Method for producing non-human primate model of cerebral infarction and pharmaceutical composition for treating cerebral infarction

Abstract

The present invention relates to a method for making a non-human primate model of cerebral infarction comprising a step of administering endothelin to the brain basal nucleus-thalamic region of a non-human primate, thereby inducing brain basal nucleus disorders, thalamic disorders and endocystic disorders, and a method for treating subacute stages-containing NeuroD1 protein or a polynucleotide encoding a NeuroD1 protein The pharmaceutical composition can be used for treating chronic cerebral infarction, transbronchial infarction or cerebral infarction with brain disorder in a transbronchial dominating region.

Description

Technical Field

[0001] The present invention relates to a non-human primate model of cerebral infarction and a method for preparing the same, a method for screening a therapeutic agent for cerebral infarction using the above-mentioned animal model, a method for treating cerebral infarction with perforator infarction or with brain disorders in the perforator-derived area, and a pharmaceutical composition for treating cerebral infarction with perforator infarction or with brain disorders in the perforator-derived area. Background Art

[0002] The main cerebral arteries are a general term for the multiple thick blood vessels that deliver oxygen and nutrients to the brain, including the internal carotid artery, middle cerebral artery, anterior cerebral artery that deliver blood to the brain, and the vertebral artery and basilar artery that deliver blood to the cerebellum and brainstem. The perforators of the fine arteries branching from the middle cerebral artery that constitutes the main cerebral arteries deliver oxygen and nutrients to the basal nuclei, thalamus, and internal capsule deep in the brain. The basal nuclei and thalamus are gray matter formed by the cell bodies of nerve cells, and the internal capsule is white matter that lacks the cell bodies of nerve cells and is mainly composed of nerve fibers that gather and run. The surface of the cerebral hemisphere is covered by a layer called the cerebral cortex, and the cerebral cortex, like the basal nuclei and thalamus, is gray matter formed by the cell bodies of nerve cells.

[0003] Cerebral infarction refers to a state in which the blood vessels in the brain are blocked and the nerve cells in the part where blood cannot be transported die. The stroke treatment guidelines recommend that treatment with a cerebral infarction treatment agent that minimizes brain damage should be used when precursor symptoms occur or within 4.5 hours of the onset of cerebral infarction. It can be seen that there are treatment methods for acute cerebral infarction, but on the other hand, there are no treatment methods for subacute to chronic cerebral infarction.

[0004] As a rodent cerebral infarction model, a model of inducing ischemia by opening the rat's cranium and administering endothelin with a strong vasoconstrictive effect to the surface of the middle cerebral artery is known (non-patent document 1). In the previous studies of various acute cerebral infarction therapeutic agents, various rodent cerebral infarction models represented by the rodent cerebral infarction model described in non-patent document 1 were used. Clinical trials of acute cerebral infarction therapeutic agents were carried out based on the effectiveness in these rodent cerebral infarction models, but there are almost no substances that have been proven to be effective as therapeutic agents in clinical trials (non-patent documents 2-3). As the reason for the deviation from the effectiveness in non-clinical trials using rodent cerebral infarction models and the effectiveness in clinical trials, the microcirculation in the cerebral blood vessels and brain structures of rodents and the size of the white matter occupied in the brain are different from those of humans. Therefore, the guidelines of the International Stroke Association recommend that when developing cerebral infarction therapeutic agents, in order to reduce the deviation between non-clinical and clinical results, primates whose cerebral blood vessels and tissue structures are closer to humans should be used for evaluation.

[0005] As a non-human primate cerebral infarction model, a model in which the middle cerebral artery, which is a common site of human vascular occlusion, is artificially occluded, i.e., a middle cerebral artery occlusion (MCAO) model. It is known that the MCAO model causes extensive obstacles in the cerebral cortex, internal capsule, and basal ganglia of the brain, which are similar to the obstacle sites in human cerebral infarction (non-patent literature 4). It is reported that in the evaluation of the change evaluation scale Non-Human Primate Stroke Scale (NHPSS) using the evaluation scale National Institutes of Health Stroke Scale (NIHSS) of the severity of human stroke neurology suitable for non-human primate models, the MCAO model reaches a high platform (plateau high) 3 to 5 days after infarction induction, and reaches a low platform (plateau low) 28 to 30 days after infarction induction (non-patent literature 4). The above results show that the non-human primate MCAO model is a model of gradual natural recovery, so it is believed that the model is not suitable for the evaluation of drugs for cerebral infarction in the chronic phase.

[0006] It is known that a method for producing a stroke model of non-human primates by using vascular disorder inducers such as endothelin to cause vascular disorder in the internal capsule region of the brain (Patent Document 1). In this stroke model, the disorder caused by vascular disorder in the internal capsule region can be recovered over time by self-healing power, so it is shown in multiple individuals that about 2 weeks after injury, the disorder such as walking, front and rear limb movements is restored to the same degree as before the injury (Patent Document 1). In addition, in Patent Document 1, although the motor dysfunction score such as NHPSS is not performed on the stroke model, its embodiment discloses that the stroke model does not maintain a high degree of motor dysfunction for a long time.

[0007] Reported that in order to use non-human primates to make white matter infarction model, the photosensitive pigment Bengal rose red was used and green light was irradiated, so that the method for producing thrombosis at the posterior crus of the internal capsule of the brain was reported (non-patent literature 5). This cerebral infarction model shows that when the motor nerve of the hand is completely damaged, it can cause persistent hand movement disorder and walking disorder, but it is also reported that if the obstacle site deviates slightly from the target site of the posterior crus of the internal capsule, persistent movement disorder cannot be induced (non-patent literature 5). This shows that the preparation method of this model is difficult to control the thrombosis site and is difficult to induce persistent movement disorder with high reproducibility.

[0008] So far, there are no reports on a cerebral infarction model that can produce a highly reproducible and long-term maintenance of severe motor dysfunction, or on the use of this model to evaluate the effectiveness of cerebral infarction therapeutic agents in the subacute to chronic stages after 72 hours of cerebral infarction. In addition, there are no reports on a cerebral infarction model that specifically induces dysfunction in the region innervated by perforators by administering a vascular disorder-inducing agent to the basal ganglia and thalamus innervated by perforators.

[0009] In the treatment of subacute to chronic cerebral infarction, drugs that can achieve neural regeneration have been highly anticipated. In the past, neural regeneration strategies based on cell transplantation and endogenous stem cell activation have been sought, but there have been no successful examples in clinical trials. As a strategy that is completely different from these two neural regeneration strategies, there is a method that utilizes direct in vivo reprogramming (DR). DR refers to the induction of differentiation from somatic cells into target cells without going through pluripotent stem cells such as iPS cells.

[0010] As one of the factors that realize DR of converting astrocytes into neurons in the brain, the transcription factor NeuroD1 is known. It is reported that by forcibly expressing NeuroD1 in astrocytes in the midbrain of mice, DR from astrocytes to neurons occurs (Patent Document 2, Non-Patent Document 6). In addition, it is also reported that NeuroD1 converts microglia into neurons and matures neural stem cells and neural precursor cells into neurons in mice (Non-Patent Documents 7-9).

[0011] It was reported that in a rodent cerebral cortical infarction model, NeuroD1 was forced to be expressed in astrocytes in the brain in the subacute period 7 to 9 days after brain injury, and DR was used to supplement nerve cells, thereby restoring the impairment of fine motor skills such as feeding behavior; and in a transient middle cerebral artery occlusion model mouse, NeuroD1 was forced to be expressed 1 week after brain injury, and microglia / macrophages were converted into nerve cells and the impairment of fine motor skills was restored (Non-patent Documents 10 to 13). It was further reported that in the cerebral cortex of primates, NeuroD1 was forced to be expressed 10 to 30 days after brain injury, and astrocytes were converted into nerve cells through DR (Non-patent Document 14). As can be seen, many cases of neurotransformation based on NeuroD1 have been reported, but as for the reported cases of functional recovery based on NeuroD1, they are limited to the restoration of fine motor function by expressing NeuroD1 in the acute to subacute phases in rodent cerebral infarction models, and there are no reports of (1) cases of restoration of gross motor skills such as walking and forelimb and hindlimb movements using NeuroD1, (2) cases of functional recovery when NeuroD1 is administered in the chronic phase, and (3) cases of functional recovery in non-human primates using NeuroD1.

[0012] Prior art literature

[0013] Patent Literature

[0014] Patent Document 1: Japanese Patent Application Publication No. 2015-231338

[0015] Patent Document 2: International Publication No. WO2014 / 015261

[0016] Non-patent literature

[0017] Non-patent document 1: Sharkey J et al., J. Cereb. Blood Flow Metab., vol. 13, pp. 865-871, 1993

[0018] Non-patent document 2: Clark WM et al., Neurology, vol. 57, pp. 1595-1602, 2001 Non-patent document 3: Diener HC et al., Stroke, vol. 31, pp. 2543-2551, 2000

[0019] Non-patent literature 4: Ramirez-Garcia G. et al., Neurosci., vol. 397, pp. 41-55, 2019

[0020] Non-patent literature 5: Hyung-Sun K. et al., Exp. Neurobiol., vol. 30, pp. 356-364, 2021

[0021] Non-patent document 6: Guo Z et al., Cell Stem Cell, vol. 14, pp. 188-202, 2014

[0022] Non-patent literature 7: Taito M et al., Neuron, vol. 101, pp. 472-485, 2019

[0023] Non-patent document 8: Kevin R et al., Brain, vol. 138, pp. 440-455, 2015

[0024] Non-patent document 9: Zhengliang G et al., Nat. Neurosci., Vol. 12, pp. 1090-1092, 2009

[0025] Non-patent literature 10: Chen YC et al., Mol. Ther., vol. 28, pp. 217-234, 2020

[0026] Non-patent literature 11: Livingston J et al., bioRxiv, 2020, doi: https: / / doi.org / 10.1101 / 2020.02.02.929091

[0027] Non-patent literature 12: Jiang MQ et al., Front. Aging Neurosci., vol. 13, 612856, 2021

[0028] Non-patent literature 13: Irie T et al., bioRxiv, 2021, doi: https: / / doi.org / 10.1101 / 2021.09.26.461831

[0029] Non-patent literature 14: Ge LJ et al. Front. Cell Dev. Biol., vol. 5, 590008, 2020 Summary of the invention

[0030] Problems to be solved by the invention

[0031] One object of the present invention is to provide a non-human primate model of cerebral infarction that can be used to screen or evaluate therapeutic agents for cerebral infarction, particularly therapeutic agents for cerebral infarction in the subacute to chronic stages, perforator infarction, or cerebral infarction with brain disorders in the perforator distribution area, and a method for preparing the animal model. Another object of the present invention is to provide a therapeutic agent or method for cerebral infarction with perforator infarction or cerebral infarction with brain disorders in the perforator distribution area.

[0032] Methods used to solve problems

[0033] The present inventors have conducted in-depth research to solve the above-mentioned problems, and found that by administering endothelin to the basal ganglia-thalamus region of non-human primates, in addition to being able to induce basal ganglia disorders and thalamus disorders, internal capsule disorders can also be induced. The non-human primates with basal ganglia disorders, thalamus disorders and internal capsule disorders obtained can become non-human primate models of cerebral infarction, and the non-human primate model of cerebral infarction is particularly useful as a non-human primate model of cerebral infarction in the subacute to chronic phase, perforator infarction, or cerebral infarction with brain disorders in the perforator-dominated area (Example 1). In addition, using this non-human primate model, it was found that NeuroD1 is effective as a therapeutic agent for perforator infarction or cerebral infarction with brain disorders in the perforator-dominated area (Examples 2 to 5, Example 11 and Example 12). The present invention is completed based on such insights.

[0034] That is, the present invention provides the following aspects.

[0035] [1] A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of the non-human primate brain, thereby inducing basal ganglia disorders, thalamus disorders, and internal capsule disorders.

[0036] [2] The method according to [1] above, wherein endothelin is administered to one or more nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamic region of the brain.

[0037] [3] The method according to [1] or [2] above, wherein endothelin is administered to at least three nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamic region of the brain.

[0038] [4] The method according to any one of [1] to [3] above, wherein endothelin is administered to at least four nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus and ventroposterior thalamic nucleus in the basal ganglia-thalamic region of the brain.

[0039] [5] The method according to any one of [1] to [4] above, wherein endothelin is administered to all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus and ventroposterior thalamic nucleus in the basal ganglia-thalamic region of the brain.

[0040] [6] The method according to any one of [1] to [5] above, wherein the cerebral infarction is in a subacute to chronic stage.

[0041] [7] The method according to any one of [1] to [6] above, wherein the cerebral infarction is chronic cerebral infarction.

[0042] [8] The method according to any one of [1] to [7] above, wherein the cerebral infarction is perforator infarction.

[0043] [9] The method according to any one of [1] to [7] above, wherein the cerebral infarction is a cerebral infarction with brain disorders in the perforator distribution area.

[0044]

[10] The method according to [8] or [9] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

[0045]

[11] The method according to [8] or [9] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a chronic cerebral infarction.

[0046]

[12] The method according to any one of [2] to

[11] above, comprising the step of determining an administration site in such a manner as to target the nerve nucleus and administering endothelin to the administration site.

[0047]

[13] A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of a non-human primate brain to thereby induce basal ganglia disorders, thalamus disorders, and internal capsule disorders, comprising:

[0048] (a) determining the administration site in such a manner as to target at least four nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and

[0049] (b) a step of administering endothelin to the administration site.

[0050]

[14] A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of a non-human primate brain to thereby induce basal ganglia disorders, thalamus disorders, and internal capsule disorders, comprising:

[0051] (a) determining the administration site in such a manner as to target all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and

[0052] (b) a step of administering endothelin to the administration site.

[0053]

[15] The method according to

[13] or

[14] , wherein the administration sites are at least 3.

[0054]

[16] The method according to any one of [1] to

[15] above, wherein the non-human primate is a cynomolgus monkey.

[0055]

[17] The method according to [1] to

[16] above, wherein the amount of endothelin administered per administration site is at least 10 μg, preferably at least 20 μg.

[0056]

[18] The method according to any one of [1] to

[17] above, wherein the endothelin is endothelin-1.

[0057]

[19] A non-human primate model of cerebral infarction with cerebral basal ganglia disorder, thalamus disorder and internal capsule disorder, which is a model produced by the method described in any one of [1] to

[18] above.

[0058]

[20] A method for preparing a non-human primate model of subacute to chronic cerebral infarction, comprising the step of administering endothelin to cynomolgus monkeys to induce cerebral basal ganglia disorders, thalamus disorders and internal capsule disorders, wherein the endothelin is administered to the following sites:

[0059] Based on the bregma, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 8-12 mm, L: 4-13 mm, D: 12-20 mm.

[0060]

[21] The method according to

[20] above, wherein the administration site of endothelin includes any one of the following (1) to (3):

[0061] (1) With bregma as the base point, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, (iii) B: 8-10 mm, L: 4-6 mm, D: 12-14 mm, and (iv) B: 8-10 mm, L: 11-13 mm, D: 14-16 mm;

[0062] (2) based on the bregma, the administration sites are located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 8-10 mm, L: 7-9 mm, D: 18-20 mm; and

[0063] (3) Based on the bregma, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 10-12 mm, L: 7-9 mm, D: 18-20 mm.

[0064]

[22] The method according to

[20] or

[21] above, wherein the administration site of endothelin includes any one of the following (1) to (3):

[0065] (1) With bregma as the base point, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm;

[0066] (2) based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, and (iii) B: 9 mm, L: 8 mm, D: 19 mm; and

[0067] (3) Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, and (iii) B: 11 mm, L: 8 mm, D: 19 mm.

[0068]

[23] The method according to any one of

[20] to

[22] above, wherein the administration site of endothelin includes the following administration sites:

[0069] Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm.

[0070]

[24] The method according to any one of

[20] to

[23] above, wherein the administration site of endothelin is the following administration site:

[0071] Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm.

[0072]

[25] The method according to any one of

[20] to

[24] above, wherein the cerebral infarction is chronic cerebral infarction.

[0073]

[26] The method according to

[20] to

[25] above, wherein the cerebral infarction is a perforator infarction or a cerebral infarction with brain disorders in the perforator distribution area.

[0074]

[27] The method according to

[20] to

[26] above, wherein the amount of endothelin administered per administration site is at least 10 μg, preferably at least 20 μg.

[0075]

[28] The method according to any one of

[20] to

[27] above, wherein the endothelin is endothelin-1.

[0076]

[29] A cynomolgus monkey model of cerebral infarction in the subacute to chronic stages, which is prepared by the method described in any one of

[20] to

[28] above.

[0077]

[30] A method for screening a therapeutic agent for cerebral infarction, comprising: a step of administering a test substance to the non-human primate model described in

[19] or

[29] above; and a step of determining the effect of the test substance on cerebral infarction in the non-human primate model.

[0078]

[31] A pharmaceutical composition for treating perforator infarction or subacute to chronic cerebral infarction, comprising NeuroD1 protein or a polynucleotide encoding NeuroD1 protein.

[0079]

[32] A pharmaceutical composition for treating cerebral infarction with brain disorders in the perforator distribution area, comprising NeuroD1 protein or a polynucleotide encoding NeuroD1 protein.

[0080]

[33] The pharmaceutical composition according to

[31] or

[32] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

[0081]

[34] The pharmaceutical composition according to any one of

[31] to

[33] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a chronic cerebral infarction.

[0082]

[35] The pharmaceutical composition according to the above

[31] , wherein the subacute to chronic cerebral infarction is chronic cerebral infarction.

[0083]

[36] The pharmaceutical composition according to any one of

[31] to

[35] above, which contains a polynucleotide encoding NeuroD1 protein, wherein the polynucleotide is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0084]

[37] The pharmaceutical composition according to any one of

[31] to

[36] above, wherein the polynucleotide encoding the NeuroD1 protein contains a polynucleotide having a sequence identity of 70% or more to the base sequence represented by SEQ ID NO: 7 or 10.

[0085]

[38] The pharmaceutical composition according to any one of

[31] to

[37] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide comprising the base sequence shown in SEQ ID NO: 7 or 10.

[0086]

[39] The pharmaceutical composition according to any one of

[31] to

[38] above, wherein the polynucleotide encoding the NeuroD1 protein is mRNA encoding the NeuroD1 protein.

[0087]

[40] The pharmaceutical composition according to

[39] above, wherein the mRNA encoding the NeuroD1 protein contains the base sequence encoding the NeuroD1 protein at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13.

[0088]

[41] The pharmaceutical composition according to

[39] or

[40] above, wherein the mRNA encoding the NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

[0089]

[42] The pharmaceutical composition according to any one of

[39] to

[41] above, wherein the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

[0090]

[43] The pharmaceutical composition according to any one of

[39] to

[42] above, wherein the mRNA encoding the NeuroD1 protein contains a poly A sequence.

[0091]

[44] The pharmaceutical composition according to

[43] above, wherein the length of the poly A sequence is 50 to 200 nucleotides.

[0092]

[45] The pharmaceutical composition according to any one of

[39] to

[44] above, wherein the mRNA encoding the NeuroD1 protein has a 5' cap structure.

[0093]

[46] The pharmaceutical composition according to any one of

[39] to

[45] above, wherein the mRNA encoding the NeuroD1 protein is mRNA containing modified nucleosides.

