WO2019163166A1 - Genetically modified non-human animal, and method for screening for therapeutic agent or prophylactic agent for spinocerebellar degeneration - Google Patents

Abstract

A genetically modified non-human animal is an animal in which, in a gene encoding T-type calcium channel Cav3.1 in one or both of the homologous chromosomes thereof, a triplet encoding arginine that is the seventh amino acid as counted from the N-terminal of an amino acid sequence represented by Arg-Ile-Met-Arg-Val-Leu-Arg-Ile-Ala-Arg as observed from the N-terminal to the C-terminal in S4 domain in repeat IV in the Cav3.1 is substituted by a triplet encoding an amino acid other than arginine.

Description

Screening method for therapeutic or prophylactic agent for genetically modified non-human animals and spinocerebellar degeneration

The present invention relates to a screening method for a therapeutic or prophylactic agent for genetically modified non-human animals and spinocerebellar degeneration.

Spinocerebellar degeneration (SCD) is a neurodegenerative disease that causes ataxia. SCD is an intractable disease for which no fundamental treatment has been established. SCD is known to have genetic diversity. To date, causative genes have been identified in various disease types that are autosomal dominant.

For example, the ATXN1 gene has been identified as a causal gene of spinocerebellar ataxia (SCA) type 1, which is one of the types of SCD. SCA1 type is caused by a CAG repeat sequence that is abnormally extended in the ATXN1 gene. Non-Patent Document 1 discloses a transgenic mouse into which an ATXN1 gene having 30 repeat sequences and 82 repeat sequences is artificially introduced. The transgenic mice showed ataxia symptoms and Purkinje cell degeneration.

Patent Document 1 discloses a transgenic mouse in which a vector containing a target gene linked to a mouse embryonic stem cell virus promoter is introduced into the germ line. In the transgenic mouse, the target gene is specifically overexpressed in cerebellar Purkinje cells, brain stem and olfactory bulb. Patent Document 1 describes that a PKCG gene, which is a causal gene of SCA14 type, was overexpressed in Purkinje cells as a target gene.

Although the elucidation of the causative gene of SCD has been promoted as described above, in about 30% of cases in which dominant inheritance is estimated, the causative gene is unknown and there are still many unexplained parts. There is a need for more detailed elucidation of the pathology of SCD.

Recently, as disclosed in Non-Patent Document 2, a CACNA1G gene was identified as a new causative gene by genetic analysis of a large family of Japanese autosomal dominant hereditary SCA. The CACNA1G gene encodes Cav3.1, which is one of low-potential action-type voltage-dependent calcium channels (T-type voltage-dependent calcium channel). As shown in FIG. 1, Cav3.1 has a structure (repeats I to IV) in which six transmembrane sites (S1 to S6) are repeated four times. S4 of each of repeats I to IV has a role as a potential sensor. According to Non-Patent Document 2, the arginine residue present in S4 of repeat IV is replaced with histidine due to mutation in the CACNA1G gene (R1716H). The mutation of the CACNA1G gene identified in Non-Patent Document 2 has also been found in other SCA families as reported in Non-Patent Document 3.

JP 2011-254701 A

In the transgenic mice disclosed in Non-Patent Document 1 and Patent Document 1, an artificially introduced gene is inserted at a position on the genome different from the endogenous causative gene and is overexpressed. For this reason, the position on the genome where the gene is inserted and the excessive expression level may affect the mouse. Furthermore, in transgenic mice, the copy number of the introduced gene may also affect the mouse. For this reason, even if a gene is introduced, there is a concern that human pathology is not sufficiently reproduced in mice.

In addition, no model animal having the above-described mutation in the CACNA1G gene has been prepared so far. A model animal having a similar mutation in the CACNA1G gene is also required in order to elucidate the pathological condition of SCA resulting from the CACNA1G gene mutation and to establish a therapeutic method.

The present invention has been made in view of the above circumstances, and includes a genetically modified non-human animal that can faithfully reproduce human pathology, and a therapeutic or preventive agent for spinocerebellar degeneration using the genetically modified non-human animal. An object is to provide a screening method.

The genetically modified non-human animal according to the first aspect of the present invention is:
In the gene encoding one or both of the T-type calcium channels Cav3.1 of homologous chromosomes, Arg-Ile-Met-Arg-Val- A triplet encoding arginine, which is the seventh amino acid from the N-terminal of the amino acid sequence represented by Leu-Arg-Ile-Ala-Arg, is substituted with a triplet encoding an amino acid other than arginine.

