative analysis revealed a 1:1 ratio (fourth row, right graph). From the above, it was confirmed that a four-copy mouse can be obtained by crossing three-copy mice together.

[0041] Example 2: MRI analysis Immediately after birth, mice (P0) were collected, fixed in paraformaldehyde, and thoracic images were taken using an Agilent Technology 7.0T MRI scanner with the following imaging conditions: 15mm x 15mm field of view, 0.50mm slice thickness, 512 x 512 resolution, 4200ms echo time (TE) and 36 ms repetition time (TR).

[0042] The results are shown in Figure 5. Figure 5 shows that the 2-copy mice had air in their lungs (black spots) and pulmonary respiration had begun at birth, but the 4-copy mice had not. It was also suggested that pulmonary respiratory failure at birth in the 4-copy mice was associated with a high mortality rate.

[0043] Example 3: Mortality and birth weight measurements The mortality rate during infancy was determined by measuring body weight and counting the number of pigs from 3 to 10 weeks of age, and calculating the weight change and mortality rate.

[0044] The results are shown in Figure 6. Figure 6 shows that the 4-copy mice had a lower weight gain and a higher mortality rate than the 2-copy mice.

[0045] Example 4: Behavioral evaluation of excess CNV models The evaluation was carried out using a three-chamber social behavior test apparatus and an open field test apparatus (Ohara Medical Industries Co., Ltd.).

[0046] Three-chamber social behavior test The chamber was divided into three diverticula, each of which could be accessed by each other. A mouse was placed in a cage that was immobile, and the test mouse was allowed to move freely around it for 10 minutes. The results are shown in the upper left of Figure 7. The data in the upper left of Figure 7 show how much social behavior the two mice exhibited during each minute. This test revealed that the four-copy mouse actively engaged in social behavior from the very beginning of the test.

[0047] Open field test Test mice were placed in a square chamber measuring 42.5 cm on each side for 10 minutes and allowed to move freely. The results are shown in the top center of Figure 7. The data in the top center of Figure 7 shows how long the test mice spent in the center of the chamber during the 10 minutes. Generally, the shorter this time, the stronger the fear. In this test, a decrease in the time spent in the chamber was observed in the 3-copy mice, but this tendency was not observed in the 4-copy mice, and in fact the value was the same as that of the 2-copy mice (wild type).

[0048] Prepulse Inhibition (PPI) Test PPI is a phenomenon in which, when a mouse hears a sound after hearing one, it unconsciously guards (prepares) for the second sound, making it sound quieter. It is generally known that such PPI is reduced in patients with schizophrenia. The results are shown in the upper right of Figure 7. This test showed that PPI was significantly reduced in the 4-copy mice compared to other mice, showing the same tendency as in patients with schizophrenia.

[0049] Fear conditioning test In this test, mice were placed in a cubic chamber, and conditioned by sound and electric shock. The results are shown in the bottom of Figure 7. In the bottom of Figure 7, the graphs for electric shock (left) and sound (right) tend to rise to the right, which means that the freezing ratio is increasing. The graph in the middle shows the results of the context test, in which the mouse was simply placed in the same chamber the next day. At this time, the mouse remembers the fear from the previous day, so it freezes just by being placed in the chamber. The graph on the right shows the results of the sound test, in which the mouse was placed in a chamber of a different shape, and sound was played 4 minutes later (horizontal line). These three steps showed that the freezing ratio of the 4-copy mouse was reduced. In other words, it was shown that the 4-copy mouse does not perceive fear as fear.

[0050] This example shows that the 4-copy mice, unlike the conventional 3-copy mice, also exhibit symptoms similar to attention-deficit hyperactivity disorder (ADHD). [Industrial Applicability]

[0051] The model animal of the present invention can be used to confirm or screen the effects of drugs for improving neurodevelopmental disorders, and can be used as a highly accurate model animal for researching the mechanisms of onset of neurodevelopmental disorders, making it particularly useful in the medical field.

Claims

1. This animal model has four syntenic regions in other animals that are syntenic to the human chromosome 15q11-13 region, and is a model animal for a group of neurodevelopmental disorders caused by genetic abnormalities.

2. The animal according to claim 1, wherein the neurodevelopmental disorder is at least one selected from the group consisting of autism spectrum disorder, intellectual development disorder, attention deficit hyperactivity disorder, and stereotypic movement disorder.

3. The animal of claim 1 , wherein at least one of the neurodevelopmental disorders is an autism spectrum disorder.

4. The animal of claim 3, which exhibits symptoms of attention deficit hyperactivity disorder.

5. The animal of claim 1 , wherein the animal is a mammal.

6. The animal of claim 5 , wherein the mammal is a rodent.

7. (1) Genotyping the offspring obtained by mating animals having three syntenic regions in another animal with respect to the human chromosome 15q11-13 region; and (2) selecting the offspring confirmed to have four syntenic regions in step (1) as a model animal for neurodevelopmental disorders caused by genetic abnormalities; A method for producing a model animal for a neurodevelopmental disorder, comprising:

8. The method according to claim 7, wherein the neurodevelopmental disorder is selected from the group consisting of autism spectrum disorder, intellectual development disorder, attention deficit hyperactivity disorder, and stereotypic movement disorder.

9. The method of claim 7, wherein the neurodevelopmental disorder is an autism spectrum disorder.

10. The method of claim 7, wherein the animal is a mammal.

11. The method of claim 10, wherein the mammal is a rodent.

12. The method according to any one of claims 7 to 11, wherein the genotyping is carried out by comparing the intensities of bands obtained by Southern blotting.

13. (1) administering a test substance to the animal according to any one of claims 1 to 6; and (2) A step of selecting the test substance as a candidate substance for improving neurodevelopmental disorders when the symptoms of neurodevelopmental disorders caused by genetic abnormalities are improved compared to a control group not administered the test substance or the animals before administration of the test substance. A method for screening drugs for improving neurodevelopmental disorders, comprising:

14. (1) administering a test substance to the animal according to any one of claims 1 to 6; and (2) A step of selecting the test substance as a candidate substance for a drug for improving autism spectrum disorder or attention deficit hyperactivity disorder when the autism spectrum disorder-like symptoms or attention deficit hyperactivity disorder-like symptoms are improved compared to a control group not administered the test substance or the mammal before administration of the test substance. A method for screening for a drug for improving autism spectrum disorder or attention deficit hyperactivity disorder, comprising:

15. The method of claim 14, wherein the autism spectrum disorder-like symptoms or attention deficit hyperactivity disorder-like symptoms are at least one of impaired social interaction, increased anxiety, tendency to fixate, tendency to be inattentive, and increased hyperactivity / impulsivity compared to wild-type animals.

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