[0094]

[47] The pharmaceutical composition according to

[46] above, wherein the modified nucleoside is N1-methylpseudouridine.

[0095]

[48] ​​The pharmaceutical composition according to

[47] above, wherein the mRNA encoding NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

[0096]

[49] The pharmaceutical composition according to any one of

[39] to

[48] above, wherein the mRNA encoding the NeuroD1 protein is encapsulated in a drug delivery system (DDS).

[0097]

[50] The pharmaceutical composition according to any one of

[39] to

[49] above, wherein the mRNA encoding the NeuroD1 protein is encapsulated in lipid nanoparticles.

[0098]

[51] The pharmaceutical composition according to any one of

[31] to

[38] above, comprising a polynucleotide encoding NeuroD1 protein, wherein the polynucleotide is DNA.

[0099]

[52] The pharmaceutical composition according to any one of

[31] to

[38] and

[51] , comprising an expression vector containing a polynucleotide encoding NeuroD1 protein.

[0100]

[53] The pharmaceutical composition according to

[52] above, wherein the expression vector is any one of an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

[0101]

[54] The pharmaceutical composition according to

[53] above, wherein the expression vector is an adeno-associated virus vector.

[0102]

[55] A method for treating perforator infarction or subacute to chronic cerebral infarction, comprising the step of administering the pharmaceutical composition described in any one of

[31] to

[54] above, NeuroD1 protein or a polynucleotide encoding NeuroD1 protein to a subject.

[0103]

[56] A method for treating cerebral infarction with brain disorders in the perforator distribution area, comprising the step of administering the pharmaceutical composition described in any one of

[31] to

[54] above, NeuroD1 protein or a polynucleotide encoding NeuroD1 protein to a subject.

[0104]

[57] The method according to the above-mentioned

[55] or

[56] , wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

[0105]

[58] The method according to any one of

[55] to

[57] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a chronic cerebral infarction.

[0106]

[59] The method according to the above-mentioned

[55] , wherein the subacute to chronic stage of cerebral infarction is chronic stage of cerebral infarction.

[0107]

[60] A method according to any one of

[55] to

[59] above, comprising the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0108]

[61] The method according to any one of

[55] to

[60] above, wherein the polynucleotide encoding the NeuroD1 protein contains a polynucleotide having a sequence identity of 70% or more with respect to the base sequence shown in SEQ ID NO: 7 or 10.

[0109]

[62] The method according to any one of

[55] to

[61] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide comprising the base sequence shown in SEQ ID NO: 7 or 10.

[0110]

[63] The method according to any one of

[55] to

[62] above, comprising the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide is mRNA encoding NeuroD1 protein.

[0111]

[64] The method according to

[63] above, wherein the mRNA encoding the NeuroD1 protein contains the base sequence encoding the NeuroD1 protein at positions 44 to 1114 or 44 to 1120 of sequence number 11 or 13.

[0112]

[65] The method according to

[63] or

[64] above, wherein the mRNA encoding the NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

[0113]

[66] The method according to any one of

[63] to

[65] above, wherein the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

[0114]

[67] The method according to any one of

[63] to

[66] above, wherein the mRNA encoding the NeuroD1 protein contains a poly A sequence.

[0115]

[68] The method according to the above-mentioned

[67] , wherein the length of the poly A sequence is 50 to 200 nucleotides.

[0116]

[69] The method according to any one of

[63] to

[68] above, wherein the mRNA encoding the NeuroD1 protein has a 5' cap structure.

[0117]

[70] The method according to any one of

[63] to

[69] above, wherein the mRNA encoding the NeuroD1 protein is mRNA containing modified nucleosides.

[0118]

[71] The method according to the above-mentioned

[70] , wherein the modified nucleoside is N1-methylpseudouridine.

[0119]

[72] The method according to

[71] above, wherein the mRNA encoding NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

[0120]

[73] The method according to any one of

[63] to

[72] above, wherein the mRNA encoding the NeuroD1 protein is encapsulated in a drug delivery system (DDS).

[0121]

[74] The method according to any one of

[63] to

[73] above, wherein the mRNA encoding the NeuroD1 protein is encapsulated in lipid nanoparticles.

[0122]

[75] The method according to any one of

[55] to

[62] above, comprising the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide is DNA.

[0123]

[76] The method according to any one of

[55] to

[62] and

[75] , comprising the step of administering an expression vector containing a polynucleotide encoding NeuroD1 protein to a subject.

[0124]

[77] The method according to the above-mentioned

[76] , wherein the expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

[0125]

[78] The method according to the above-mentioned

[77] , wherein the expression vector is an adeno-associated virus vector.

[0126]

[79] A NeuroD1 protein or a polynucleotide encoding the NeuroD1 protein, which is used for treating perforator infarction or subacute to chronic cerebral infarction.

[0127]

[80] A NeuroD1 protein or a polynucleotide encoding the NeuroD1 protein, for use in treating cerebral infarction with brain disorders in the perforator distribution area.

[0128]

[81] The protein or polynucleotide for the above use according to

[79] or

[80] , wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

[0129]

[82] The protein or polynucleotide for the above use according to any one of

[79] to

[81] , wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a chronic cerebral infarction.

[0130]

[83] The protein or polynucleotide for the above application according to

[79] , wherein the subacute to chronic cerebral infarction is chronic cerebral infarction.

[0131]

[84] A polynucleotide for the above application according to any one of

[79] to

[83] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0132]

[85] A polynucleotide for the above application according to any one of

[79] to

[84] above, wherein the polynucleotide encoding the NeuroD1 protein contains a polynucleotide having a sequence identity of 70% or more relative to the base sequence shown in sequence number 7 or 10.

[0133]

[86] The polynucleotide for the above application according to any one of

[79] to

[85] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide containing the base sequence shown in sequence number 7 or 10.

[0134]

[87] The polynucleotide for the above use according to any one of

[79] to

[86] , wherein the polynucleotide is mRNA encoding NeuroD1 protein.

[0135]

[88] The polynucleotide for the above application according to

[87] , wherein the mRNA encoding the NeuroD1 protein contains the base sequence encoding the NeuroD1 protein at positions 44 to 1114 or positions 44 to 1120 of sequence number 11 or 13.

[0136]

[89] The polynucleotide for the above use according to

[87] or

[88] , wherein the mRNA encoding the NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

[0137]

[90] The polynucleotide for the above use according to any one of

[87] to

[89] above, wherein the mRNA encoding the NeuroD1 protein is an mRNA of a polynucleotide consisting of the base sequence from base 1 to base 1231 of sequence number 11 or 13.

[0138]

[91] The polynucleotide for the above use according to any one of

[87] to

[90] , wherein the mRNA encoding the NeuroD1 protein contains a poly A sequence.

[0139]

[92] The polynucleotide for the above application according to

[91] , wherein the length of the poly A sequence is 50 to 200 nucleotides.

[0140]

[93] The polynucleotide for the above use according to any one of

[87] to

[92] , wherein the mRNA encoding the NeuroD1 protein has a 5' cap structure.

[0141]

[94] The polynucleotide for the above use according to any one of

[87] to

[93] above, wherein the mRNA encoding the NeuroD1 protein is a polynucleotide containing modified nucleosides.

[0142]

[95] The polynucleotide for the above application according to

[94] , wherein the modified nucleoside is N1-methylpseudouridine.

[0143]

[96] The polynucleotide for the above use according to

[95] , wherein the mRNA encoding NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

[0144]

[97] The polynucleotide for the above use according to any one of

[87] to

[96] , wherein the mRNA encoding NeuroD1 protein is encapsulated in a drug delivery system (DDS).

[0145]

[98] The polynucleotide for the above use according to any one of

[87] to

[97] , wherein the mRNA encoding the NeuroD1 protein is encapsulated in lipid nanoparticles.

[0146]

[99] The polynucleotide for the above application according to any one of

[79] to

[86] above, wherein the polynucleotide is DNA.

[0147]

[100] The polynucleotide for the above use according to any one of

[79] to

[86] and

[99] , wherein the polynucleotide is an expression vector containing a polynucleotide encoding NeuroD1 protein.

[0148]

[101] The polynucleotide for the above application according to

[100] , wherein the expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

[0149]

[102] The polynucleotide for the above application according to

[101] , wherein the expression vector is an adeno-associated virus vector.

[0150]

[103] Use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating perforator infarction or subacute to chronic cerebral infarction.

[0151]

[104] Use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating cerebral infarction with brain disorders in the perforator distribution area.

[0152]

[105] The use according to any one of the above

[103] or

[104] , wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

[0153]

[106] The use according to any one of

[103] to

[105] above, wherein the perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a chronic cerebral infarction.

[0154]

[107] The use according to the above-mentioned

[103] , wherein the subacute to chronic stage of cerebral infarction is chronic stage of cerebral infarction.

[0155]

[108] The use according to any one of

[103] to

[107] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8.

[0156]

[109] The use according to any one of

[103] to

[108] above, wherein the polynucleotide encoding the NeuroD1 protein contains a polynucleotide having a sequence identity of 70% or more with respect to the base sequence shown in SEQ ID NO: 7 or 10.

[0157]

[110] The use according to any one of

[103] to

[109] above, wherein the polynucleotide encoding the NeuroD1 protein is a polynucleotide containing the base sequence shown in SEQ ID NO: 7 or 10.

[0158]

[111] The use according to any one of

[103] to

[110] above, wherein the polynucleotide is mRNA encoding NeuroD1 protein.

[0159]

[112] The use according to

[111] above, wherein the mRNA encoding the NeuroD1 protein contains the base sequence encoding the NeuroD1 protein at positions 44 to 1114 or positions 44 to 1120 of sequence number 11 or 13.

[0160]

[113] The use according to

[111] or

[112] above, wherein the mRNA encoding NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

[0161]

[114] The use according to any one of

[111] to

[113] above, wherein the mRNA encoding the NeuroD1 protein is an mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of sequence number 11 or 13.

[0162]

[115] The use according to any one of

[111] to

[114] above, wherein the mRNA encoding the NeuroD1 protein contains a poly A sequence.

[0163]

[116] The use according to the above-mentioned

[115] , wherein the length of the poly A sequence is 50 to 200 nucleotides.

[0164]

[117] The use according to any one of

[111] to

[116] above, wherein the mRNA encoding the NeuroD1 protein has a 5' cap structure.

[0165]

[118] The use according to any one of

[111] to

[117] above, wherein the mRNA encoding the NeuroD1 protein is mRNA containing modified nucleosides.

[0166]

[119] The use according to the above-mentioned

[118] , wherein the modified nucleoside is N1-methylpseudouridine.

[0167]

[120] The use according to the above-mentioned

[119] , wherein the mRNA encoding NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

[0168]

[121] The use according to any one of

[111] to

[120] above, wherein the mRNA encoding NeuroD1 protein is encapsulated in a drug delivery system (DDS).

[0169]

[122] The use according to any one of

[111] to

[121] above, wherein the mRNA encoding NeuroD1 protein is encapsulated in lipid nanoparticles.

[0170]

[123] The use according to any one of

[103] to

[110] above, wherein the polynucleotide is DNA.

[0171]

[124] The use according to any one of

[103] to

[110] and

[123] , wherein the polynucleotide is an expression vector containing a polynucleotide encoding NeuroD1 protein.

[0172]

[125] According to the use described in

[124] above, the expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

[0173]

[126] The use according to the above-mentioned

[125] , wherein the expression vector is an adeno-associated virus vector.

[0174]

[127] The pharmaceutical composition according to the above

[50] , wherein the lipid nanoparticles contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof as the cationic lipid.

[0175]

[128] The pharmaceutical composition according to the above

[50] , wherein the lipid nanoparticles contain 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azadiyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0176]

[129] The pharmaceutical composition according to

[127] or

[128] above, wherein the lipid nanoparticles further contain DSPC, cholesterol and DMG-PEG2000.

[0177]

[130] The method according to the above-mentioned

[74] , wherein the lipid nanoparticles contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof as a cationic lipid.

[0178]

[131] The method according to the above-mentioned

[74] , wherein the lipid nanoparticles contain 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azepinediyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0179]

[132] The method according to the above-mentioned

[130] or

[131] , wherein the lipid nanoparticles further contain DSPC, cholesterol and DMG-PEG2000.

[0180]

[133] The polynucleotide for the above application according to

[98] , wherein the lipid nanoparticles contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof as a cationic lipid.

[0181]

[134] According to the polynucleotide for the above application described in

[98] , the lipid nanoparticles contain 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azepinediyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0182]

[135] The polynucleotide for the above application according to

[133] or

[134] , wherein the lipid nanoparticles further contain DSPC, cholesterol and DMG-PEG2000.

[0183]

[136] The use according to the above-mentioned

[122] , wherein the lipid nanoparticles contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof as a cationic lipid.

[0184]

[137] The use according to the above-mentioned

[122] , wherein the lipid nanoparticles contain 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azepinediyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0185]

[138] The use according to

[136] or

[137] above, wherein the lipid nanoparticles further contain DSPC, cholesterol and DMG-PEG2000.

[0186] This specification incorporates the disclosures of Japanese Patent Applications No. 2022-106308, 2022-211994, and 2023-041345 on which the priority of this application is based.

[0187] Effects of the Invention

[0188] According to the present invention, a non-human primate model of cerebral infarction with cerebral basal ganglia disorder, thalamic disorder and internal capsule disorder can be prepared. The non-human primate model of cerebral infarction is particularly useful as a non-human primate model of cerebral infarction in the subacute to chronic phase, perforator infarction or cerebral infarction with brain disorder in the perforator distribution area. In addition, the present invention can provide a therapeutic agent, a therapeutic pharmaceutical composition or a therapeutic method that is particularly useful for treating perforator infarction or cerebral infarction with brain disorder in the perforator distribution area. BRIEF DESCRIPTION OF THE DRAWINGS

[0189] Figure 1 (A to C) are photographs showing the results of TTC staining of coronal sections of the brain of cynomolgus monkey individuals treated with endothelin-1 in Research Experiment 5 of Example 1. A shows lesions in the caudate nucleus, putamen, and globus pallidus, B shows lesions in the putamen, globus pallidus, and internal capsule, and C shows lesions in the ventrolateral and ventroposterior thalamic nuclei, and the internal capsule. The circular frame indicates the lesion.

[0190] Figure 2 (A to C) are photographs showing the results of TTC staining of coronal sections of the brain of cynomolgus monkey individuals treated with endothelin-1 in Research Experiment 6 of Example 1. A shows lesions in the caudate nucleus, putamen, and internal capsule, B shows lesions in the internal capsule and ventrolateral nucleus of the thalamus, and C shows lesions in the ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus. The circular frame indicates the lesion.

[0191] Figure 3 The results of mRS evaluation over time of cynomolgus monkeys treated with endothelin-1 in Experiment 6 of Example 1 are shown. The horizontal axis represents the number of days after endothelin-1 injection, and the vertical axis represents the mRS value (maximum value 6).

[0192] Figure 4 The results of the evaluation of the NHPSS motor system score over time in cynomolgus monkeys treated with endothelin-1 in Experiment 6 of Example 1 are shown. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents the NHPSS (motor system score) value (maximum value 32).

[0193] Figure 5 The results obtained by evaluating the time-dependent changes in motor dysfunction after endothelin-1 treatment in the AAV-NeuroD1 group (AAV-NeuroD1) and the control group (AAV-control) of Example 5 using mRS are shown. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents the value of mRS (maximum value 6). The white circles and black circles represent the average values ​​of mRS in the AAV-NeuroD1 group and the control group at each evaluation time, respectively. The error bar represents the standard error (SEM). The AAV vector was administered on the 21st day after the injection of endothelin-1.

[0194] Figure 6 The results obtained by evaluating the motor system score of NHPSS for the time-dependent changes in motor dysfunction after endothelin-1 treatment in the AAV-NeuroD1 group (AAV-NeuroD1) and the control group (AAV-control) of Example 5 are shown. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents the value of NHPSS (motor system score) (maximum value 32). The white circles and black circles represent the average values ​​of NHPSS (motor system score) of the AAV-NeuroD1 group and the control group at each evaluation time, respectively. The error bar represents the standard error (SEM). The AAV vector was administered on the 21st day after the injection of endothelin-1.

[0195] Figure 7 The figure shows the changes in body weight of the AAV-NeuroD1 group (AAV-NeuroD1) and the control group (AAV-control) of Example 5. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents body weight. The white circles and black circles represent the average values ​​of body weight of the AAV-NeuroD1 group and the control group at each evaluation time, respectively. The error bars represent the standard error (SEM).

[0196] Figure 8 The figure shows the transition of feeding behavior of the AAV-NeuroD1 group (AAV-NeuroD1) and the control group (AAV-control) of Example 5. The horizontal axis represents the number of weeks after the injection of endothelin-1, and the vertical axis represents the feeding score. The white circle and the black circle represent the average value of the feeding score of the AAV-NeuroD1 group and the control group at each evaluation time, respectively. The error bar represents the standard error (SEM).

[0197] Fig. 9A : FIG. 9 shows images of the brain of cynomolgus monkeys treated with endothelin-1 obtained in Example 6 over a period of days. Fig. 9A A horizontal section T2-weighted image of Example 6(a) is shown. Fig. 9A (a) to (e) show the results on the 1st day, 7th day, 21st day, 41st day, and 69th day after the injection of endothelin-1, respectively. L represents the left brain, and R represents the right brain. Endothelin-1 was injected into the left brain. (iii) and (iv) in the figure show the endothelin-1 injection sites (iii) and (iv) described in Example 2.

[0198] Fig. 9B : FIG. 9 shows images of the brain of cynomolgus monkeys treated with endothelin-1 obtained in Example 6 over a period of days. Fig. 9B A horizontal cross-sectional flare image of Example 6(a) is shown. Fig. 9B(a) to (e) show the results on the 1st day, 7th day, 21st day, 41st day, and 69th day after the injection of endothelin-1, respectively. L represents the left brain, and R represents the right brain. Endothelin-1 was injected into the left brain. (iii) and (iv) in the figure show the endothelin-1 injection sites (iii) and (iv) described in Example 2.

[0199] Fig. 9C : FIG. 9 shows images of the brain of cynomolgus monkeys treated with endothelin-1 obtained in Example 6 over a period of days. Fig. 9C A horizontal section perfusion image of Example 6(b) is shown. Fig. 9C (a) to (e) show the results on the 1st day, 7th day, 21st day, 41st day, and 69th day after the injection of endothelin-1, respectively. L represents the left brain, and R represents the right brain. Endothelin-1 was injected into the left brain. (iii) and (iv) in the figure show the endothelin-1 injection sites (iii) and (iv) described in Example 2.

[0200] Fig.9D : FIG. 9 shows images of the brain of cynomolgus monkeys treated with endothelin-1 obtained in Example 6 over a period of days. Fig.9D The coronal DTI of Example 6(c) is shown. Fig.9D (a) to (e) show the results on the 1st day, 7th day, 21st day, 41st day, and 69th day after the injection of endothelin-1, respectively. L indicates the left brain, and R indicates the right brain. Endothelin-1 was injected into the left brain.