In this case, the amino acids other than arginine are
Histidine,
It is good as well.

In addition, the density of Purkinje cells
Significantly smaller than the wild type,
It is good as well.

Also, the density of Purkinje cell dendritic spines is
Significantly smaller than the wild type,
It is good as well.

In addition, the genetically modified non-human animal according to the first aspect of the present invention,
A mouse,
It is good as well.

In addition, the genetically modified non-human animal according to the first aspect of the present invention,
A spinal cerebellar degeneration model animal,
It is good as well.

The screening method for a therapeutic or prophylactic agent for spinocerebellar degeneration according to the second aspect of the present invention comprises:
The genetically modified non-human animal according to the first aspect of the present invention is used.

According to the present invention, human pathology can be reproduced more faithfully.

It is a figure which shows the structure of Cav3.1 typically. It is a figure which shows the position of the base substituted in the genome of a knock-in mouse. It is a figure which shows a part of base sequence of the gene which codes Cav3.1 in a wild type and knock-in mouse. It is a figure which shows the result of the rotarod test which concerns on Example 2. FIG. (A) And (B) is a figure which shows time until a 6-month-old mouse | mouth and a 14-month-old mouse | mouth fall from a turntable, respectively. It is a figure which shows the result of the beam walking test which concerns on Example 3. FIG. (A) And (B) is a figure which shows time until a 6-month-old mouse | mouth and a 10-month-old mouse | mouth reach a goal point, respectively. It is a figure which shows the result of the footprint test which concerns on Example 4. FIG. (A) is a figure which shows the footprint of a mouse | mouth. (B) is a figure which shows the stride of a mouse | mouth. It is a figure which shows the result of the open field test which concerns on Example 5. FIG. (A) is a diagram showing an image of a cerebellar slice according to Example 6. FIG. (B) is a diagram showing the density of Purkinje cells. (A) is a figure which shows the image of the dendrite spine which concerns on Example 7. FIG. (B) is a figure which shows the density of a dendritic spine.

Embodiments according to the present invention will be described with reference to the accompanying drawings. In addition, this invention is not limited by the following embodiment and drawing.

(Embodiment 1)
In the genetically modified non-human animal according to the present embodiment, the gene encoding one or both of the homologous chromosomes of the T-type calcium channel Cav3.1 is modified. The non-human animal is a vertebrate other than a human, preferably a mammal, more preferably a rodent. Specifically, examples of non-human animals include mice, rats, guinea pigs, hamsters, rabbits, chickens and monkeys. Considering versatility and convenience, the non-human animal is preferably a mouse. Below, the case where a mouse | mouth is used as a non-human animal which concerns on this Embodiment is demonstrated.

CACNA1G gene encoding Cav3.1 is on chromosome 17 in humans. Cav3.1 is expressed in the central nervous system, particularly in the cerebellum and thalamus. Human Cav3.1 is registered as, for example, ID: NP_934196.1 in the database of the National Center for Biotechnology Information (NCBI). The S4 region of repeat IV is from the 1686th amino acid to the 1707th amino acid from the N-terminal of the amino acid sequence registered with the ID. Within the S4 region of repeat IV in Cav3.1, there are four elements represented by Arg-Ile-Met-Arg-Val-Leu-Arg-Ile-Ala-Arg (SEQ ID NO: 1) from the N-terminus toward the C-terminus There is an amino acid sequence containing the following arginine residues. The amino acid sequence shown in SEQ ID NO: 1 is widely conserved in animals such as humans, gorillas, monkeys, rabbits, mice, chickens and frogs.

In Cav3.1, in order to open calcium channels, S4 of each of repeats I to IV needs to be activated simultaneously. Since arginine is an amino acid having a positive charge in the side chain, the four arginines in the amino acid sequence shown in SEQ ID NO: 1 are important for voltage sensing and S4 activation.

SCA classified as type 42 (SCA type 42) exhibits progressive ataxia and cerebellar atrophy. In patients with SCA42 type, arginine, which is the seventh amino acid from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, is substituted with histidine. Replacing arginine with histidine changes the electrophysiological properties of Cav3.1. As a result, SCA42 type is considered to develop.

The ortholog in the mouse of the CACNA1G gene is the Cacna1g gene. The Cacna1g gene is on chromosome 11. In mice, the amino acid sequence shown in SEQ ID NO: 1 is encoded by exon 29 of the Cacna1g gene.