[0201] Fig.10 The results of the daily changes in motor dysfunction after endothelin-1 treatment in the NeuroD1 mRNA-LNP group (ND1mRNA) and the control group (control mRNA) of Example 12 (a) using mRS are shown. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents the value of mRS (maximum value 6). The white circle represents the average value of mRS in the NeuroD1 mRNA-LNP group at each evaluation time. It should be noted that mRS was evaluated every 2 weeks after 28 days after the injection of endothelin-1, and the result on the 56th day represents the average value of the results obtained by evaluating each individual on the 57th, 58th or 59th day. The error bar represents the standard error (SEM). The black circle represents the mRS of one individual in the control group at each evaluation time. It should be noted that the result on the 56th day represents the result evaluated on the 58th day. mRNA-LNP was administered on the 21st day after the injection of endothelin-1.

[0202] Fig.11The analysis results using DTI of Example 12(b) are shown. A and B show diffusion tensor tractography (DTT) obtained by tensor analysis based on DTI images of the brain of cynomolgus monkey individuals treated with mRNA-LNP. R in the figure represents the right brain (non-impaired side. No mRNA-LNP was administered), and L represents the left brain (impaired side. mRNA-LNP was administered). The measured FA values ​​are plotted in the image. A shows the results of the control group (control mRNA) on the 114th day after the injection of endothelin-1, and B shows the representative results of FA values ​​of three individuals in the NeuroD1 mRNA-LNP group (ND1 mRNA) on the 114th to 115th days after the injection of endothelin-1. C shows the quantitative results of FA values ​​using DTI. The vertical axis of the graph of C represents the value (relative value) obtained by dividing the FA value of the impaired side by the FA value of the non-impaired side. White circles represent the relative values ​​of the NeuroD1 mRNA-LNP group, and bars represent their averages. Error bars represent standard error (SEM). Black circles represent the relative values ​​of one individual in the control group.

[0203] Fig.12 The results of the analysis of the volume of the lateral ventricle of Example 12 (c) are shown. A and B show T2-weighted images of the brain of a cynomolgus monkey individual treated with mRNA-LNP. R in the figure represents the right brain (non-impaired side. No mRNA-LNP was administered), and L represents the left brain (impaired side. mRNA-LNP was administered). A shows the results of the control group (control mRNA) on the 114th day after the injection of endothelin-1, and B shows the representative results of three individuals analyzed on the 114th to 115th days after the injection of endothelin-1 in the NeuroD1 mRNA-LNP group (ND1 mRNA). C shows the quantitative results of the volume of the lateral ventricle. The vertical axis of the graph of C represents the value (relative value) obtained by dividing the volume of the lateral ventricle on the impaired side by the volume of the lateral ventricle on the non-impaired side. The white circles represent the relative values ​​of the NeuroD1 mRNA-LNP group, and the bars represent their average values. The error bars represent the standard error (SEM). The black circles represent the relative values ​​of one individual in the control group.

[0204] Fig.13The results of the dopamine transporter expression analysis of Example 12 (d) are shown. A and B are photographs showing the results of immunostaining of dopamine transporters in coronal sections of the brains of cynomolgus monkeys treated with mRNA-LNP. R in the figure represents the right brain (non-impaired side. No mRNA-LNP was administered), and L represents the left brain (impaired side. mRNA-LNP was administered). The arrow indicates the striatum. A shows the results of the control group (control mRNA) on the 118th day after endothelin-1 treatment (brain ischemia induction), and B shows the representative results of immunostaining of dopamine transporters in three individuals of the NeuroD1 mRNA-LNP group (ND1 mRNA) on the 119th to 122nd day after the injection of endothelin-1. C shows the results obtained by analyzing the expression intensity of the dopamine transporter. The vertical axis of the graph of C represents the value (relative value) obtained by dividing the fluorescence staining intensity on the impaired side by the fluorescence staining intensity on the non-impaired side. The white circles represent the relative values ​​of the NeuroD1 mRNA-LNP group, and the bars represent their average values. Error bars indicate standard error (SEM). Black circles indicate relative values ​​of one individual in the control group.

[0205] Fig.14 The results obtained by daily changes of motor dysfunction after endothelin-1 treatment in the NeuroD1mRNA-LNP (ND1-2-L7) group (ND1 mRNA) and the control group (control mRNA) of Example 14 using mRS are shown. The horizontal axis represents the number of days after the injection of endothelin-1, and the vertical axis represents the value of mRS (maximum value 6). The white circle represents the average value of mRS in the NeuroD1mRNA-LNP group at each evaluation time. It should be noted that mRS was evaluated every 2 weeks after the 28th day after the injection of endothelin-1. The black circle represents the average value of mRS in the control group at each evaluation time. It should be noted that the result on the 70th day represents the average value of the results obtained by evaluating on the 69th or 70th day. mRNA-LNP was administered on the 21st day after the injection of endothelin-1. DETAILED DESCRIPTION

[0206] The present invention is described in detail below.

[0207] 1. Preparation of non-human primate models

[0208] The present invention provides a method for preparing a non-human primate model of cerebral infarction (hereinafter also referred to as "the method for preparing an animal model of the present invention"), which includes the steps of inducing basal ganglia disorders, thalamic disorders and internal capsule disorders by administering endothelin to the basal ganglia-thalamic region of the brain of non-human primates.

[0209] a) Non-human primates

[0210] In this specification, "non-human primate" refers to any primate (primate) animal other than humans. The non-human primate (monkey) used in the present invention is preferably a non-human primate that can evaluate brain dysfunction represented by basal ganglia disorder, thalamus disorder and internal capsule disorder by motor dysfunction scores using modified Rankin Scale (modified Rankin Scale, mRS, refer to Table 1 described later), Non-Human Primate Stroke Scale (NHPSS (Ben R et al., J. Neurol. Res., vol. 25 (1), pp. 68-78, 2003), refer to Table 2 described later). As non-human primates to which endothelin is administered according to the present invention, for example, apes, monkeys belonging to the family Cercopithecidae, and monkeys belonging to the family Capuchinidae can be listed. "Ape" refers to a monkey belonging to the superfamily Hominoidea of ​​the order Primates (primates). As apes, for example, chimpanzees, gorillas, orangutans, bonobos, gibbons, etc. can be listed. As monkeys belonging to the family of cercopithecidae, it is not limited to the following, for example, monkeys such as Japanese monkeys, rhesus monkeys, crab-eating monkeys, Formosan macaques, pig-tailed monkeys, and captive monkeys belonging to the genus Macaca (Macaques); monkeys such as Hamadryas baboons and gelada baboons belonging to the genus Baboons (Baboons); monkeys such as mandrills and drill baboons belonging to the genus Mandrill. As monkeys belonging to the family of Capuchinidae, it is not limited to the following, for example, monkeys such as black-capped capuchins belonging to the genus Capuchin (Capuchins); monkeys such as common squirrel monkeys belonging to the genus Squirrel Monkey (Squirrel Monkey); monkeys such as common marmosets belonging to the genus Marmoset (Marmoset). In one embodiment, the non-human primate used in the method for preparing an animal model of the present invention is a crab-eating macaque. In one embodiment, the non-human primate used in the method for preparing an animal model of the present invention is a crab-eating macaque over 3 years old. The non-human primate used in the method for preparing an animal model of the present invention may be male or female, and in one embodiment, is preferably male. In one embodiment, the non-human primate used in the method for preparing an animal model of the present invention is a male cynomolgus monkey.

[0211] The "crab-eating macaque" (Macaca fascicularis) is a primate classified as a member of the genus Macaca in the family Cercopithecidae of the order Mammalia. Like humans, crab-eating macaques walk on two legs. Therefore, the course of their blood vessels, such as the large bends in the internal carotid artery, is very similar to that of humans. In addition, they have advanced brain functions, and can observe not only motor functions, but also consciousness levels and cognitive functions, such as their vigilance toward humans.

[0212] b) Endothelin

[0213] "Endothelin" (mature form) is a peptide consisting of 21 amino acid residues and having two disulfide bonds in the molecule. Endothelin is generated by processing a precursor consisting of 203 amino acid residues. Endothelin has three peptide isomers with very similar amino acid sequences and structures called endothelin-1, endothelin-2 and endothelin-3 encoded by different genes. Almost all mammals have these three endothelins, and the functions of these three endothelins are conserved between species. Furthermore, it is also known that the amino acid sequence of endothelin-1 is highly conserved between species. The three endothelins have transient vasodilation and subsequent persistent vasoconstriction. It is known that ischemia can be induced by opening the rat's cranium and administering endothelin to the surface of the middle cerebral artery (non-patent document 1). The endothelin used in the present invention is, for example, endothelin-1 or endothelin-2. In one embodiment, the endothelin administered to non-human primates is human endothelin-1 or human endothelin-2. The sequence of the endothelin used in the present invention is not limited as long as it has endothelin activity (vasoconstrictive effect). For example, endothelin-1 can be a peptide containing an amino acid sequence having a sequence identity of more than 90%, preferably more than 95%, relative to the amino acid sequence shown in sequence number 9, and more preferably the amino acid sequence shown in sequence number 9.

[0214] c) Administration of endothelin to the basal ganglia-thalamus region of non-human primates and induction of brain disorders

[0215] In this specification, "basal ganglia-thalamus area" refers to the area composed of basal ganglia and thalamus. The basal ganglia is a collection of nerve nuclei composed of striatum, globus pallidus, subthalamic nucleus and substantia nigra. The basal ganglia and cerebellum form the cerebral cortex and circular circuits to control the activities of the cerebral cortex, especially the frontal lobe. The striatum is composed of the caudate nucleus and putamen, and there is an internal capsule between them. The globus pallidus is divided into outer segments and inner segments. The substantia nigra is composed of the reticular part and the compact part. Since the putamen and globus pallidus look like convex lenses, they are called lenticular nuclei. The basal ganglia are divided into the caudate nucleus and the lenticular nucleus (putamen and globus pallidus) by the internal capsule, and the thalamus exists on the inner side compared to the basal ganglia. The basal ganglia are related to daily life and habits. The thalamus is a two-body structure that is symmetrical in the center of the cerebral hemisphere and located in the upper part of the midbrain, and is composed of multiple nerve nuclei. The left and right thalamus can be divided into the lateral part and the medial part. The lateral part is composed of the ventroposterior nucleus of the thalamus, the ventrolateral nucleus of the thalamus, the ventroanterior nucleus of the thalamus, the lateral dorsal nucleus of the thalamus, the posterolateral nucleus of the thalamus, the pulvinar nucleus of the thalamus, and the lateral geniculate nucleus of the thalamus. The medial part is composed of the anterior thalamic nucleus group, the medial thalamic nucleus group, and the medial geniculate nucleus of the thalamus. The thalamus plays a role in transmitting sensory inputs such as vision, hearing, and body sensation to the cerebral cortex. In addition, information related to movement regulation is also transmitted from the cerebellum and the basal nucleus of the brain to the movement-related areas via the thalamus.

[0216] In the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing brain disorders. Endothelin has a strong vasoconstrictive effect. By administering endothelin, the blood vessels around the administration site can be contracted to induce ischemia and induce infarction (obstruction focus). In the present invention, "inducing disorder" refers to inducing ischemia around the administration site by administering endothelin, causing nerve cell necrosis and impairing the function of the necrotic site. In one embodiment, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing basal ganglia disorders, thalamus disorders and internal capsule disorders. In the present invention, "administering endothelin to the basal ganglia-thalamus region of the brain" refers to administering endothelin with the basal ganglia-thalamus region as the target (that is, in a way that endothelin reaches the basal ganglia-thalamus region of the brain). In the present invention, for example, the record of "administering endothelin" to the nerve nucleus also refers to administering endothelin with the nerve nucleus as the target (that is, in a way that endothelin reaches the nerve nucleus).

[0217] In this specification, "administration site" refers to a position determined by three-dimensional coordinates consisting of a position on the skull surface (on the cranium) and a depth from the dura mater just below the administration position on the skull when endothelin is administered (preferably injected) into the basal ganglia-thalamus region of the brain.

[0218] In one embodiment, the method for preparing an animal model of the present invention comprises the step of administering endothelin to a nerve nucleus selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus in the basal ganglia-thalamus region of the brain. In one embodiment, the method for preparing an animal model of the present invention comprises the step of administering endothelin to a nerve nucleus selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus in the basal ganglia-thalamus region of the brain of a non-human primate, at least 1, at least 2, at least 3, at least 4 or all 5 (putamen, caudate nucleus, globus pallidus, ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus) . In one embodiment, the method for preparing an animal model of the present invention comprises the step of administering endothelin to at least 1, at least 2, at least 3, at least 4 or at least 5 administration sites (typically, injection sites) targeted within the basal ganglia-thalamus region of the brain of a non-human primate. In one embodiment, the method for preparing an animal model of the present invention comprises administering endothelin to at least one, at least two, at least three, at least four or at least five administration sites (typically, injection sites) in the basal ganglia-thalamus region of the brain of a non-human primate, selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus. In one embodiment, endothelin can be administered to the basal ganglia, ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus of a non-human primate, thereby inducing brain disorders. In one embodiment, endothelin can be administered to the putamen, globus pallidus and thalamus (e.g., ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus) of a non-human primate, thereby inducing brain disorders. In one embodiment, endothelin can be administered to the putamen, caudate nucleus, globus pallidus and thalamus (e.g., ventrolateral nucleus of the thalamus and ventroposterior nucleus of the thalamus) of a non-human primate, thereby inducing brain disorders.

[0219] In one embodiment, the animal model preparation method of the present invention includes the steps of determining the administration site by targeting the basal ganglia-thalamus region of the brain of a non-human primate, for example, at least one, at least two, at least three, at least four or all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus and ventroposterior thalamic nucleus, and administering endothelin to the administration site.

[0220] In one embodiment, the animal model preparation method of the present invention includes the steps of determining the administration site by targeting all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus and ventroposterior thalamic nucleus in the basal ganglia-thalamic region of the brain of non-human primates, and administering endothelin to the administration site.

[0221] In one embodiment, the animal model preparation method of the present invention can be: a method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing basal ganglia disorders, thalamus disorders and internal capsule disorders, comprising:

[0222] (a) determining the administration site in such a manner as to target at least four nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and

[0223] (b) a step of administering endothelin to the administration site.

[0224] In one embodiment, the animal model preparation method of the present invention can be: a method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing basal ganglia disorders, thalamus disorders and internal capsule disorders, comprising:

[0225] (a) determining the administration site in such a manner as to target all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and

[0226] (b) a step of administering endothelin to the administration site.

[0227] In one embodiment, the animal model preparation method of the present invention can be: a method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing basal ganglia disorders, thalamus disorders and internal capsule disorders, comprising:

[0228] (a) determining at least three or four administration sites in a manner that targets at least four or all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and

[0229] (b) a step of administering endothelin to the administration site.

[0230] The administration site (typically, injection site) of endothelin can be appropriately determined by a person skilled in the art using known information, brain images obtained by computed tomography (CT) or magnetic resonance imaging (MRI), or the method described in this specification using the bregma as the base point.

[0231] In one embodiment, the location of the administration site (typically, injection site) of endothelin for the basal ganglia-thalamus region of the brain, such as the putamen, caudate nucleus, globus pallidus, ventrolateral nucleus of the thalamus, and ventroposterior nucleus of the thalamus, can be determined by using the intersection of the sagittal suture and the coronal suture located in front of the skull surface, i.e., the anterior fontanel (Bregma), as the base point (B: 0 mm, ML coordinate: 0 mm (center)). More specifically, the location of the administration site (typically, injection site) can be determined by using the coordinates of the administration site on the specified skull (equivalent to the hole on the skull for administration), and the coordinates are represented by the distance B in the anterior-posterior axis direction and the distance L (left) or R (right) in the left-right axis direction on the imaginary horizontal plane with the anterior fontanel as the base point, and the depth (vertical distance) D from the dura mater directly below the administration site on the skull. With respect to the distance B in the anterior-posterior axis direction, the distance in the front is represented by a positive number, and the distance in the back is represented by a negative number. In the present invention, typically, a needle can be inserted from a dosing position on the skull (the position of a hole on the skull) specified by the distance in the front-back left-right axis direction toward a vertically downward direction (i.e., the direction of gravity), and endothelin is administered to the brain through the needle. Therefore, in the present invention, the depth D of the prescribed dosing site refers to the depth in the brain from the dura mater directly below the dosing position on the skull toward a vertically downward direction (i.e., the direction of gravity). In the present invention, by using the anterior fontanelle as the base point of the dosing site (typically, the injection site), the deviation between individuals can be reduced, and the dosing site in the prescribed nerve nucleus can be determined as a more stable position. In the present invention, the position of the dosing site is determined in a state where the head of a non-human primate is fixed. The fixation of the head of a non-human primate can use, for example, a brain positioning and fixing device. Specifically, after the ear rods of the brain positioning and fixing device are inserted into the two ears of the non-human primate to fix the head so that it cannot move left and right, the head is fixed using a palate rod and an eye socket rod so that it cannot move up and down. By inserting the ear rods to fix the head, the head of the non-human primate can be positioned and fixed with good reproducibility. Thus, when administering the drug, the head of the non-human primate is fixed in a manner such that the top of the head is on the upper side, the straight line connecting the two ears is horizontal, and the face is facing forward.

[0232] Regarding the administration site (typically, injection site) of the prescribed position determined with the bregma as the base point, which areas in the brain, such as which nerve nuclei can be administered endothelin (that is, which areas or which nerve nuclei are the administration targets), it can be confirmed in advance. Regarding the administration site of the prescribed position, the area or nerve nucleus that becomes the administration target can be determined by, for example, injecting a staining solution into the administration site and investigating the stained area in the brain just after injection. As the staining solution used, it is not limited to the following, and Evans blue (CAS number: 314-13-6) can be cited. Endothelin can be administered to the administration site that has been confirmed to be the target of administration with the target area or nerve nucleus.

[0233] In one embodiment, the administration site of endothelin may include: based on the bregma, at least 3 administration sites (e.g., 3, 4, or 5) in total, located at (i) B: 0-4 mm, L: 7-9 mm, D: 15-21 mm and (ii) B: 4-14 mm, L: 3-14 mm, D: 12-23 mm. These administration sites are particularly suitable for monkeys belonging to the genus Macaca, such as cynomolgus monkeys, but are not limited to these species. The administration of endothelin to each administration site can be performed simultaneously, successively, or multiple times. The administration of endothelin to one administration site can be performed only once or multiple times. For example, after endothelin is administered to a certain administration site, endothelin can be administered to other administration sites and / or the same administration site at intervals. In one embodiment, after endothelin is administered to a certain administration site, endothelin can be further administered to other administration sites and / or the same administration site the next day or later.

[0234] In one embodiment, the method for preparing an animal model of the present invention is a method comprising administering endothelin to a cynomolgus monkey at an administration site of endothelin recorded in this specification with the bregma as the base point, thereby inducing basal ganglia disorder, thalamic disorder and internal capsule disorder. In one embodiment, the method for preparing an animal model of the present invention comprises the steps of specifying an administration site on the skull of a cynomolgus monkey and administering endothelin to an administration site directly below the administration site, and the administration site of the endothelin includes an administration site recorded in this specification with the bregma as the base point. In these embodiments, the method for preparing an animal model of the present invention can be a method for preparing a non-human primate model of cerebral infarction in the subacute to chronic stage, for example, in the chronic stage (for example, perforator infarction or cerebral infarction with brain disorders in the perforator distribution area).

[0235] When using the cynomolgus monkey as an example, the administration site located at B: 0-4mm, L: 7-9mm, and D: 15-21mm based on the bregma is the target of the thalamus. Similarly, the administration site located at B: 4-14mm, L: 3-14mm, and D: 12-23mm based on the bregma is the target of the basal ganglia (particularly the striatum and globus pallidus).

[0236] In one embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) and (iii) B: 8-12 mm (e.g., 9 mm or 11 mm), L: 4-13 mm (e.g., 5 mm, 8 mm, 12 mm or 13 mm), D: 12-20 mm (e.g., 13 mm, 15 mm or 19 mm) (at least 3 sites in total, e.g., 3, 4 or 5 administration sites).

[0237] In one embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) and (iii) B: 8-12 mm (e.g., 9 mm or 11 mm), L: 7-14 mm (e.g., 8 mm or 13 mm), D: 18-20 mm (e.g., 19 mm) (at least 3 sites in total, e.g., 3 or 4 administration sites).

[0238] In a more preferred embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (iii) B: 8-10 mm (e.g., 9 mm), L: 4-6 mm (e.g., 5 mm), D: 12-14 mm (e.g., 13 mm) and (iv) B: 8-10 mm (e.g., 9 mm), L: 11-13 mm (e.g., 12 mm), D: 14-16 mm (e.g., 15 mm) (at least 4 administration sites in total).

[0239] In one embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) and (iii) B: 8-10 mm (e.g., 9 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) (at least 3 administration sites in total).

[0240] In one embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) and (iii) B: 10-12 mm (e.g., 11 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) (at least 3 administration sites in total).

[0241] In a more preferred embodiment, the administration sites of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 2mm, L: 8mm, D: 19mm, (ii) B: 5mm, L: 8mm, D: 19mm, (iii) B: 9mm, L: 5mm, D: 13mm and (iv) B: 9mm, L: 12mm, D: 15mm (at least 4 administration sites in total).

[0242] In a more preferred embodiment, the administration sites of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 2mm, L: 8mm, D: 19mm, (ii) B: 5mm, L: 8mm, D: 19mm and (iii) B: 9mm, L: 8mm, D: 19mm (at least 3 administration sites in total).

[0243] In a more preferred embodiment, the administration sites of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 2mm, L: 8mm, D: 19mm, (ii) B: 5mm, L: 8mm, D: 19mm and (iii) B: 11mm, L: 8mm, D: 19mm (at least 3 administration sites in total).

[0244] In a further preferred embodiment, the administration site of endothelin can be: based on the anterior fontanelle, the administration sites are respectively located at (i) B: 2mm, L: 8mm, D: 19mm, (ii) B: 5mm, L: 8mm, D: 19mm, (iii) B: 9mm, L: 5mm, D: 13mm and (iv) B: 9mm, L: 12mm, D: 15mm (at least 4 administration sites in total).

[0245] In one embodiment, the administration site of endothelin may include: based on the anterior fontanelle, the administration sites located at (i) B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm), (ii) B: 8-10 mm (e.g., 9 mm), L: 4-6 mm (e.g., 5 mm), D: 12-14 mm (e.g., 13 mm), (iii) B: 8-10 mm (e.g., 9 mm), L: 12-14 mm (e.g., 13 mm), D: 18-20 mm (e.g., 19 mm), (iv) B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) and (v) B: 8-10 mm (e.g., 9 mm), L: 7-9 mm (e.g., 8 mm), D: 18-20 mm (e.g., 19 mm) (at least 5 administration sites in total). In one embodiment, in one individual, endothelin may be administered to the administration sites of (i), (ii) and (iii), and then further administered to the administration sites of (iv) and (v) on the next day.

[0246] When explaining the example of cynomolgus monkeys, the administration site located at B: 1-3 mm (e.g., 2 mm), L: 7-9 mm (e.g., 8 mm), and D: 18-20 mm (e.g., 19 mm) based on the bregma is targeted at the ventrolateral nucleus of the thalamus and the ventroposterior nucleus of the thalamus. Similarly, the administration site located at B: 4-6 mm (e.g., 5 mm), L: 7-9 mm (e.g., 8 mm), and D: 18-20 mm (e.g., 19 mm) based on the bregma is targeted at the globus pallidus. The administration site located at B: 8-10 mm (e.g., 9 mm), L: 4-6 mm (e.g., 5 mm), and D: 12-14 mm (e.g., 13 mm) based on the bregma is targeted at the caudate nucleus. In addition, the administration site located at B: 8-10 mm (e.g. 9 mm), L: 11-13 mm (e.g. 12 mm), and D: 14-16 mm (e.g. 15 mm) based on the anterior fontanelle targets the putamen. Similarly, the administration site located at B: 8-10 mm (e.g. 9 mm), L: 12-14 mm (e.g. 13 mm), and D: 18-20 mm (e.g. 19 mm) based on the anterior fontanelle targets the putamen (particularly the deep part of the putamen). These administration sites are adjacent to the internal capsule, so administration of endothelin to these administration sites may also cause obstruction of the internal capsule.

[0247] The above administration sites are particularly suitable for use in monkeys belonging to the genus Macaca, such as cynomolgus monkeys, but are not limited to these species. Endothelin administration to these administration sites may be performed simultaneously, sequentially, or in multiple portions.

[0248] In the method for preparing an animal model of the present invention, endothelin can be administered only to the left brain in order to induce motor dysfunction in the right half of the body, and endothelin can be administered only to the right brain in order to induce motor dysfunction in the left half of the body. The above-mentioned endothelin administration site based on the anterior fontanelle shows an example of administering endothelin to the left brain, but the method of administering endothelin to the corresponding site of the right brain that is bilaterally symmetrical (that is, the administration site having the same distance B and depth D in the anterior-posterior axis direction relative to the above-mentioned endothelin administration site in the left brain, but the left-right axis direction is the same distance The distance is determined by replacing L (left) with the R (right) direction in the left and right axis directions. In addition, a method of administering endothelin to both the left and right brains by appropriately adjusting the dosage of endothelin in order to induce motor dysfunction in the left and right halves of the body can also be included in the present invention.

[0249] In the animal model preparation method of the present invention, the dosage of endothelin at each administration site (typically, injection site) (typically, injection amount) can be, for example, at least 0.1 μg, at least 1 μg, at least 4 μg or at least 10 μg, preferably at least 20 μg, at least 30 μg, at least 50 μg, at least 55 μg or at least 60 μg, and preferably less than 200 μg, less than 150 μg, less than 100 μg, less than 90 μg or less than 70 μg. In one embodiment, the dosage of endothelin per administration site (typically, injection site) can be 0.1 μg to 200 μg, 10 μg to 200 μg, 20 μg to 200 μg, 30 μg to 200 μg, 0.1 μg to 150 μg, 10 μg to 150 μg, 20 μg to 150 μg, 30 μg to 150 μg, 10 μg to 10 ... In one embodiment, the dosage of endothelin per administration site (typically, injection site) is preferably 20 μg to 70 μg, 20 μg to 60 μg or 30 μg to 60 μg, and more preferably 50 μg to 60 μg.

[0250] The dosage (typically, injection amount) of endothelin per these administration sites (typically, injection sites) is particularly suitable for use in monkeys belonging to the genus Macaca, such as cynomolgus monkeys, but is not limited to these species. The dosage (typically, injection amount) of endothelin administered or injected can be appropriately determined by those skilled in the art according to the species used (e.g., the weight of the species).

[0251] In the animal model preparation method of the present invention, endothelin can be administered in the form of a solution (e.g., an aqueous solution) in an aqueous medium (water, saline, buffer, a physiologically acceptable organic solvent, etc.). In one embodiment, endothelin is administered in the form of an endothelin solution diluted with an aqueous acetic acid solution (e.g., 0.05-5% acetic acid, preferably 0.1-1% acetic acid). When endothelin is administered in the form of a solution, administration can be performed by injection, but is not limited to this. Injection is not limited to the following, and can be performed using any intracerebral injection means such as a syringe such as a microsyringe, a cannula for administration, and / or a microinjection tube.

[0252] The amount (volume) of the endothelin solution administered or injected is not limited to the following, and each administration site can be 2 μL or more, preferably 4 μL or more, more preferably 10 μL or more, 12 μL or more, 20 μL or more, or 30 μL or more. The amount of the endothelin solution administered or injected can also be 100 μL or less, 50 μL or less, 40 μL or less, or 30 μL or less per administration site. In one embodiment, the amount of the endothelin solution administered or injected can be 2 μL to 100 μL, 4 μL to 50 μL, 10 μL to 100 μL, 12 μL to 40 μL, 12 μL to 30 μL, 20 μL to 40 μL, 20 μL to 50 μL, 25 μL to 50 μL, 30 μL to 50 μL, or 30 μL to 100 μL per administration site, and can be, for example, 30 μL. In one embodiment, when at least 20 μg of endothelin is administered to each administration site (typically, injection site), the amount of endothelin solution administered or injected may be 10 μL or more or 12 μL or more, for example, 10 μL to 50 μL per administration site. In one embodiment, when at least 50 μg of endothelin is administered to each administration site (typically, injection site), the amount of endothelin solution administered or injected may be 20 μL or more or 30 μL or more, for example, 20 μL to 50 μL per administration site.

[0253] The amount (volume) of the endothelin solution administered or injected is particularly suitable for use in monkeys belonging to the genus Macaca, such as cynomolgus monkeys, but is not limited to these species. The amount (volume) of the endothelin solution administered or injected can be appropriately determined by those skilled in the art according to the species used.

[0254] The concentration of endothelin in the solution administered or injected is not limited to the following, and can be at least 0.1 μg / μL, preferably at least 0.5 μg / μL or at least 1 μg / μL, and preferably 10 μg / μL or less, 5 μg / μL or less, or 3 μg / μL or less. In one embodiment, the endothelin concentration can be, for example, 0.1 μg / μL to 10 μg / μL, preferably 0.5 μg / μL to 5 μg / μL, more preferably 0.5 μg / μL to 3 μg / μL, further preferably 1 μg / μL to 3 μg / μL, for example, 2 μg / μL.

[0255] In one embodiment, the method for preparing an animal model of the present invention may include the step of administering to the administration site (typically, the injection site) 10 μL or more, preferably 10 μL to 50 μL of an endothelin solution with a concentration of 0.5 to 3 μg / μL, preferably 1 to 3 μg / μL, for example, 2 μg / μL. In one embodiment, the method for preparing an animal model of the present invention may include the step of administering to the administration site 12 μL or more, preferably 12 μL to 40 μL of an endothelin solution with a concentration of 0.5 to 3 μg / μL, preferably 1 to 3 μg / μL, for example, 2 μg / μL. In one embodiment, the method for preparing an animal model of the present invention may include the step of administering to the administration site 20 μL or more, preferably 20 μL to 50 μL of an endothelin solution with a concentration of 0.5 to 3 μg / μL, preferably 1 to 3 μg / μL, for example, 2 μg / μL. In one embodiment, the method for preparing an animal model of the present invention may include administering to each administration site 30 μL or more, preferably 30 μL to 50 μL or 30 μL to 100 μL of an endothelin solution with a concentration of 0.5 to 3 μg / μL, preferably 1 to 3 μg / μL, for example, 2 μg / μL. In one embodiment, the method for preparing an animal model of the present invention may include administering to each administration site 20 μL to 40 μL, preferably 25 μL to 35 μL, for example, 30 μL of an endothelin solution with a concentration of 0.5 to 3 μg / μL, preferably 1 to 3 μg / μL, for example, 2 μg / μL. These can be appropriately determined by a person skilled in the art according to the species used.

[0256] By appropriately selecting the concentration and dosage (typically, injection amount) of endothelin, the effect of endothelin can be easily spread from the administration site to adjacent brain regions (eg, internal capsule, subthalamic nucleus, etc.).

[0257] The animal model preparation method of the present invention can induce basal ganglia disorders, thalamic disorders and internal capsule disorders by administering endothelin as described above to the basal ganglia-thalamic region of non-human primates. In one embodiment, basal ganglia disorders, thalamic disorders and internal capsule disorders can be induced by administering endothelin as described above to the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus and / or ventroposterior thalamic nucleus.

[0258] In the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain to induce cerebral ischemia, and necrosis occurs around the administration site, thereby inducing a disorder.

[0259] In the present invention, "basal ganglia disorder" refers to a disorder occurring in the basal ganglia. When a disorder occurs in the basal ganglia, as observed in Parkinson's disease, not only is it difficult to carry out conscious movements, but also daily natural movements and actions become difficult.

[0260] The putamen, which constitutes the basal nucleus of the brain, forms the dorsal striatum together with the caudate nucleus, and also forms the outermost part of the lenticular nucleus. It is generally believed that the putamen has a role in reinforcement learning. The caudate nucleus, which constitutes the basal nucleus of the brain, exists near the center of the brain and on both sides of the thalamus. It has been shown that the caudate nucleus is closely related to learning and memory, especially feedback processing. The globus pallidus, which constitutes the basal nucleus of the brain, is divided into an outer segment and an inner segment, both of which contain large GABAergic projection neurons. Disorders of these nerve nuclei can cause disorders of the basal nucleus of the brain.

[0261] For the purposes of the present invention, "thalamic disorder" refers to a disorder produced in the thalamus of the brain. When the administration of endothelin to the basal ganglia-thalamus region of the brain includes administration to the thalamus (e.g., ventrolateral nucleus of the thalamus and / or ventroposterior nucleus of the thalamus), the administration can induce thalamic disorder. In one embodiment, when the administration of endothelin to the basal ganglia-thalamus region of the brain is only to the basal ganglia of the brain, thalamic disorder can also be induced. The thalamus has 2 left and right parts in the central part of the cerebral hemisphere and the upper part of the midbrain, and each thalamus can be divided into a lateral part and a medial part. The "ventrolateral nucleus of the thalamus" is an area in the lateral part of the thalamus, which receives GABA input from the globus pallidus and substantia nigra and is mainly projected to the motor area of ​​the cerebral cortex. The "ventroposterior nucleus of the thalamus" is an area in the medial part of the thalamus, which mainly receives sensory information from the ascending sensory conduction pathway, conveys it and projects it to the sensory area of ​​the cerebral cortex.

[0262] In the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing disorders in the internal capsule in addition to the disorders at the site of endothelin administration.

[0263] For the purposes of the present invention, "internal capsule disorder" refers to a disorder occurring in the internal capsule of the brain. The internal capsule is not a nucleus (nerve cell body), but rather a collection of nerve fibers. The internal capsule is divided into anterior crus and posterior crus. Nerve fibers connecting the thalamus and the frontal lobe run in the anterior crus of the internal capsule, and nerve fibers connecting the temporal lobe, parietal lobe, and occipital lobe to the brainstem run in the posterior crus of the internal capsule. When an internal capsule disorder occurs, hemiplegia (hemiplegia) characteristically occurs on the opposite side of the disorder site. In the acute phase when an internal capsule disorder occurs, partial muscle spasms occur first, and motor paralysis of the upper and lower limbs occurs. Disorders of the posterior crus of the internal capsule are also accompanied by weakened or absent sensation in the trunk, limbs, and head.

[0264] In one embodiment, the animal model preparation method of the present invention can further induce subthalamic nucleus disorder by administering endothelin to the basal ganglia-thalamus region of non-human primates. The subthalamic nucleus is one of the nerve nuclei constituting the basal ganglia of the brain, which receives inhibitory output from the globus pallidus external ganglion and excitatory input to the globus pallidus external ganglion and the globus pallidus internal ganglion-substantia nigra pars reticulata.

[0265] In the method for preparing an animal model of the present invention, by administering endothelin to the basal ganglia-thalamus region of the brain of non-human primates, in addition to inducing basal ganglia disorders and thalamus disorders, internal capsule disorders are induced because the effect of endothelin administered to the basal ganglia-thalamus region of the brain spreads to the internal capsule. As a situation where the effect of endothelin spreads to the internal capsule, for example, endothelin administered with the basal ganglia-thalamus region as a target may also be directly administered to the internal capsule, endothelin administered to the basal ganglia-thalamus region may diffuse to the internal capsule, and necrosis caused by endothelin administered to the basal ganglia-thalamus region of the brain may extend to the internal capsule. In the method for preparing an animal model of the present invention, internal capsule disorders can be induced by allowing the effect of endothelin to directly or indirectly spread to the internal capsule. Similarly, the disorder of the surrounding areas other than the administration site such as the subthalamic nucleus is induced by administering endothelin to the basal ganglia-thalamus region of the brain, which is also due to the effect of the administered endothelin spreading to the area.

[0266] If there is a disorder in the basal ganglia and its constituent nerve nuclei, thalamus, internal capsule, etc., 2,3,5-triphenyltetrazolium can be used to treat TTC is reduced by hydrogen produced in the mitochondria to form water-insoluble triphenylformazan (TPF), which is red in color. Therefore, tissues without damage are stained red, while tissues with damage are not stained.

[0267] The present invention also provides a non-human primate model of cerebral infarction with basal ganglia disorder, thalamus disorder and internal capsule disorder prepared using the above-mentioned animal model preparation method of the present invention (hereinafter also referred to as "the non-human primate model of the present invention").

[0268] The non-human primate model of the present invention has cerebral basal ganglia disorders, thalamic disorders and internal capsule disorders induced (made) in the animal model preparation method of the present invention, and is useful as a model (animal model) of cerebral infarction. In one embodiment, the non-human primate model of the present invention can also be used as an acute cerebral infarction model. In one embodiment, the non-human primate model of the present invention can maintain the disorder for a long period of time (e.g., 2 weeks to 3 months or longer), so it can be used as a model of cerebral infarction in the subacute to chronic phase. In one embodiment, the non-human primate model of the present invention is useful as a model of cerebral infarction in the subacute to chronic phase. In one embodiment, the non-human primate model of the present invention is particularly useful as a model of cerebral infarction in the chronic phase. In one embodiment, in the animal model preparation method of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing cerebral infarction in the acute to chronic phase. In one embodiment, in the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing cerebral infarction in the subacute to chronic phase. In one embodiment, in the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing cerebral infarction in the chronic phase.

[0269] In non-human primates represented by cynomolgus monkeys, the acute phase refers to a period of less than 72 hours after the onset of cerebral infarction, during which symptoms may worsen. The subacute phase refers to the period from 72 hours to less than 2 weeks after the onset of cerebral infarction, and the chronic phase refers to the period after 2 weeks after the onset of cerebral infarction. In the present invention, the subacute phase to the chronic phase for the evaluation of cerebral infarction in non-human primate models or the treatment of non-human primates, etc., can be 72 hours after the onset of cerebral infarction, for example, 2 weeks after the onset of cerebral infarction, and can also be 3 weeks after or 1 month after the onset of cerebral infarction, for example, 3 weeks after the onset of cerebral infarction to 6 months after the onset of cerebral infarction, or 1 month after 6 months after the onset of cerebral infarction, or 3 weeks after the onset of cerebral infarction to 3 months after the onset of cerebral infarction, or 1 month after 3 months after the onset of cerebral infarction. In this specification, the "subacute phase to chronic phase" in cerebral infarction in non-human primates represented by cynomolgus monkeys refers to the period after 72 hours after the onset of cerebral infarction, and the "chronic phase" refers to the period after 2 weeks after the onset of cerebral infarction. In the present specification, "one week later" means seven days later, "two weeks later" means fourteen days later, and "three weeks later" means twenty-one days later.