Therefore, the genetically modified mouse according to the present embodiment is a triplet in which the triplet encoding arginine, which is the seventh amino acid from the N-terminal of the amino acid sequence shown in SEQ ID NO: 1, encodes an amino acid other than arginine in the Cacna1g gene. Has been replaced. As the amino acid other than arginine, histidine is preferable as in the mutation related to SCA42 type. The amino acids other than arginine are not particularly limited as long as Cav3.1 is expressed on the cell membrane and has a certain activity even if it is not equivalent to the wild-type activity. For example, the amino acid other than arginine may be lysine having a positive charge in the side chain, or an amino acid having no charge in the side chain.

In the case of a wild type mouse, the base sequence of DNA encoding the amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2. On the other hand, in the genetically modified mouse, the base sequence shown in SEQ ID NO: 2 is replaced with the base sequence shown in SEQ ID NO: 3, for example, in one or both of the homologous chromosomes. Arginine, which is the seventh amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1, is encoded by the seventh triplet “CGC” counted from the 5 ′ end of SEQ ID NO: 2, In this nucleotide sequence, the triplet is replaced with “CAC” encoding histidine. In the genetically modified mouse, the seventh triplet “CGC” counted from the 5 ′ end of SEQ ID NO: 2 may be replaced with “CAT” encoding histidine. The base sequence in the Cacna1g gene shown in SEQ ID NO: 2 and SEQ ID NO: 3 is the base sequence of the coding strand.

Next, a method for producing the genetically modified mouse will be described. In order to modify the Cacna1g gene as described above, the genome of a one-cell fertilized egg must be modified, and an individual into which the target mutation has been introduced must be selected from the obtained individuals. The genetically modified mouse is preferably a knock-in mouse in which a mutant gene is introduced into the Cacna1g gene by homologous recombination to express the mutant protein.

Knock-in can be performed by a known method. Preferably, a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeat and Criss associated protein) system (also referred to as “CRISPR / Cas9”) is used for the knock-in.

In CRISPR / Cas9, a guide RNA having a base sequence complementary to the target base sequence adjacent to the PAM sequence and Cas9 nuclease are used. The Cas9 nuclease that recognizes the guide RNA bound to the target base sequence of the Cacna1g gene cleaves the double-stranded DNA at the 5 'end side from the PAM sequence. Instead of guide RNA, single-stranded oligo DNA (ssODN) may be used as a template for DNA repair. The ssODN has a target base into which a mutation has been introduced and a homologous sequence (arm sequence) of 40 to 80 bases adjacent to the 5 'end side and 3' end side of the target base.

In the Cacna1g gene, when the triplet encoding arginine which is the seventh amino acid from the N-terminus of the amino acid sequence shown in SEQ ID NO: 1 is replaced with the triplet “CAC” encoding histidine, the base shown in SEQ ID NO: 4 The sequence ssODN is preferred.

The double-strand DNA breakage causes a loss of much genetic information or canceration, so it is repaired very rapidly in the cell. In non-homologous end-joining repair, which is one of the main pathways for repair, where the cleaved ends are joined together, the triplet encoding the arginine in the Cacna1g gene using ssODN as a template has high probability to the triplet encoding histidine. Replaced.

Note that not only Cas9 nuclease but also a programmable endonuclease such as TALEN and zinc finger nuclease (ZFN) may be used for the cleavage of double-stranded DNA.

In CRISPR / Cas9, a vector expressing Cas9 nuclease and guide RNA is introduced into a fertilized mouse egg. The introduction method is not particularly limited, and examples thereof include known methods such as a microinjection method, an electroporation method, a calcium phosphate method, and a lipofection method. When ssODN is used, a CRISPR / Cas9 vector expressing Cas9 nuclease may be introduced into a fertilized egg together with ssODN.

A heterozygous male and female are obtained by crossing a chimeric mouse obtained by returning a fertilized egg introduced with a CRISPR / Cas9 vector or the like into the womb of a wild type pseudopregnant mouse and a wild type mouse. Next, by mating heterozygous males and females, homozygous mice in which a mutation has been introduced into the Cacna1g gene can be obtained. In heterozygous mice, a mutation has been introduced into one of the Cacna1g genes of the homologous chromosome. In homozygous mice, mutations have been introduced into both Cacna1g genes of homologous chromosomes.