[0270] The non-human primate model of the present invention is also useful as an animal model of perforator infarction. In one embodiment, in the method for preparing an animal model of the present invention, perforator infarction can be induced by administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate. Perforator infarction can be a perforator infarction with brain disorders in a specific area, for example, i) perforator infarction with brain disorders in the basal ganglia-thalamus region of the brain, ii) perforator infarction with brain disorders in the basal ganglia and / or thalamus of the brain, iii) perforator infarction with brain disorders in the basal ganglia-thalamus region and internal capsule of the brain, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule of the brain. In one embodiment, the non-human primate model of the present invention can also be used as an animal model of i) perforator infarction with brain disorders in the basal ganglia-thalamus region, ii) perforator infarction with brain disorders in the basal ganglia and / or thalamus, iii) perforator infarction with brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule. It should be noted that perforator infarction with brain disorders in a specific region includes not only perforator infarction with brain disorders only in the specific region, but also perforator infarction with brain disorders outside the specific region.

[0271] In one embodiment, the non-human primate model of the present invention can be used as an animal model of perforator infarction in the subacute to chronic phase. In one embodiment, the non-human primate model of the present invention is a perforator infarction in the subacute to chronic phase, and can be used as an animal model of perforator infarction with i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, in the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing perforator infarction in the subacute to chronic phase (for example, i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule).

[0272] In one embodiment, the non-human primate model of the present invention is particularly useful as an animal model of chronic perforator infarction. In one embodiment, the non-human primate model of the present invention is a chronic perforator infarction, and can be used as an animal model of perforator infarction with i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, in the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the non-human primate, so that chronic perforator infarction (e.g., i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule) can be induced.

[0273] Diseases that cause brain damage due to blockage or rupture of cerebral blood vessels are called strokes. Among them, diseases caused by blockage of blood vessels that nourish the brain are called cerebral infarction. Cerebral infarction is sometimes also called ischemic stroke. Cerebral infarction can be roughly divided into four types, including cardiogenic cerebral embolism, atherothrombotic cerebral infarction, lacunar cerebral infarction, and infarction that cannot be classified into these (unclassifiable). "Perforators" are fine arteries that branch off from the middle cerebral artery that constitutes the main cerebral artery, and they innervate the basal ganglia-thalamus region and internal capsule of the brain. It should be noted that the "innervation" of a brain region by a blood vessel means that the blood vessel is used to supply blood to the brain region, and the brain region is located in the vascular territory of the blood vessel. "Perforator infarction" refers to a disease in which the thalamus, caudate nucleus, putamen, globus pallidus and / or internal capsule innervated by the perforator undergo infarction, and the function of the infarcted area is impaired. As nerve nuclei that are prone to infarction in perforator infarction, the putamen and caudate nucleus are known. As perforator infarction, what is widely known is cerebral infarction caused by simple occlusion of deep perforators, i.e., lacunar cerebral infarction. On the other hand, as a type of perforator infarction, in recent years, it has been proposed that thrombosis based on atherosclerotic plaques near the branching part of the large-diameter perforator mother artery affects the entire perforator, and it is called perforator artery atheromatous disease (BAD). As perforator infarction, striatal capsule infarction, internal capsule infarction, anterior choroidal artery infarction, thalamic infarction, etc. are also known. Perforator infarction is also known as cerebral infarction in which infarction occurs in the area innervated by the perforator (perforator innervation area). "Perforator innervation area" refers to the area innervated by the perforator, which refers to the basal ganglia-thalamic area and internal capsule of the brain.

[0274] The main symptoms of lacunar infarction are the same as those of conventional cerebral infarction, namely hemiplegia (motor paralysis), hemiplegia (sensory disturbance), and slurred speech (articulation disorder), but the range of necrosis is usually less than 15 mm when the perforator is blocked.

[0275] BAD is one of the types of infarction that cannot be classified into the above categories. Atherothrombotic cerebral infarction is a cerebral infarction in which the large blood vessels are blocked due to arteriosclerosis. BAD is a type that is exactly between lacunar cerebral infarction and atherothrombotic cerebral infarction. The large blood vessels are not narrowed, but the roots of the perforators branching from them are blocked, so that the cerebral infarction occurs in a larger area than in lacunar cerebral infarction. The symptoms of BAD vary depending on the location of the cerebral infarction. The main symptoms are motor paralysis, sensory disorders, and dysphonia, which are the same as lacunar cerebral infarction. The course of symptoms is similar to that of atherothrombotic cerebral infarction. Usually, the scope of cerebral infarction gradually expands and progresses within a few days after the onset of the disease.

[0276] In one embodiment, the non-human primate model of the present invention can be used as an animal model of lacunar infarction and / or BAD. In one embodiment, in the method for preparing an animal model of the present invention, lacunar infarction and / or BAD can be induced by administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate. In one embodiment, the non-human primate model of the present invention can be used as an animal model of lacunar infarction and / or BAD in the subacute to chronic phase. In one embodiment, in the method for preparing an animal model of the present invention, lacunar infarction and / or BAD can be induced by administering endothelin to the basal ganglia-thalamus region of the brain of a non-human primate. In one embodiment, the non-human primate model of the present invention is particularly useful as an animal model of lacunar infarction and / or BAD in the chronic phase. In one embodiment, in the method for preparing an animal model of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of a non-human primate, thereby inducing chronic lacunar infarction and / or BAD.

[0277] Lacunar infarction or BAD can be lacunar infarction or BAD with brain disorder in a specific area, for example, i) lacunar infarction or BAD with brain disorder in the basal ganglia-thalamus area, ii) lacunar infarction or BAD with brain disorder in the basal ganglia and / or thalamus, iii) lacunar infarction or BAD with brain disorder in the basal ganglia-thalamus area and internal capsule, or iv) lacunar infarction or BAD with brain disorder in the basal ganglia, thalamus and internal capsule. Perforator infarction with brain disorder in a specific area can be lacunar infarction and BAD with brain disorder in a specific area.

[0278] The non-human primate model of the present invention can also be used as an animal model of cerebral infarction with brain damage in the perforator distribution area. In this specification, "cerebral infarction with brain damage in the perforator distribution area" refers to cerebral infarction in which brain damage (brain damage) caused by infarction mainly occurs in the perforator distribution area. Cerebral infarction with brain damage in the perforator distribution area not only includes cerebral infarction in which brain damage occurs in the perforator distribution area due to infarction generated in the perforator distribution area, but also includes cerebral infarction in which brain damage occurs secondarily in the perforator distribution area, such as cellular necrosis caused by infarction generated around the perforator distribution area extending to the perforator distribution area, thereby impairing the function of the part. In this specification, brain damage refers to brain functional damage (functional damage) caused by cell necrosis caused by infarction. Brain damage generally includes cell necrosis in the area with the brain damage. "Cerebral infarction with brain damage in the perforator distribution area" can be cerebral infarction with cell necrosis in the perforator distribution area.

[0279] Examples of cerebral infarction with brain disorders in the perforator distribution area include perforator infarction (eg, lacunar infarction, BAD, or perforator infarction with brain disorders in a specific region).

[0280] As cerebral infarction with brain disorders in the perforator distribution area, including cerebral infarction with disorders in part or all of the perforator distribution area, for example, there can be cited cerebral infarction with brain disorders in the perforator distribution area, and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, the non-human primate model of the present invention can also be used as an animal model of cerebral infarction with brain disorders in the perforator distribution area, and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, in the animal model preparation method of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing cerebral infarction with a brain disorder in the perforator-innervated area (for example, a brain disorder in the perforator-innervated area, and the brain disorder is i) a brain disorder including a brain disorder in the basal ganglia-thalamus region, ii) a brain disorder including a brain disorder in the basal ganglia and / or thalamus, iii) a brain disorder including a brain disorder in the basal ganglia-thalamus region and internal capsule, or iv) a brain disorder including a brain disorder in the basal ganglia, thalamus and internal capsule).

[0281] In one embodiment, the non-human primate model of the present invention can be used as an animal model of cerebral infarction in the subacute to chronic stage with brain disorders in the perforator distribution area. In one embodiment, the non-human primate model of the present invention can also be used as an animal model of cerebral infarction in the subacute to chronic stage with brain disorders in the perforator distribution area, and the brain disorders are i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, in the animal model preparation method of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing subacute to chronic cerebral infarction with brain disorders in the perforator distribution area (for example, subacute to chronic cerebral infarction with brain disorders in the perforator distribution area and the brain disorders include i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule).

[0282] In one embodiment, the non-human primate model of the present invention can be used as an animal model of cerebral infarction in the chronic stage with brain disorders in the perforator distribution area. In one embodiment, the non-human primate model of the present invention can also be used as an animal model of cerebral infarction in the chronic stage with brain disorders in the perforator distribution area, and the brain disorders are i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, in the animal model preparation method of the present invention, endothelin is administered to the basal ganglia-thalamus region of the brain of non-human primates, thereby inducing chronic cerebral infarction with brain disorders in the perforator-derived area (for example, chronic cerebral infarction with brain disorders in the perforator-derived area and the brain disorders include i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule).

[0283] For perforator infarction or cerebral infarction with brain disorders in the perforator distribution area, disorders are often observed in the internal capsule in addition to the basal ganglia. The basal ganglia and thalamus are gray matter composed of the cell bodies of nerve cells, while the internal capsule is a white matter that lacks the cell bodies of nerve cells and is mainly composed of nerve fibers that gather and run. As one of the nerve fiber bundles that form the internal capsule, the corticospinal tract can be listed. The corticospinal tract is a bundle of axons (nerve fibers) that runs from the motor area of ​​the cerebral cortex through the spinal cord to the lower motor neurons, and the corticospinal tract is almost entirely composed of axons of upper motor neurons.

[0284] When the internal capsule of the perforator distribution area is obstructed, the corticospinal tract is damaged. Damage to the corticospinal tract can be evaluated, for example, by diffusion tensor image (DTI) analysis using magnetic resonance imaging (MRI). In one embodiment, the non-human primate model of the present invention is accompanied by corticospinal tract damage.

[0285] When the basal ganglia of the perforator-derived area are impaired, the expression of dopamine transporters in the striatum is mainly observed to be decreased. Dopamine transporters are present in the striatal terminals of the nerves (dopamine nerves) from the substantia nigra to the striatum in the brain, and have the function of reuptake of dopamine released into the striatum. Abnormalities in the basal ganglia of the brain and the dopaminergic system can lead to motor dysfunctions unique to basal ganglia disorders such as voluntary movements, postural muscle tension and abnormal walking. The decreased expression of dopamine transporters can be evaluated, for example, by immunostaining using anti-dopamine transporter antibodies. In one embodiment, the non-human primate model of the present invention is accompanied by decreased expression of dopamine transporters.

[0286] The non-human primate model of the present invention can also be used as an animal model of cerebral infarction with localized brain disorders. Cerebral infarction with localized brain disorders does not include cerebral infarction that causes brain disorders in a wide range of the brain, such as cerebral infarction caused by infarction of the middle cerebral artery, resulting in disorders of the cerebral cortex, internal capsule, basal ganglia, etc. In one embodiment, the non-human primate model of the present invention can be used as an animal model of cerebral infarction with localized brain disorders in the subacute to chronic stages. In one embodiment, the non-human primate model of the present invention can be used as an animal model of cerebral infarction with localized brain disorders in the chronic stage.

[0287] For cerebral infarction, as a cause of serious sequelae in the chronic stage, astrocytes in the central nervous system of mammals react around the injured part to form a scab-like tissue called a glial scar, which hinders nerve regeneration. The chronic stage cerebral infarction model with glial scar formation is very useful for developing a therapeutic agent for cerebral infarction in the chronic stage. In one embodiment, the non-human primate model of the present invention shows neuronal cell shedding and astrocyte aggregation in the chronic stage of cerebral infarction.

[0288] d) Methods for confirming and evaluating cerebral infarction

[0289] The non-human primate model of the present invention is a non-human primate model of cerebral infarction and / or a non-human primate model of perforator infarction. For example, it can be judged by histological methods such as TTC staining and hematoxylin-eosin (HE) staining, or the infarction focus (obstruction focus) of the non-human primate model can be judged by using computed tomography (CT) or MRI in the same way as the diagnosis method of human cerebral infarction, such as perforator infarction. It can also be judged by the same method that it is an animal model of cerebral infarction with brain disorders in the perforator distribution area. In addition to the above method, the non-human primate model of cerebral infarction in the subacute to chronic stage can also be confirmed based on the persistence of the disorder. For example, when the motor dysfunction lasts for at least 4 weeks from the onset of cerebral infarction, the non-human primate model of cerebral infarction can be judged to be useful as a non-human primate model of cerebral infarction in the subacute to chronic stage. The method for confirming the persistence of the disorder is not limited to this and can also be changed according to the evaluation method.

[0290] The indicators used to evaluate the state of motor dysfunction (symptoms, severity, persistence of the disorder, etc.) of the non-human primate model of the present invention are not particularly limited as long as they can evaluate the state of motor dysfunction of the animal, and for example, NHPSS and / or mRS (for monkeys) described in this specification can be used for evaluation. The motor dysfunction induced in the non-human primate model of the present invention can be a disorder of gross motor skills.

[0291] Evaluation using mRS can be implemented by scoring based on Table 1 described later. The mRS (for clinical use in humans) recorded in Table 1 is an indicator widely used as an indicator of physical impairment in the diagnosis and treatment of stroke. The mRS (for monkeys) recorded in Table 1, which is modified in a manner used for monkeys, can be used to evaluate the state of motor dysfunction in the non-human primate model of the present invention (symptoms, severity, persistence of impairment, etc.). Evaluation using NHPSS can be implemented by scoring based on the benchmark of Table 2 described later. Evaluation using NHPSS can be based on the scores of 1 to 4 items in the motor system, consciousness disorder, muscle control system and perceptual system, and preferably the item includes at least the motor system. As for the perceptual system, fluctuations in reactivity between individuals are prone to occur, so scoring may not be performed. Since motor dysfunction is mainly observed in the subacute and chronic stages, evaluation using NHPSS in these periods can be performed based only on the motor system score.

[0292] When using mRS to judge the severity of motor dysfunction, for example, when mRS is 3 or more, it can be judged as having moderate to severe motor dysfunction. NHPSS can also be used to judge the severity. For example, in the motor system score of NHPSS (the total score of item A in Table 2 below; the same below), when the score on the obstacle side is 12 or more, it can be judged as having moderate to severe motor dysfunction. In this specification, motor dysfunction with high severity refers to moderate to severe motor dysfunction.

[0293] The severity of the motor dysfunction in the non-human primate model of the present invention is, for example, an mRS of 2 or more, preferably an mRS of 3-5.

[0294] The severity of the motor dysfunction in the non-human primate model of the present invention is, for example, a score of 4 or more on the impaired side in the motor system score of the NHPSS, preferably a score of 12 or more, more preferably 14 or more, and particularly preferably 16 on the impaired side.

[0295] In one embodiment, in the method for preparing an animal model of the present invention, the severity of the motor dysfunction induced by the administration of endothelin is an mRS of 2 or more. In one embodiment, in the method for preparing an animal model of the present invention, the severity of the motor dysfunction induced by the administration of endothelin is an mRS of 3 to 5. In one embodiment, in the method for preparing an animal model of the present invention, the severity of the motor dysfunction induced by the administration of endothelin is a score of 4 or more on the impaired side of the motor system score of NHPSS. In one embodiment, in the method for preparing an animal model of the present invention, the severity of the motor dysfunction induced by the administration of endothelin is a score of 12 or more on the impaired side of the motor system score of NHPSS. In one embodiment, in the method for preparing an animal model of the present invention, the severity of the motor dysfunction induced by the administration of endothelin is an mRS of 3 to 5 and a score of 12 or more on the impaired side of the motor system score of NHPSS.

[0296] In one embodiment, the severity of the motor dysfunction in the non-human primate model of the present invention is an mRS of 2 or more. In one embodiment, the severity of the motor dysfunction in the animal model obtained by the preparation method of the present invention is an mRS of 3 to 5. In one embodiment, the severity of the motor dysfunction in the non-human primate model of the present invention is a score of 4 or more on the impaired side of the motor system score of NHPSS. In one embodiment, the severity of the motor dysfunction in the non-human primate model of the present invention is a score of 12 or more on the impaired side of the motor system score of NHPSS. In one embodiment, the severity of the motor dysfunction in the non-human primate model of the present invention is an mRS of 3 to 5 and a score of 12 or more on the impaired side of the motor system score of NHPSS.

[0297] In the non-human primate model of the present invention, the mRS score is relatively stable after 2 weeks after the onset of cerebral infarction. Therefore, when using mRS to judge the persistence of motor dysfunction, for example, the persistence of motor dysfunction can be judged based on the mRS score after 2 weeks after the onset of cerebral infarction and after any specified time. Similarly, when using the motor system score of NHPSS to judge the persistence of motor dysfunction, for example, the persistence of motor dysfunction can be judged based on the motor system score of NHPSS after 2 weeks after the onset of cerebral infarction and after any specified time.

[0298] [Table 1]

[0299]

[0300] 1 Mild paralysis: No obvious paralysis is seen, but slight paralysis symptoms are visible.

[0301] 2 Paralysis: When obvious paralysis is seen.

[0302] 3 Mild voluntary motor deficits: No obvious voluntary motor deficits are observed, but symptoms of voluntary motor deficits are slightly visible.

[0303] 4 Decreased voluntary movements: When a significant decrease in voluntary movements is seen.

[0304] 5 Level of consciousness (Monkey): Judged based on the level of responsiveness to humans.

[0305] 6 Sitting / Supine Position: At least one of the sitting or supine positions.

[0306] 7 Mildly decreased level of consciousness: There is no obvious decrease in the level of consciousness, but symptoms of decreased level of consciousness are slightly visible.

[0307] 8 Reduced level of consciousness: When a significant decrease in level of consciousness is seen.

[0308] [Table 2]

[0309] <nhpss>

[0310]

[0311]

[0312] 2. Screening methods for therapeutic agents for cerebral infarction

[0313] The present invention also provides a method for screening a therapeutic agent for cerebral infarction using the non-human primate model of the present invention (hereinafter also referred to as "the method for screening a therapeutic agent for cerebral infarction of the present invention"). More specifically, the method for screening a therapeutic agent for cerebral infarction of the present invention comprises the steps of administering a test substance to the non-human primate model of the present invention and determining the effect of the test substance on cerebral infarction in the non-human primate model.

[0314] In one embodiment, the method for screening a therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction, preferably cerebral infarction in the subacute to chronic stages. In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for perforator infarction (e.g., perforator infarction with brain disorders in the basal ganglia-thalamus region, perforator infarction with brain disorders in the basal ganglia and / or thalamus, perforator infarction with brain disorders in the basal ganglia-thalamus region and internal capsule, or perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule, and / or, lacunar cerebral infarction and / or BAD) or cerebral infarction with brain disorders in the perforator-derived area (e.g., cerebral infarction with brain disorders in the perforator-derived area and the brain disorders include i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule). In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for perforator infarction in the subacute to chronic stage (especially the chronic stage) (for example, i) brain disorder in the basal ganglia-thalamus region of the brain, ii) brain disorder in the basal ganglia and / or thalamus, iii) brain disorder in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorder in the basal ganglia, thalamus and internal capsule, and / or lacunar cerebral infarction and / or BAD) or cerebral infarction with brain disorder in the perforator distribution area in the subacute to chronic stage (especially the chronic stage) (for example, cerebral infarction with brain disorder in the perforator distribution area and the brain disorder is a brain disorder including i) brain disorder in the basal ganglia-thalamus region of the brain, ii) brain disorder in the basal ganglia and / or thalamus, iii) brain disorder in the basal ganglia-thalamus region and internal capsule, or iv) brain disorder in the basal ganglia, thalamus and internal capsule). In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for cerebral infarction other than perforator infarction and cerebral infarction with brain disorders in the perforator distribution area in the subacute phase to the chronic phase (especially the chronic phase). The screening method of the present invention can be a screening method for a cerebral infarction therapeutic agent for cerebral infarction of the same object as the object to which the treatment method of the present invention described later is applied.

[0315] The screening method of the cerebral infarction therapeutic agent of the present invention is particularly useful for evaluating and screening cerebral infarction therapeutic agents for perforator infarction in the subacute to chronic stages (especially the chronic stage) of humans (for example, i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule, and / or lacunar cerebral infarction and / or BAD) or cerebral infarction in the subacute to chronic stages (especially the chronic stage) of humans with brain disorders in the perforator distribution area (for example, brain disorders in the perforator distribution area and the brain disorders include i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule). Similarly, the screening method for cerebral infarction therapeutic agents of the present invention is also useful for evaluating and screening cerebral infarction therapeutic agents for cerebral infarction other than perforator infarction (i.e., cardiogenic cerebral embolism and atherothrombotic cerebral infarction) in the subacute to chronic stages (especially the chronic stage) of human cerebral infarction.

[0316] Cerebral infarction can be divided into cerebral cortical infarction, brainstem infarction, subcortical infarction (such as perforator infarction) and cerebellar infarction according to its onset site. In this specification, cerebral infarction can be cerebral cortical infarction, brainstem infarction, subcortical infarction (such as perforator infarction) and / or cerebellar infarction.

[0317] The screening method for the therapeutic agent for cerebral infarction of the present invention can also be used to screen for a therapeutic agent for cerebral infarction with a high degree of severity. In one embodiment, the screening method for the therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction for cerebral infarction with an mRS of 2 or more. In one embodiment, the screening method for the therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction for cerebral infarction with an mRS of 3 to 5. In one embodiment, the screening method for the therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction for cerebral infarction with a score of 4 or more on the impaired side in the motor system score of NHPSS. In one embodiment, the screening method for the therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction for cerebral infarction with a score of 12 or more on the impaired side in the motor system score of NHPSS. In one embodiment, the screening method for the therapeutic agent for cerebral infarction of the present invention is a method for screening a therapeutic agent for cerebral infarction for cerebral infarction with an mRS of 3 to 5 and a score of 12 or more on the impaired side in the motor system score of NHPSS.

[0318] The screening method of the cerebral infarction therapeutic agent of the present invention can also be used to screen a cerebral infarction therapeutic agent for cerebral infarction with localized brain disorders. In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for cerebral infarction with localized brain disorders. In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for cerebral infarction with localized brain disorders in the subacute stage to the chronic stage. In one embodiment, the screening method of the cerebral infarction therapeutic agent of the present invention is a method for screening a cerebral infarction therapeutic agent for cerebral infarction with localized brain disorders in the chronic stage.

[0319] In the screening method for the therapeutic agent for cerebral infarction of the present invention, the test substance administered to the non-human primate model of the present invention can be a nucleic acid (DNA or RNA (for example, messenger RNA (messenger RNA; mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide, etc.)). The nucleic acid can be a natural nucleic acid or an artificial nucleic acid (for example, a nucleic acid containing natural nucleic acid bases and / or artificial nucleic acid bases). Furthermore, the test substance can be any substance represented by peptides, proteins, cells, low molecular weight compounds, and medium molecular weight compounds, and examples thereof include substances with neural regeneration activity, various transcription factors, various neurotrophic factors, nerve cell growth factors, nerve cell growth promoting substances, direct reprogramming factors, neural stem cells, nerve cells, mesenchymal stem cells, etc. In the screening method for the therapeutic agent for cerebral infarction of the present invention, the means of administering the test substance is not limited to the following, and examples thereof include oral administration, injection (intravenous injection, subcutaneous injection, intramuscular injection, etc.), direct administration to the site of brain disorder (for example, disorder focus), and transplantation. The non-human primate model of the present invention can be administered with the test substance at any time. When the purpose is to screen for a cerebral infarction therapeutic agent for cerebral infarction in the subacute to chronic phase, the test substance is preferably administered to the non-human primate model of the present invention in the subacute to chronic phase (e.g., 3 weeks after the onset of cerebral infarction). Similarly, when the purpose is to screen for a cerebral infarction therapeutic agent for cerebral infarction in the chronic phase, the test substance is preferably administered to the non-human primate model of the present invention in the chronic phase (e.g., 2 weeks or 3 weeks after the onset of cerebral infarction).

[0320] The screening method of the cerebral infarction therapeutic agent of the present invention includes a step of determining the effect of the test substance administered to the non-human primate model of the present invention. The determination of the effect of the test substance administered on the cerebral infarction of the non-human primate model of the present invention can be implemented as follows: based on the observation of the individual mobility of the animal model, the rotational movement ability, the maintenance of the sitting posture, the standing action, the jumping movement ability, the ability to try to take objects, the ability to take feed, the ability to grasp objects and other motor functions, the diagnosis performed by CT or MRI, the electromyogram or NHPSS and / or mRS are evaluated, etc., to determine whether there is improvement compared with before the administration of the test substance, and thus implement it. The test substance determined to be improved compared with before the administration of the test substance can be identified as a cerebral infarction therapeutic agent. In addition, the result of the administration of the non-human primate model of the present invention in the subacute phase to the chronic phase is that the test substance determined to be improved compared with before the administration of the test substance can be identified as a cerebral infarction therapeutic agent in the subacute phase to the chronic phase. The result of administration of the non-human primate model of the present invention to perforator infarction (e.g., lacunar infarction and / or BAD) is that the test substance that is determined to be improved compared to before administration of the test substance can be identified as a therapeutic agent for cerebral infarction of perforator infarction (e.g., lacunar infarction and / or BAD). The result of administration of the non-human primate model of the present invention to perforator infarction (e.g., lacunar infarction and / or BAD) in the subacute to chronic stage is that the test substance that is determined to be improved compared to before administration of the test substance can be identified as a therapeutic agent for cerebral infarction of perforator infarction (e.g., lacunar infarction and / or BAD) in the subacute to chronic stage. The result of administration of the non-human primate model of the present invention to cerebral infarction with brain disorders in the perforator distribution area is that the test substance that is determined to be improved compared to before administration of the test substance can be identified as a therapeutic agent for cerebral infarction with brain disorders in the perforator distribution area.

[0321] 3. Pharmaceutical compositions and treatment methods, etc.

[0322] The present invention provides a pharmaceutical composition for treating cerebral infarction (a pharmaceutical composition for treating cerebral infarction), which contains a cerebral infarction therapeutic agent identified by the screening method for cerebral infarction therapeutic agents of the present invention, or a cerebral infarction therapeutic agent for perforator infarction, cerebral infarction with brain disorders in the perforator distribution area, or cerebral infarction in the subacute to chronic stages. The present invention also provides a pharmaceutical composition for treating perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase), containing NeuroD1 protein or a polynucleotide encoding NeuroD1 protein, a pharmaceutical composition for treating cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase), a pharmaceutical composition for treating cerebral infarction with brain disorders in the perforator distribution area (e.g., subacute to chronic phase or cerebral infarction in the chronic phase), a pharmaceutical composition for treating cerebral infarction with localized brain disorders (e.g., subacute to chronic phase or cerebral infarction in the chronic phase), a pharmaceutical composition for treating cerebral infarction with an mRS of 2 or more (e.g., subacute to chronic phase or cerebral infarction in the chronic phase), and a pharmaceutical composition for treating cerebral infarction with an NIHSS of 5 or more (e.g., subacute to chronic phase or cerebral infarction in the chronic phase). Although described in detail later, in this specification, "polynucleotide encoding NeuroD1 protein" refers to a polynucleotide containing a base sequence encoding NeuroD1 protein. In this specification, "base sequence encoding NeuroD1 protein" refers to a base sequence that can be translated into NeuroD1 protein. It should be noted that "base sequence encoding NeuroD1 protein" can be, for example, CDS (coding sequence; sequence from start codon to stop codon (including start codon and stop codon)) encoding NeuroD1 protein. In a typical embodiment, the polynucleotide encoding NeuroD1 protein encodes NeuroD1 protein in an expressible state. In the present invention, "encoding in an expressible state" means that the base sequence encoding NeuroD1 protein is configured in a functional state (for example, in the same frame) under the control of a sequence that can be transcribed into mRNA or can perform or promote mRNA translation (for example, a promoter, an enhancer, a Kozak sequence, a transcription response sequence, a terminator, a poly A sequence addition signal, a cap structure at the 5' end of mRNA, a poly A sequence at the 3' end of mRNA, etc.). In the present specification, pharmaceutical compositions for treating perforator infarction, treating cerebral infarction in the subacute to chronic stages, treating cerebral infarction with brain disorders in the perforator distribution area, treating cerebral infarction with localized brain disorders, treating cerebral infarction with an mRS of 2 or above, or treating cerebral infarction with an NIHSS of 5 or above, for example, which contain NeuroD1 protein or a polynucleotide encoding NeuroD1 protein, are also referred to as "pharmaceutical compositions of the present invention".Furthermore, the present invention provides a pharmaceutical composition for treating perforator infarction, a pharmaceutical composition for treating cerebral infarction in the subacute to chronic stages, a pharmaceutical composition for treating cerebral infarction with brain disorders in the perforator distribution area, a pharmaceutical composition for treating cerebral infarction with localized brain disorders, a pharmaceutical composition for treating cerebral infarction with an mRS of 2 or above, and / or a method for producing a pharmaceutical composition for treating cerebral infarction with an NIHSS of 5 or above, which comprises the step of producing the pharmaceutical composition using NeuroD1 protein or a polynucleotide encoding NeuroD1 protein.

[0323] "NeuroD1" (Neuronal differentiation 1) is a basic helix-loop-helix (bHLH) transcription factor belonging to the NeuroD family. NeuroD1 protein can activate the transcription of genes containing a specific DNA sequence known as E-box. That is, NeuroD1 protein has transcription factor activity. NeuroD1 has the core function of inducing differentiation from neural stem cells to neural cells. It is known that by ectopically expressing NeuroD1 in glial cells such as astrocytes and NG2 cells, glial cells can be converted into nerves (Patent Document 2). Glial cells can be divided into four types of cells: astrocytes, oligodendrocytes, ependymal cells, and microglia.

[0324] The NeuroD1 protein may be the following protein (i) or (ii):

[0325] (i) a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8, or an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, for example 99.4% or more or 99.5% or more to the amino acid sequence shown in SEQ ID NO: 6 or 8;

[0326] (ii) A protein consisting of an amino acid sequence having 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10 or 1 to 35) amino acids inserted, deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 6 or 8.

[0327] The NeuroD1 protein used in the pharmaceutical composition of the present invention preferably has transcription factor activity. It should be noted that SEQ ID NO: 6 or 8 is the amino acid sequence of mouse NeuroD1 protein or human NeuroD1 protein, respectively, and is the polypeptide sequence encoded by the CDS of mouse NeuroD1 gene or human NeuroD1 gene, respectively.

[0328] In one embodiment, NeuroD1 is human NeuroD1. The human NeuroD1 protein is the protein of (i) or (ii) below:

[0329] (i) a protein consisting of the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the amino acid sequence of SEQ ID NO: 8 and having transcription factor activity;

[0330] (ii) A protein consisting of an amino acid sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) amino acids inserted, deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 8 and having transcription factor activity.

[0331] In one embodiment, NeuroD1 is mouse NeuroD1. Mouse NeuroD1 protein is the protein of (i) or (ii) below:

[0332] (i) a protein consisting of the amino acid sequence of SEQ ID NO: 6 or an amino acid sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the amino acid sequence of SEQ ID NO: 6 and having transcription factor activity;

[0333] (ii) A protein consisting of an amino acid sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) amino acids inserted, deleted, substituted and / or added in the amino acid sequence shown in SEQ ID NO: 6 and having transcription factor activity.

[0334] Regarding whether a certain protein has transcription factor activity, a person skilled in the art can easily judge by, for example, preparing a recombinant vector in which a polynucleotide encoding the protein is integrated into a vector having an arbitrary promoter, introducing the vector into a cell to express the protein, measuring the expression level of a gene (also referred to as a regulated gene in this specification) whose expression (transcription) is regulated by the protein acting as a transcription factor, and comparing it with a cell into which the vector has not been introduced, and evaluating whether the expression level of the regulated gene has changed (increased or decreased), thereby making a judgment. For example, in a cell into which a vector containing a polynucleotide encoding NeuroD1 has been introduced, when the expression level of a regulated gene whose expression is hyperregulated by NeuroD1 acting as a transcription factor increases, it can be judged that the NeuroD1 protein has transcription factor activity.

[0335] The NeuroD1 protein contained in the pharmaceutical composition of the present invention can be encapsulated in any drug delivery system (DDS) that can deliver proteins to target cells. In one embodiment, the pharmaceutical composition of the present invention contains NeuroD1 protein and the protein is encapsulated in DDS. In one embodiment, the pharmaceutical composition of the present invention may contain NeuroD1 protein and the protein is encapsulated in a drug delivery carrier. In one embodiment, the pharmaceutical composition of the present invention may contain NeuroD1 protein and the protein is encapsulated in nanoparticles such as lipid nanoparticles and exosomes, but is not limited thereto.

[0336] In one embodiment, the polynucleotide encoding NeuroD1 protein is a polynucleotide selected from the group consisting of the following (i) to (iv):

[0337] (i) a polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8, or an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, for example 99.4% or more or 99.5% or more to the amino acid sequence shown in SEQ ID NO: 6 or 8;

[0338] (ii) a polynucleotide comprising a base sequence encoding a protein consisting of an amino acid sequence having 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10 or 1 to 35) amino acids inserted, deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 6 or 8;

[0339] (iii) a polynucleotide comprising the base sequence represented by SEQ ID NO: 5, 7 or 10, or a base sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, further preferably 99% or more, for example 99.4% or more or 99.5% or more to the base sequence represented by SEQ ID NO: 5, 7 or 10; and

[0340] (iv) A polynucleotide comprising a base sequence having 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10 or 1 to 35) bases inserted, deleted, substituted and / or added in the base sequence shown in SEQ ID NO: 5, 7 or 10.

[0341] The NeuroD1 protein encoded by these polynucleotides preferably has transcription factor activity. It should be noted that the base sequence shown in sequence number 5 is the CDS of the mouse NeuroD1 gene, and the base sequence shown in sequence number 7 or 10 is the CDS of the human NeuroD1 gene. The base sequence shown in sequence number 10 is a base sequence that differs from the base sequence shown in sequence number 7 only in the stop codon.

[0342] In one embodiment, the polynucleotide encoding the human NeuroD1 protein is a polynucleotide selected from the group consisting of the following (i) to (iv):

[0343] (i) a polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 8 or an amino acid sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the amino acid sequence set forth in SEQ ID NO: 8 and having transcription factor activity;

[0344] (ii) a polynucleotide comprising a base sequence encoding a protein having transcription factor activity consisting of an amino acid sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) amino acids inserted, deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 8;

[0345] (iii) a polynucleotide comprising the base sequence shown in SEQ ID NO: 7 or 10, or a base sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the base sequence shown in SEQ ID NO: 7 or 10 and encoding a protein having transcription factor activity; and

[0346] (iv) A polynucleotide comprising a base sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) bases inserted, deleted, substituted and / or added in the base sequence shown in SEQ ID NO: 7 or 10 and encoding a protein having transcription factor activity.

[0347] In one embodiment, the polynucleotide encoding the mouse NeuroD1 protein is a polynucleotide selected from the group consisting of the following (i) to (iv):

[0348] (i) a polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 6 or an amino acid sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the amino acid sequence set forth in SEQ ID NO: 6 and having transcription factor activity;

[0349] (ii) a polynucleotide comprising a base sequence encoding a protein having an amino acid sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) amino acids inserted, deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 6 and having transcription factor activity;

[0350] (iii) a polynucleotide comprising the base sequence shown in SEQ ID NO: 5 or a base sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the base sequence shown in SEQ ID NO: 5 and encoding a protein having transcription factor activity; and

[0351] (iv) A polynucleotide comprising a base sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) bases inserted, deleted, substituted and / or added in the base sequence shown in SEQ ID NO: 5 and encoding a protein having transcription factor activity.

[0352] A polynucleotide encoding NeuroD1 protein, for example, a polynucleotide encoding NeuroD1 protein in an expressible state, may be codon-optimized. Codon optimization may be performed using methods known in the art.

[0353] In one embodiment, the polynucleotide encoding NeuroD1 protein is a polynucleotide selected from the group consisting of the following (i) to (iii):

[0354] (i) a polynucleotide comprising a base sequence having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more to the base sequence of SEQ ID NO: 10 and encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8;

[0355] (ii) a polynucleotide comprising a base sequence having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more to the base sequence of SEQ ID NO: 7 and encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8; and

[0356] (iii) A polynucleotide containing a base sequence having a sequence identity of 60% or more, preferably 70% or more, more preferably 80% or more, and further preferably 90% or more or 95% or more to the base sequence shown in sequence number 5 and encoding a protein consisting of the amino acid sequence shown in sequence number 6.

[0357] In this specification, "sequence identity" refers to the ratio (%) of residues that are consistent between sequences when comparing a biological sequence (base sequence or amino acid sequence, etc.) as a benchmark and a target biological sequence (usually the ratio of consistent residues to the entire length of the target sequence). "Sequence identity" can be calculated, for example, using EMBOSS Needle (Weizhong L et al., Nucleic Acids Res., vol. 43, pp. W580-W584, 2015) in the form of a value of identity obtained using the prepared default parameters. The above parameters are as follows.

[0358] Gap Open Penalty=10

[0359] Gap Extend Penalty = 0.5

[0360] Matrix=EBLOSUM62

[0361] End Gap Penalty = false

[0362] The pharmaceutical composition of the present invention contains NeuroD1 protein or a polynucleotide encoding NeuroD1 protein. In one embodiment, the pharmaceutical composition of the present invention contains a polynucleotide selected from the group consisting of the following (i) to (xiii):

[0363] (i) a polynucleotide comprising a base sequence encoding a protein having transcription factor activity and consisting of an amino acid sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the amino acid sequence of SEQ ID NO: 6 or 8;

[0364] (ii) a polynucleotide comprising a base sequence encoding a protein having transcription factor activity consisting of an amino acid sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) amino acids inserted, deleted, substituted and / or added to the amino acid sequence of SEQ ID NO: 6 or 8;

[0365] (iii) a polynucleotide comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8;

[0366] (iv) a polynucleotide comprising the base sequence shown in SEQ ID NO: 5, 7 or 10, or a base sequence having a sequence identity of 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more to the base sequence shown in SEQ ID NO: 5, 7 or 10 and encoding a protein having transcription factor activity; and

[0367] (v) A polynucleotide comprising a base sequence having 1 to 10 (e.g., 1 to 2, 1 to 3, 1 to 5 or 1 to 7) bases inserted, deleted, substituted and / or added in the base sequence shown in SEQ ID NO: 5, 7 or 10 and encoding a protein having transcription factor activity.