In order to confirm whether or not a mutation has been introduced into the Cacna1g gene, DNA may be extracted from the mouse and a known method such as PCR (Polymerase Chain Reaction) and RFLP (Restriction Fragment Length Polymorphism) may be used.

Next, the pathological features of the genetically modified mouse according to this embodiment will be described below. The genetically modified mice show signs of neurodegeneration, particularly Purkinje cell degeneration. For example, in the genetically modified mouse, the density of Purkinje cells is significantly smaller than that of the wild type. Purkinje cell density can be evaluated by staining cerebellar sections collected from wild-type and genetically modified mice with hematoxylin and eosin (H & E) according to a known method, and counting Purkinje cells present in the visual field under the microscope.

In addition, the density of dendritic spines in Purkinje cells is significantly smaller in the genetically modified mice than in the wild type. Dendritic spine density is determined by preparing cerebellar slices from cerebellum collected from wild-type and genetically modified mice, staining Purkinje cells according to known methods, and counting dendritic spines present in the visual field under the microscope Can be evaluated.

Here, “significantly small” means small with a statistically significant difference. For example, in the case of Purkinje cell density, the number of Purkinje cells is counted for a plurality of individual cerebellar sections of wild type and genetically modified mice, and the average value of Purkinje cell density in the cerebellar section is calculated, and wild type and genetically modified What is necessary is just to test the difference of the average number of mice | mouth by statistical methods, such as t test. The genetically modified mouse is several months old or more, preferably 6 months old or more, 7 months old or more, 8 months old or more, preferably 9 months old or more, 10 months old or more, 11 months old or more, or 12 months old or more The above pathological features appear.

In addition, the genetically modified mouse according to the present embodiment exhibits ataxia symptoms as shown in Examples 2 to 4 below. More specifically, ataxia symptoms include, for example, a significant decrease in motor function, a decrease in balance, a decrease in motor coordination, and gait disturbance compared to the wild type. In addition, the spontaneous activity of the genetically modified mouse is improved compared to the wild type.

The use of the genetically modified mouse is not particularly limited. Due to the pathological features and ataxia symptoms, the genetically modified mice are suitable as model mice for neurodegenerative diseases, particularly SCD, SCA and SCA42 types. For example, the genetically modified mouse is useful for elucidating the onset mechanism of SCD and SCA, particularly SCA42 type, and for understanding the disease state. Moreover, since Cav3.1 is a T-type calcium channel, it is useful for elucidating the role of the Ca2 + signal pathway in the degeneration of Purkinje cells.

The above-mentioned genetically modified mouse can be used as an overactive model mouse since spontaneous activity is improved. Since overactivity is one of the symptoms such as autism, the genetically modified mouse is also useful for elucidating the mechanism such as autism and understanding the disease state.

The use of the above-mentioned genetically modified mouse is preferably a screening method for therapeutic or prophylactic drugs of SCD, SCA and SCA42 type (SCD etc.). The screening method using the genetically modified mouse includes, for example, an administration step of administering a test substance to the genetically modified mouse, and an evaluation step of evaluating an index related to SCD and the like of the genetically modified mouse. In the administration step, it is preferable to use a genetically modified mouse that is 10 months of age or more in terms of more faithfully reproducing the pathology such as SCD.

In the administration of the test substance to the genetically modified mouse, a test substance such as a compound, antibody, peptide, DNA aptamer, RNA aptamer, siRNA, miRNA and antisense nucleic acid is administered to the genetically modified mouse. The method of administration is not particularly limited, but it is preferable to select an appropriate method for the test substance. Examples of the administration method include oral administration, intraperitoneal administration, intravenous injection, and subcutaneous injection. The test substance may be administered once or multiple times. What is necessary is just to determine the density | concentration of a test substance suitably with the kind of test substance. Moreover, you may administer the test substance of the several density | concentration which diluted the density | concentration of the test substance several steps.

Examples of the index in the evaluation step include pathological evaluation such as Purkinje cell morphology, Purkinje cell density and dendritic spine density, and ataxia index and behavior index. In the evaluation step, an index related to SCD or the like may be compared between the genetically modified mouse to which the test substance is administered and the genetically modified mouse to which the test substance is not administered. A test substance that has improved SCD or the like by an index may be selected as a candidate for a therapeutic or prophylactic agent such as SCD.

As described in detail above, the genetically modified mouse can reproduce human SCD and SCA, particularly SCA42 type pathology more faithfully from its pathological characteristics as shown in the following examples.