[0368] (vi) a polynucleotide comprising a base sequence having a sequence identity of 70% or more to the base sequence shown in SEQ ID NO: 7 or 10;

[0369] (vii) a polynucleotide comprising a base sequence having a sequence identity of 70% or more to the base sequence of SEQ ID NO: 7 or 10 and encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8;

[0370] (viii) a polynucleotide comprising a base sequence having a sequence identity of 70% or more to the base sequence of SEQ ID NO: 7 and encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8;

[0371] (ix) a polynucleotide comprising a base sequence having a sequence identity of 70% or more to the base sequence of SEQ ID NO: 10 and encoding a protein consisting of the amino acid sequence of SEQ ID NO: 8;

[0372] (x) a polynucleotide comprising the base sequence shown in SEQ ID NO: 7 or 10;

[0373] (xi) a polynucleotide comprising the base sequence shown in SEQ ID NO: 7;

[0374] (xii) a polynucleotide comprising the base sequence shown in SEQ ID NO: 10; and

[0375] (xiii) a polynucleotide comprising a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114, the base sequence at positions 44 to 1120, or the base sequence at positions 1 to 1231 of sequence number 11 or 13, or consisting of the base sequence, and preferably encoding a protein (preferably a protein having transcription factor activity) consisting of an amino acid sequence having a sequence identity of 90% or more (e.g., 95% or more) with respect to the amino acid sequence shown in sequence number 8.

[0376] Polynucleotides encoding NeuroD1 protein, for example, polynucleotides encoding NeuroD1 protein in an expressible state are not limited to the following, and may be DNA or RNA such as mRNA, or may be a combination thereof (chimeric nucleic acid). In addition, the polynucleotide may be a natural nucleic acid or an artificial nucleic acid (e.g., a nucleic acid containing a natural nucleic acid base and / or an artificial nucleic acid base).

[0377] In one embodiment, a polynucleotide encoding NeuroD1 protein in an expressible state may be included in an expression vector. The expression vector containing a polynucleotide encoding NeuroD1 protein in the present invention contains a polynucleotide (e.g., DNA or RNA) containing a base sequence encoding NeuroD1 protein in an expressible state under the control of a sequence capable of performing or promoting transcription to mRNA or translation of mRNA, that is, the expression vector contains a polynucleotide encoding NeuroD1 protein in an expressible state. In the present invention, sequences capable of performing or promoting transcription to mRNA or translation of mRNA (e.g., promoters, enhancers, Kozak sequences, transcription response sequences, terminators, poly A sequence addition signals, cap structures at the 5' end of mRNA, poly A sequences at the 3' end of mRNA, etc.) are not particularly limited as long as they can function in the organism of the subject of treatment. The promoter may be any promoter such as a constitutive promoter, a cell-specific promoter, a tissue-specific promoter, a transient promoter, or an inducible promoter, depending on the purpose of use. For example, promoters that can function in the human body include, but are not limited to, human GFAP promoter, CMV promoter, EF-1α promoter, PGK promoter, and MSCV promoter. Human GFAP promoter may contain the base sequence shown in SEQ ID NO: 14. In order to deliver a polynucleotide encoding NeuroD1 protein to astrocytes, it is particularly preferred to use the GFAP promoter.

[0378] In the present invention, the expression vector can be any vector such as a viral vector, a plasmid vector, etc. The expression vector used in the present invention is not limited to the following, and can be an adeno-associated virus (AAV) vector, an adenovirus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector, and an AAV vector is particularly preferred. When the expression vector is a viral vector, the expression vector contains a polynucleotide containing a base sequence encoding NeuroD1 protein under the control of a sequence (promoter, etc.) that can perform or promote transcription or translation of mRNA in the form of a nucleic acid vector packaged in a viral particle.

[0379] In the present invention, as long as NeuroD1 can be expressed in host cells, the serotype of the AAV vector is not particularly limited, and AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, etc. can be used. In one embodiment, the serotype of AAV may be AAV9.

[0380] The expression vector can be manufactured using methods known in the art. As an example of a method for manufacturing an AAV vector, an AAV vector plasmid is prepared in which an arbitrary promoter sequence (e.g., human GFAP promoter sequence) and an arbitrary nucleic acid molecule sequence (e.g., a base sequence encoding NeuroD1 protein) are inserted into a commercially available vector plasmid (e.g., pAAV-CMV Vector (Takara Biotech)). In addition, a plasmid containing the Rep gene and Cap gene of AAV that supplements the auxiliary function of AAV and a plasmid containing auxiliary virus additional genes (e.g., regions VA, E2A, E4) that promote AAV replication are prepared. These three plasmids are co-transfected into packaging cells to produce recombinant AAV (i.e., AAV vector) in the cells. The packaging cells are then cultured to generate AAV vectors, and the AAV vectors are purified using standard techniques known in the art. As examples of packaging cells used, mammalian cells can be cited, specifically 293T cells, A549 cells, HeLa cells, and HEK293 cells, but are not limited to these. As a purification method, cesium chloride density gradient centrifugation, digestion of DNA and RNA using Benzonase, sucrose gradient centrifugation, iodixanol density gradient centrifugation, ultrafiltration, diafiltration, affinity chromatography, ion exchange chromatography, polyethylene glycol precipitation, ammonium sulfate precipitation, etc. can be used.

[0381] In one embodiment, the pharmaceutical composition of the present invention comprises an AAV vector, a retroviral vector, a Sendai virus vector or a lentiviral vector containing a polynucleotide encoding NeuroD1 protein. In one embodiment, the pharmaceutical composition of the present invention comprises an AAV vector containing a polynucleotide encoding NeuroD1 protein. In one embodiment, the pharmaceutical composition of the present invention comprises an AAV9 vector containing a polynucleotide encoding NeuroD1 protein.

[0382] "mRNA" refers to an RNA containing a base sequence encoding (at least one) polypeptide (e.g., a naturally occurring polypeptide, a polypeptide having an insertion, deletion, substitution and / or addition of amino acids relative to a naturally occurring polypeptide, or a non-naturally occurring polypeptide) under the control of a sequence that can carry out or promote the translation of the mRNA (e.g., a Kozak sequence, a start codon, a cap structure at the 5' end of the mRNA, a 5' untranslated region (UTR), a 3'UTR, a poly A sequence at the 3' end of the mRNA, etc.). The mRNA can be translated, for example, in vitro or in vivo to produce the encoded polypeptide. In one embodiment, the mRNA can be translated in vivo to produce the encoded polypeptide.

[0383] In one embodiment, the polynucleotide encoding NeuroD1 protein can be mRNA encoding NeuroD1 protein. In the present invention, "mRNA encoding NeuroD1 protein" refers to an RNA containing a polynucleotide containing a base sequence encoding NeuroD1 protein under the control of a sequence capable of performing or promoting the translation of mRNA. The mRNA encoding NeuroD1 protein in the present invention typically contains a polynucleotide containing a base sequence encoding NeuroD1 protein in a functional state under the control of a sequence capable of performing or promoting the translation of mRNA, that is, contains a polynucleotide encoding NeuroD1 protein in an expressible state. In one embodiment, the mRNA encoding NeuroD1 protein in the present invention is a polynucleotide encoding NeuroD1 protein containing a functional sequence for expressing NeuroD1 protein (for example, including CDS, 5'UTR, 3'UTR, 5' cap structure and poly A sequence, but not limited thereto).

[0384] The polynucleotides described in this specification may record a representative DNA sequence in the form of a polynucleotide defined by a base sequence containing "T", and when the polynucleotide is RNA (e.g., mRNA), "T" is replaced by "U". Therefore, for example, when a DNA sequence is represented by a base sequence defined by a specific sequence number, for RNA (e.g., mRNA) containing the base sequence defined by the specific sequence number, the base sequence can be understood as an RNA sequence in which each "T" of the DNA sequence is replaced by "U". When the bases "adenine (A)", "thymine (T)", "guanine (G)", "cytosine (C)" and "uracil (U)" constituting the base sequence (e.g., the base sequence shown in sequence numbers 1 to 3, 5, 7, 10 to 14), and the nucleosides and nucleotides containing them are not specifically described in the sequence table, each base, nucleoside and nucleotide may be a natural type or a modified type corresponding thereto, and / or may have other modifications (methylation, etc.).

[0385] The mRNA may contain a CDS, a 5'UTR, a 3'UTR, a 5' cap structure and a poly A sequence. The CDS contained in the mRNA encoding the NeuroD1 protein may be codon optimized. The stop codon located at the 3' end of the CDS is not particularly limited as long as it can stop the translation from the mRNA to the NeuroD1 protein, and any stop codon of TAA, TGA, or TAG may be used. In addition, multiple stop codons may be further used following the stop codon contained in the CDS (for example, TGATAG may be used following the stop codon TAA contained in the CDS). The UTR contained in the mRNA encoding the NeuroD1 protein is not particularly limited as long as it can express the NeuroD1 protein, for example, it may be the 5'UTR and / or 3'UTR from the NeuroD1 gene, or it may be the 5'UTR and / or 3'UTR from a heterologous gene (for example, the human α-globin gene, the human β-globin gene, etc.). The 5'UTR and the 3'UTR may be from the same heterologous gene, or they may be from different heterologous genes. UTR can be a natural type, or a modified type in which the sequence is changed relative to the natural UTR by insertion, deletion, substitution and / or addition of one or several (e.g., 2, 3, 4, 5 or 6) nucleotides. The "UTR from a gene" in this specification includes not only natural UTRs, but also such modified UTRs. Regarding 5'UTR and / or 3'UTR, multiple UTRs can be directly connected or connected with a spacer sequence. The 5'UTR may contain a portion of a Kozak sequence (e.g., the 5' side sequence of the start codon in the Kozak sequence). As a 5' cap structure, for example, Cap-0, Cap-1, Cap-2, etc. can be listed (Anand R et al., Nucleic Acids Res., vol.44(16), pp.7511-7526, 2016). Cap-0 is a structure in which guanosine is bound to the 5' end of mRNA via a triphosphate bond (5'-5' bond) and the 7-position of the guanine base of guanosine is methylated, and is also called a 7-methylguanylate cap. Cap-1 is a structure in which the 2-position of the ribose of the first nucleotide at the 5' end of mRNA (the nucleotide that forms a 5'-5' bond with the guanosine of Cap-0) is methylated based on Cap-0, and Cap-2 is a structure in which the 2-position of the ribose of the first and second nucleotides from the 5' end of mRNA is methylated based on Cap-0. The length of the poly A sequence is not particularly limited as long as it has functions for mRNA stabilization, nuclear transport, translation, etc., and can be, for example, 20 to 1000 nucleotides (for example, 30 to 500, 50 to 200, 60 to 150, 70 to 130, 70 to 120, 70 to 90, 70 to 85, 70 to 80, 75 to 85, at least 70, at least 75 or 79 nucleotides).The poly A sequence may be a sequence in which a plurality of poly A sequences are linked via a spacer sequence consisting of nucleotides other than A (International Publication No. WO2016 / 005004, etc.).

[0386] In one embodiment, the mRNA encoding NeuroD1 protein is the following mRNA (i) or (ii):

[0387] (i) an mRNA comprising a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 6 or 8, or an amino acid sequence having a sequence identity of 90% or more, preferably 95% or more, more preferably 98% or more, still more preferably 99% or more, for example 99.4% or more or 99.5% or more to the amino acid sequence shown in SEQ ID NO: 6 or 8; or

[0388] (ii) mRNA containing a base sequence encoding a protein consisting of an amino acid sequence having 1 to 50 (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10 or 1 to 35) amino acids inserted, deleted, substituted and / or added to the amino acid sequence shown in SEQ ID NO: 6 or 8. The protein (NeuroD1 protein) encoded by the polynucleotide preferably has transcription factor activity.

[0389] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a polypeptide having a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the protein consisting of the amino acid sequence shown in SEQ ID NO: 8. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0390] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence shown in SEQ ID NO: 7 or 10 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence shown in SEQ ID NO: 7 or 10 and encoding a polypeptide having a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the protein consisting of the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence shown in SEQ ID NO: 10 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence shown in SEQ ID NO: 10 and encoding a polypeptide having a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) with respect to the protein consisting of the amino acid sequence shown in SEQ ID NO: 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in SEQ ID NO: 10. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence shown in sequence number 7 and encoding a protein consisting of the amino acid sequence shown in sequence number 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence shown in sequence number 7 and encoding a polypeptide having a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) with respect to the protein consisting of the amino acid sequence shown in sequence number 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 7. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0391] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of sequence number 11 or 13, and encoding a polypeptide having a sequence identity of 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) with respect to the protein consisting of the amino acid sequence shown in sequence number 8. The protein preferably has transcription factor activity. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of sequence number 11 or 13, and encoding a protein consisting of the amino acid sequence shown in sequence number 8. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 11 or 13. The base sequence from positions 44 to 1114 of SEQ ID NO: 11 or 13 is the CDS of the human NeuroD1 gene, and the base sequence from positions 44 to 1120 of SEQ ID NO: 11 or 13 contains the CDS of the human NeuroD1 gene and a plurality of stop codons.

[0392] In one embodiment, the mRNA encoding the NeuroD1 protein contains a 5'UTR and / or a 3'UTR. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a 5'UTR and / or a 3'UTR from a heterologous gene (e.g., a human α-globin gene, a human β-globin gene, etc.). In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a 5'UTR and / or a 3'UTR from a human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR from a human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing the 5'UTR and / or 3'UTR from the human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, and further containing the 5'UTR and / or 3'UTR from the human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a 5'UTR consisting of the base sequence from positions 4 to 43 of sequence number 11 or 13 and / or a 3'UTR consisting of the base sequence from positions 1121 to 1231 of sequence number 11 or 13. In one embodiment, the mRNA encoding the NeuroD1 protein contains a polynucleotide consisting of the base sequence from positions 4 to 1231 of sequence number 11 or 13. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence from positions 4 to 1231 of sequence number 11 or 13 and encoding a protein consisting of the amino acid sequence shown in sequence number 8.

[0393] In one embodiment, the mRNA encoding the NeuroD1 protein contains a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a poly A sequence consisting of 50 to 200 nucleotides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a poly A sequence consisting of 70 to 90 nucleotides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a poly A sequence consisting of 75 to 85 nucleotides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a poly A sequence consisting of 120 nucleotides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a poly A sequence consisting of 79 nucleotides.

[0394] In one embodiment, the length of the poly A sequence of the mRNA encoding the NeuroD1 protein is 30 to 500 nucleotides (e.g., 40 to 200, 50 to 200, 50 to 150, 50 to 100, 50 to 90, 60 to 150, 60 to 100, 60 to 90, 70 to 130, 70 to 120, 70 to 100, 70 to 90, 70 to 85, 70 to 80, 75 to 130, 75 to 120, 75 to 100, 75 to 90, preferably 74 to 84, 75 to 85, 75 to 83 or 76 to 82 nucleotides). In one embodiment, the length of the poly A sequence of the mRNA encoding the NeuroD1 protein is 74 to 84 nucleotides. In one embodiment, the length of the poly A sequence of the mRNA encoding the NeuroD1 protein is 75 to 85 nucleotides.

[0395] In one embodiment, the mRNA encoding the NeuroD1 protein contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further contains a 5'UTR and / or a 3'UTR and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further contains a 5'UTR and / or a 3'UTR and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, and further contains a 5'UTR and / or a 3'UTR and a poly A sequence.

[0396] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence from a human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence from a human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence from positions 44 to 1114 or positions 44 to 1120 of sequence number 11 or 13, and further containing the 5'UTR and / or 3'UTR from the human α-globin gene and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a 5'UTR consisting of the base sequence from positions 4 to 43 of sequence number 11 or 13 and / or a 3'UTR consisting of the base sequence from positions 1121 to 1231 of sequence number 11 or 13 and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from positions 4 to 1231 of sequence number 11 or 13 and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence and a poly A sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence number 11 or 13 and encoding a protein consisting of the amino acid sequence shown in sequence number 8.

[0397] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 7 or 10, and further containing a 5'UTR consisting of the base sequence from the 4th to the 43rd of sequence number 11 or 13 and / or a 3'UTR consisting of the base sequence from the 1121st to the 1231st of sequence number 11 or 13 and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from the 4th to the 1231st of sequence number 11 or 13 and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence from the 4th to the 1231st of sequence number 11 or 13 and encoding a protein consisting of the amino acid sequence shown in sequence number 8 and a poly A sequence.

[0398] mRNA can be manufactured using methods known in the art. As an example of a method for manufacturing mRNA, a plasmid is prepared in which any UTR (e.g., 5'UTR and 3'UTR of human α-globin) and any base sequence of a nucleic acid molecule (e.g., a base sequence encoding NeuroD1 protein) are inserted into a commercially available plasmid (e.g., a high-copy plasmid for E. coli (e.g., pUC18, pCU18, etc.)). The prepared plasmid can be amplified, for example, with E. coli and purified using methods known in the art. For the purified plasmid, for example, linearization can be performed using methods known in the art, such as methods using restriction enzymes and buffers, but are not limited to these. The linearized reaction product can be purified, for example, using a method using PureLink (registered trademark) (Thermo Fisher Scientific). The purification method can be changed according to the size of the linearized reaction product. The purified linear reaction product can be used directly as a DNA template, or a DNA template can be generated by providing the linearized reaction product to a polymerase chain reaction (PCR). The generated DNA template can be transcribed using an in vitro transcription (IVT) system. The system typically contains a transcription buffer, nucleoside triphosphates (NTPs), an RNase inhibitor, and a polymerase (e.g., T7 RNA polymerase). In the present invention, NTPs may be natural or non-natural (modified). The DNA template may be removed using methods known in the art, such as treatment with deoxyribonuclease I (DNase I), but is not limited thereto. Purification of the transcribed RNA may, for example, be performed using a silica column. When the RNA contains a poly A sequence, purification may be performed using, for example, an Oligo dT column. mRNA may be produced by a capping reaction and / or a poly A addition reaction. The capping reaction may be performed using methods known in the art, such as adding a 5' cap structure to the 5' end of the sequence transcribed by IVT or adding a 5' cap structure to the 5' end during IVT. The capping method can be performed using methods known in the art, such as a method using vaccinia capping enzyme and 2'O-methyltransferase, a method using CleanCap (registered trademark) (TriLink BioTechnologies), etc., but is not limited to these. When the sequence obtained by IVT transcription does not contain a poly A sequence, a poly A addition reaction can be performed. The constructed mRNA can be purified using methods known in the art.