In addition, some of the above-mentioned genetically modified mice, for example, organs, tissues, and cells can also be used in screening methods for therapeutic or prophylactic drugs such as SCD.

The following examples further illustrate the present invention, but the present invention is not limited to the examples.

(Example 1: Production of knock-in mouse)
A knock-in mouse having a mutation in the Cacna1g gene was prepared as follows. FIG. 2 shows a part of the nucleotide sequence of exon 29 of Cacna1g gene of C57BL / 6J mouse. In the knock-in mouse, bases G (coding strand) and C (non-coding strand) indicated by arrows in FIG. , Protein: R1716H). FIG. 3 shows the allele of the wild-type mouse and the knock-in mouse in the region containing the base to be substituted. In wild-type mice, “G” of “CGC” encoding arginine is replaced with “A” of “CAC” encoding histidine in knock-in mice.

As the ssODN, an oligo DNA having a total of 120 bases having a base after substitution, an arm sequence of 60 bases adjacent to the 5 'end of the base and 59 bases adjacent to the 3' end of the base was used. As shown in FIG. 2, a PAM sequence is present on the 3 'end side from the base to be substituted. Cas9 nuclease binds to the PAM sequence.

CRISPR / Cas9 vector and ssODN were injected into fertilized eggs of C57BL / 6J mice, and mutations were introduced by homologous recombination. In accordance with a conventional method, fertilized eggs were transplanted into pseudopregnant ICR mice to obtain knock-in mice.

The base sequence of the Cacna1g gene in the genome of the obtained heterozygote (+ / R1716H mouse) and homozygote (R1716H / R1716H mouse) was confirmed by PCR and RFLP. The base sequences of the forward primer and the reverse primer used for PCR are shown in SEQ ID NOs: 5 and 6, respectively.

(Example 2: Rotarod test)
In order to evaluate the motor function of knock-in mice, an accelerated rotarod test was performed. After 2 days of training, 4 trials per day were conducted for 4 consecutive days. After the first trial was performed on all mice, the second trial was performed, and the trial interval in the same mouse was 20 minutes or longer. The turntable was constantly accelerated from 5 rpm to 40 rpm in 5 minutes, and the time until the mouse fell from the turntable was measured. The subjects of evaluation were 6-month-old and 14-month-old wild-type mice (+ / + mice) and R1716H / R1716H mice. The mean values on the 4th day were compared between + / + mice and R1716H / R1716H mice.

4A and 4B show the average values of the time taken to fall from the turntable at 6 months and 14 months of age, respectively. At 6 months of age, there was no significant difference between + / + and R1716H / R1716H mice. On the other hand, at the age of 14 months, the time to drop from the turntable in the R1716H / R1716H mice was significantly shortened. From these results, it was shown that the motor function was reduced by the mutation.

(Example 3: Beam walking test)
A beam walking test was performed to evaluate balance and motor coordination. A wooden beam having a diameter of 1 cm and a length of 90 cm was placed at a height of 50 cm from the ground. The starting point was brightened with a lighting device, and a black home cage was placed at the goal point. The time taken for the mouse to move from the start point to the goal point on the beam and reach the goal point was measured. The training was performed twice on the first day, the trial was performed twice on the second day, and the shortest time was recorded. The evaluation targets were 6 and 10 month old + / + mice, + / R1716H mice and R1716H / R1716H mice.

The average values of the time required to reach the goal point at 6 months and 10 months are shown in FIGS. 5 (A) and 5 (B), respectively. At 6 months of age, there was no significant difference between + / +, + / R1716H and R1716H / R1716H mice. On the other hand, at the age of 10 months, the time required to reach the goal point in R1716H / R1716H mice tended to be longer than that in + / + mice. From this result, it was shown that the balance sensation and motor coordination are reduced by the mutation.

(Example 4: Footprint test)
A footprint test was conducted to observe how to walk. A black ink was applied to the front leg of the mouse, and a black ink was applied to the back leg, and the mouse was allowed to walk in a 110 cm long tube with half paper, and the stride was measured from the footprint as shown in FIG. . The subjects of observation were 12-month-old + / + mice and R1716H / R1716H mice.

The average value of the mouse stride is shown in FIG. 6 (B). There was a tendency for the stride to be shorter in R1716H / R1716H mice compared to + / + mice. From these results, it was suggested that the gait disturbance was caused by the mutation.

(Example 5: Open field test)
An open field test was performed using a behavior analysis apparatus SCANET (Merquest). In order to compare exploratory behavior, without starting training, the measurement was started at the same time as placing the mouse on the behavior analysis device, and the total travel distance was measured every 10 minutes for 30 minutes. The total movement distance was evaluated as the number of times that the x-axis and the y-axis each crossed a sensor having a width of 6 mm. The subjects of evaluation were 6-month-old + / + mice, + / R1716H mice, and R1716H / R1716H mice.

Figure 7 shows the average value of the total travel distance. Compared to + / + mice, the distance traveled was significantly longer in R1716H / R1716H mice. This result suggested that there was an overactivity tendency due to mutation.

(Example 6: Purkinje cell density)
The density of Purkinje cells was measured and compared in 10 month old + / + mice and R1716H / R1716H mice. Mice were anesthetized by intraperitoneal injection of somnopentyl (1.5 mg / g body weight). The whole body was perfused and fixed by intracardiac perfusion with phosphate buffered saline containing 4% PFA. The fixed brain was slowly dehydrated with 10%, 20% and 30% sucrose and frozen in OCT-compound. A 10 μm brain section was prepared using a cryostat (Leica CM3050S) and attached to a slide glass. Brain sections were subjected to H & E staining, and the number of cerebellar Purkinje cells was counted under a microscope. The density of Purkinje cells was measured by dividing the number of Purkinje cells by the length of the line connecting the centers of the cell bodies. An optical microscope (manufactured by Nikon) was used for imaging the cerebellar slice.

FIG. 8 (A) shows cerebellar sections imaged under a microscope for + / + mice and R1716H / R1716H mice. As shown in FIG. 8 (B), the density of Purkinje cells in R1716H / R1716H mice was significantly smaller compared to + / + mice.

(Example 7: Density of Purkinje cell dendritic spine)
The density of Purkinje cell dendritic spines was measured and compared in 6-7 month old + / + and R1716H / R1716H mice. After the mouse was inhaled with CO ₂ gas and anesthetized, the brain was collected from the mouse, and a 250 μm cerebellar slice was prepared by vibratome. Neurobiotin was injected into Purkinje cell bodies with a patch pipette, and cerebellar slices were fixed with 4% PFA, stained with anti-biotina Alexa 488, and observed under a microscope. A confocal microscope (LSM800 Airyscan) manufactured by Zeiss was used for imaging dendritic spines. 63xoil was used for the objective lens. The number of dendritic spines was counted using Imaris (manufactured by Bitplane).

FIG. 9 (A) shows dendritic spines imaged for + / + mice and R1716H / R1716H mice. As shown in FIG. 9 (B), the density of dendritic spines in R1716H / R1716H mice was significantly lower compared to + / + mice.

The knock-in mice according to the above examples exhibited ataxia symptoms of SCD in humans and showed signs of degeneration of cerebellar Purkinje cells.

The present invention is capable of various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for explaining the present invention and do not limit the scope of the present invention. In other words, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

This application is based on Japanese Patent Application No. 2018-31706 filed on Feb. 26, 2018. The specification, claims, and entire drawings of Japanese Patent Application No. 2018-31706 are incorporated herein by reference.

The present invention is suitable for a model animal that reproduces a human disease state, particularly an SCD model animal.

Claims (7)

1. In the gene encoding one or both of the T-type calcium channels Cav3.1 of homologous chromosomes, Arg-Ile-Met-Arg-Val- A triplet encoding arginine, which is the seventh amino acid from the N-terminal of the amino acid sequence represented by Leu-Arg-Ile-Ala-Arg, is substituted with a triplet encoding an amino acid other than arginine;
   Genetically modified non-human animals.
2. Amino acids other than arginine are
   Histidine,
   The genetically modified non-human animal according to claim 1.
3. Purkinje cell density is
   Significantly smaller than the wild type,
   The genetically modified non-human animal according to claim 1 or 2.
4. Purkinje cell dendritic spine density is
   Significantly smaller than the wild type,
   The genetically modified non-human animal according to any one of claims 1 to 3.
5. A mouse,
   The genetically modified non-human animal according to any one of claims 1 to 4.
6. A spinal cerebellar degeneration model animal,
   The genetically modified non-human animal according to any one of claims 1 to 5.
7. Using the genetically modified non-human animal according to any one of claims 1 to 6,
   A screening method for a therapeutic or prophylactic agent for spinocerebellar degeneration.

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