[0399] In one embodiment, the mRNA encoding the NeuroD1 protein may have a cap structure and may contain a 5' terminal sequence suitable for the capping method. For example, when using the capping method using CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), the 5' end may contain the base sequence AGG. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence (AGG) at positions 1 to 3 of sequence number 11 or 13 from the 5' side, a 5'UTR, a base sequence encoding a protein composed of the amino acid sequence shown in sequence number 8, a 3'UTR, and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing, from the 5' side, the base sequence (AGG) at positions 1 to 3 of SEQ ID NO: 11 or 13, a 5'UTR, a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, a 3'UTR, and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing, from the 5' side, the base sequence (AGG) at positions 1 to 3 of SEQ ID NO: 11 or 13, a 5'UTR, the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, a 3'UTR, and a poly A sequence.

[0400] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence (AGG) at positions 1 to 3 of SEQ ID NO: 11 or 13 from the 5' side, the 5'UTR from the human α-globin gene, a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and the 3'UTR and poly A sequence from the human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence (AGG) at positions 1 to 3 of SEQ ID NO: 11 or 13 from the 5' side, the 5'UTR from the human α-globin gene, a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, the 3'UTR and poly A sequence from the human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing, from the 5' side, the base sequence (AGG) at positions 1 to 3 of SEQ ID NO: 11 or 13, the 5'UTR derived from the human α-globin gene, the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, the 3'UTR derived from the human α-globin gene, and a poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing, from the 5' side, the base sequence (AGG) from the 1st to the 3rd base sequence of SEQ ID NO: 11 or 13, the 5'UTR consisting of the base sequence from the 4th to the 43rd base sequence of SEQ ID NO: 11 or 13, the base sequence from the 44th to the 1114th or the 44th to the 1120th base sequence of SEQ ID NO: 11 or 13, the 3'UTR consisting of the base sequence from the 1121st to the 1231st base sequence of SEQ ID NO: 11 or 13, and the poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from the 1st to the 1231st base sequence of SEQ ID NO: 11 or 13 and the poly A sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence (AGG) at positions 1 to 3 of sequence number 11 or 13 from the 5' side, a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence number 11 or 13 and encoding a protein consisting of the amino acid sequence shown in sequence number 8, and a poly A sequence.

[0401] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in SEQ ID NO: 11 or 13 or consisting of the base sequence.

[0402] In the present invention, the mRNA encoding NeuroD1 protein can be a modified mRNA. For example, by using modified NTPs when manufacturing mRNA, modified mRNA can be manufactured. Modified mRNA may have modifications (e.g., chemical modifications) located on the nucleic acid bases, sugar moieties, and / or phosphoric acid of the nucleotide. Modified mRNA, for example, contains modified nucleosides. "Modified nucleosides" refer to modified nucleosides. For example, nucleosides that have been modified by methylation, atom exchange, saturation of double bonds, deamination, or replacement of oxygen atoms with sulfur atoms, etc. Modified nucleosides can be pseudouridine, N1-methylpseudouridine (also known as 1-methylpseudouridine), 5-methylcytidine, 7-methylguanosine, N6-methyladenosine, 1-methyladenosine, dihydrouridine, inosine, 4-thiouridine, etc., but are not limited to these. Polynucleotides in which "part or all" of a specific type of nucleosides are replaced with modified nucleosides include: polynucleotides in which a part of such nucleosides contained in the original polynucleotide sequence (specifically, mRNA encoding NeuroD1 protein) are replaced with modified nucleosides and the other parts are not replaced with modified nucleosides, and polynucleotides in which all such nucleosides contained in the original polynucleotide sequence are replaced with modified nucleosides. In the sequence of a polynucleotide containing modified nucleosides, "a part" related to the ratio of a specific type of nucleosides replaced with modified nucleosides includes one or more nucleosides, and the replacement ratio of a specific type of nucleosides relative to the total number of residues may be, for example, 60-99%, 70-99%, 80-99%, 90-99%, 95-99%, 60-99.9%, 70-99.9%, 80-99.9%, 90-99.9% or 95-99.9%, but is not limited to these. In one embodiment, the mRNA encoding NeuroD1 protein in the present invention may contain modified nucleosides. In one embodiment, the mRNA encoding the NeuroD1 protein of the present invention may contain N1-methylpseudouridine. In one embodiment, in the mRNA encoding the NeuroD1 protein of the present invention, a part or all of the uridine may be replaced by N1-methylpseudouridine. In one embodiment, in the mRNA encoding the NeuroD1 protein of the present invention, all of the uridine may be replaced by N1-methylpseudouridine.

[0403] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11, and containing a modified nucleoside, preferably N1-methylpseudouridine.

[0404] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and part or all, preferably all uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, which has a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and part or all, preferably all uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, and part or all, preferably all uridines are substituted with N1-methylpseudouridine.

[0405] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR, and containing a modified nucleoside, preferably N1-methylpseudouridine, having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and encoding a base sequence consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further containing 5'UTR and / or 3'UTR, and containing modified nucleosides, preferably N1-methylpseudouridine.

[0406] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA comprising the base sequence of bases 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further comprising a 5'UTR and / or a 3'UTR, and wherein some or all, preferably all, uridines are substituted with N1-methylpseudouridine.

[0407] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR and a poly A sequence, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR and a poly A sequence, and containing a modified nucleoside, preferably N1-methylpseudouridine, having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR and a poly A sequence, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further containing a 5'UTR and / or 3'UTR and a poly A sequence, and containing modified nucleosides, preferably N1-methylpseudouridine.

[0408] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR and a poly A sequence, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or a 3'UTR and a poly A sequence, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA comprising the base sequence of bases 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further comprising a 5'UTR and / or 3'UTR and a poly A sequence, and wherein some or all, preferably all, uridines are substituted with N1-methylpseudouridine.

[0409] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine, having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, further containing a 5'UTR and / or a 3'UTR from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further containing the 5'UTR and / or 3'UTR from the human α-globin gene, and containing modified nucleosides, preferably N1-methylpseudouridine.

[0410] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 and further containing a 5'UTR and / or 3'UTR from a human α-globin gene, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 and further containing a 5'UTR and / or 3'UTR from a human α-globin gene, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 and further containing a 5'UTR and / or 3'UTR from a human α-globin gene, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence of bases 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 and further containing the 5'UTR and / or 3'UTR derived from the human α-globin gene, and in which part or all, preferably all, uridine is substituted with N1-methylpseudouridine.

[0411] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine, having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, and containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence from a human α-globin gene, and containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11, further containing the 5'UTR and / or 3'UTR and poly A sequence from the human α-globin gene, and containing modified nucleosides, preferably N1-methylpseudouridine.

[0412] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence derived from a human α-globin gene, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and further containing a 5'UTR and / or 3'UTR and a poly A sequence derived from a human α-globin gene, and a part or all, preferably all, of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 and further containing the 5'UTR and / or 3'UTR and poly A sequence from the human α-globin gene, and in which part or all, preferably all, uridine is substituted with N1-methylpseudouridine.

[0413] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11 and containing modified nucleosides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11 and containing N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11, and a part or all of the uridine is replaced by N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11, and all uridine is replaced by N1-methylpseudouridine. The mRNA encoding the NeuroD1 protein in which all uridine is replaced by N1-methylpseudouridine can be, for example, a polynucleotide containing the base sequence shown in sequence number 13.

[0414] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence and a poly A sequence that have a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence numbers 11 or 13 and encodes a protein consisting of the amino acid sequence shown in sequence number 8, and has a modified nucleoside. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence and a poly A sequence that have a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence numbers 11 or 13 and encodes a protein consisting of the amino acid sequence shown in sequence number 8, and has a N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence and a poly A sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence at positions 4 to 1231 of SEQ ID NO: 11 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and a part or all of the uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence and a poly A sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence at positions 4 to 1231 of SEQ ID NO: 11 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8, and all of the uridines are N1-methylpseudouridine.

[0415] In one embodiment, the mRNA encoding NeuroD1 protein has a 5' cap structure (eg, Cap-0, Cap-1, or Cap-2). In one embodiment, the 5' cap structure contained in the mRNA encoding NeuroD1 protein is Cap-1.

[0416] In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (e.g., the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), further contains a 5'UTR and / or a 3'UTR, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (e.g., the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and further contains a 5'UTR and / or 3'UTR and a poly A sequence, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (e.g., the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), further contains the 5'UTR and / or 3'UTR from the human α-globin gene, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and further contains the 5'UTR and / or 3'UTR from the human α-globin gene and a poly A sequence, and has a 5' cap structure.

[0417] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), contains modified nucleosides, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), further contains a 5'UTR and / or a 3'UTR, contains modified nucleosides, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and further contains a 5'UTR and / or a 3'UTR and a poly A sequence, contains modified nucleosides, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and further contains the 5'UTR and / or 3'UTR from the human α-globin gene, contains modified nucleosides, preferably N1-methylpseudouridine, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 (for example, the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13, or a base sequence having a sequence identity of 70% or more (for example, 80% or more, 90% or more, or 95% or more) with respect to the base sequence of positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 11 or 13), and further contains the 5'UTR and / or 3'UTR from the human α-globin gene and a poly A sequence, contains modified nucleosides, preferably N1-methylpseudouridine, and has a 5' cap structure.

[0418] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from positions 1 to 1231 of sequence number 11 or 13 and a poly A sequence, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from positions 4 to 1231 of sequence number 11 or 13 and a poly A sequence, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence from positions 4 to 1231 of sequence number 11 or 13 and encoding a protein consisting of the amino acid sequence shown in sequence number 8 and a poly A sequence, and having Cap-1 as a 5' cap structure.

[0419] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from positions 1 to 1231 of SEQ ID NO: 11 or 13 and a poly A sequence, and containing modified nucleosides, preferably N1-methylpseudouridine, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from positions 4 to 1231 of SEQ ID NO: 11 or 13 and a poly A sequence, and containing modified nucleosides, preferably N1-methylpseudouridine, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence number 11 or 13, encodes a protein consisting of the amino acid sequence shown in sequence number 8, and a poly A sequence, contains modified nucleosides, preferably N1-methylpseudouridine, and has Cap-1 as a 5' cap structure.

[0420] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a polynucleotide consisting of the base sequence from positions 1 to 1231 of sequence number 11 or 13 and a poly A sequence, in which a part or all, preferably all uridines are substituted with N1-methylpseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a polynucleotide consisting of the base sequence from positions 4 to 1231 of sequence number 11 or 13 and a poly A sequence, in which a part or all, preferably all uridines are substituted with N1-methylpseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of sequence number 11 or 13, encoding a protein consisting of the amino acid sequence shown in sequence number 8, and a poly A sequence, in which a part or all, preferably all, of the uridine is substituted with N1-methylpseudouridine, and having Cap-1 as a 5' cap structure.

[0421] In one embodiment, the mRNA encoding NeuroD1 protein comprises a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and an mRNA of a poly A sequence, wherein:

[0422] (i) 7-methylguanosine is bonded to the 5' end of the polynucleotide consisting of the base sequence from bases 1 to 1231 of SEQ ID NO: 11 via a triphosphate bond (5'-5' bond),

[0423] (ii) the adenosine at position 1 of sequence number 11 is 2'-O-methyladenosine, and

[0424] (iii) A portion or all of the uridine is replaced by N1-methylpseudouridine.

[0425] In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 13 and a poly A sequence, wherein:

[0426] (i) 7-methylguanosine is bonded to the 5' end of the polynucleotide consisting of the base sequence from bases 1 to 1231 of SEQ ID NO: 13 via a triphosphate bond (5'-5' bond), and

[0427] (ii) The adenosine at position 1 of SEQ ID NO: 13 is 2'-O-methyladenosine.

[0428] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11, in which a part or all of the uridine is replaced by N1-methyl pseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 11, in which all of the uridine is replaced by N1-methyl pseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA containing the base sequence shown in sequence number 13, and has Cap-1 as a 5' cap structure.

[0429] In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence shown in SEQ ID NO: 11, wherein:

[0430] (i) 7-methylguanosine is bonded to the 5' end of the base sequence represented by SEQ ID NO: 11 via a triphosphate bond (5'-5' bond),

[0431] (ii) the adenosine at position 1 of sequence number 11 is 2'-O-methyladenosine, and

[0432] (iii) A portion or all of the uridine is replaced by N1-methylpseudouridine.

[0433] In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA containing the base sequence shown in SEQ ID NO: 13, wherein:

[0434] (i) 7-methylguanosine is bonded to the 5' end of the base sequence represented by SEQ ID NO: 13 via a triphosphate bond (5'-5' bond), and

[0435] (ii) The adenosine at position 1 of SEQ ID NO: 13 is 2'-O-methyladenosine.

[0436] In one embodiment, the mRNA encoding the NeuroD1 protein may be an mRNA that does not contain a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the base sequence shown in sequence number 11 and containing modified nucleosides. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the base sequence shown in sequence number 11 and containing N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the base sequence shown in sequence number 11, and a part or all of the uridine is replaced by N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the base sequence shown in sequence number 11, and all uridine is replaced by N1-methylpseudouridine. The mRNA encoding the NeuroD1 protein in which all uridine is replaced by N1-methylpseudouridine can be, for example, a polynucleotide consisting of the base sequence shown in sequence number 13.

[0437] In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein, the mRNA may be encapsulated in DDS. In one embodiment, the pharmaceutical composition of the present invention may contain mRNA encoding NeuroD1 protein and the mRNA may be encapsulated in a "drug delivery system" (DDS).

[0438] As the DDS that can be used in the present invention, it is preferred that the DDS can control the in vivo distribution of the mRNA encoding the NeuroD1 protein, improve the effect of the mRNA, and inhibit side effects. In one embodiment, the DDS can be a drug delivery carrier. As drug delivery carriers, nanoparticles such as lipid nanoparticles, polymer particles (for example, nanomicelle carriers formed by block copolymers formed by connecting polyethylene glycol and biocompatible cationic polymers) can be listed. In one embodiment, the pharmaceutical composition of the present invention contains the mRNA encoding the NeuroD1 protein and the mRNA is encapsulated in the drug delivery carrier. In one embodiment, the pharmaceutical composition of the present invention contains the mRNA encoding the NeuroD1 protein and the mRNA is encapsulated in the nanoparticles.

[0439] "Lipid nanoparticles" (lipid nanoparticles; LNP) are nanoparticles composed of lipids with a diameter ranging from 10nm to 1000nm (10nm and less than 1000nm), and can be used as DDS for encapsulating nucleic acid drugs, etc. As pharmaceutical compositions containing protein-encoding mRNA and the mRNA encapsulated in lipid nanoparticles, for example, RNA vaccines encapsulated in LNP are known (for example, LRBaden et al., The New England Journal of Medicine, vol 384, No.5, pp.403-416, 2021, Fernando et al., The New England Journal of Medicine, vol 383, No.27, pp 2603-2615, 2020). Lipid nanoparticles contain any lipid such as cationic lipids, PEGylated lipids, sterols, neutral phospholipids, etc. In one embodiment, the lipid nanoparticles contain cationic lipids. In one embodiment, the lipid nanoparticles contain neutral phospholipids, sterols, and PEGylated lipids. In one embodiment, the lipid nanoparticles contain cationic lipids, neutral phospholipids, sterols and PEGylated lipids. As PEGylated lipids, for example, DSPE-mPEG, ALC-0159, DMG-mPEG, DMG-PEG2000, etc. can be cited. As sterols, for example, cholesterol can be cited. As neutral phospholipids, for example, distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE), etc. can be cited. As cationic lipids (or ionized lipids), for example, DLin-MC3-DMA used in lipid nanoparticles encapsulating siRNA can be cited. In one embodiment, the lipid nanoparticles contain DSPC, cholesterol and DMG-PEG2000. In one example, lipid nanoparticles containing RNA can be composed of an outer lipid layer and a cationic lipid core surrounding the RNA and wrapped therein, wherein the outer lipid layer is composed of PEGylated lipids (e.g., DSPE-mPEG, ALC-0159, DMG-mPEG, DMG-PEG2000), sterols (e.g., cholesterol), neutral phospholipids (e.g., distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE, etc.)) and cationic lipids (or ionizable lipids) (e.g., DLin-MC3-DMA used in lipid nanoparticles encapsulating siRNA).Such RNA-containing lipid nanoparticles can typically be manufactured as follows: lipid-containing ethanol and RNA-containing low pH buffer are rapidly mixed with a mixer (e.g., microfluidic mixing), thereby promoting RNA encapsulation, and then adjusting the pH to gradually increase it to near neutrality (e.g., pH 7.4), removing the ethanol to form lipid nanoparticles. The above-mentioned PEGylated lipids, sterols, neutral phospholipids, and cationic lipids constituting the lipid nanoparticles can be manufactured using methods known in the art.

[0440] Other examples of cationic lipids that can be used in lipid nanoparticles include, for example, compound 1 (Ex1) represented by formula (I) (i.e., 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl)) (see Example 15) or a salt thereof, or compound 2 (Ex2) represented by formula (II) (i.e., 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azadiyl]di(heptanoic acid)bis(2-nonylundecyl)) (see Example 16) or a salt thereof.

[0441]

[0442]

[0443] In addition, the salt of the compound in the present invention refers to a pharmaceutically acceptable salt, and may form an acid addition salt depending on the type of the substituent. Specifically, examples include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, mandelic acid, tartaric acid, dibenzoyltartaric acid, ditoluoyltartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, aspartic acid, and glutamic acid.

[0444] In one embodiment, the pharmaceutical composition of the present invention contains mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles.

[0445] In one embodiment, the pharmaceutical composition of the present invention contains an mRNA selected from the group consisting of the following (i) to (xx), and the mRNA is encapsulated in lipid nanoparticles:

[0446] (i) mRNA containing a base sequence encoding NeuroD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 8;

[0447] (ii) an mRNA comprising a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence of SEQ ID NO: 7 or 10 and encoding a NeuroD1 protein consisting of the amino acid sequence of SEQ ID NO: 8;

[0448] (iii) an mRNA comprising a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) to the base sequence shown at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13 and encoding a NeuroD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 8;

[0449] (iv) mRNA comprising the base sequence shown in SEQ ID NO: 7 or 10;

[0450] (v) an mRNA comprising the base sequence shown at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13;

[0451] (vi) an mRNA comprising or consisting of the base sequence represented by SEQ ID NO: 11 or 13;

[0452] (vii) an mRNA comprising or consisting of the base sequence represented by SEQ ID NO: 11, wherein a part or all of the uridine is substituted with N1-methylpseudouridine;

[0453] (viii) an mRNA comprising the base sequence shown in SEQ ID NO: 11, in which a part or all of the uridines are substituted with N1-methylpseudouridine, and which has Cap-1 as a 5' cap structure;

[0454] (ix) mRNA containing the base sequence shown in SEQ ID NO: 11, in which all uridines are substituted with N1-methylpseudouridines, and having Cap-1 as a 5' cap structure (mRNA containing the base sequence shown in SEQ ID NO: 13, and having Cap-1 as a 5' cap structure);

[0455] (x) an mRNA that is any one of the mRNAs (i) to (v) above and further contains a 5'UTR and / or a 3'UTR;

[0456] (xi) an mRNA which is any one of the mRNAs (i) to (v) above and further contains a poly A sequence;

[0457] (xii) an mRNA which is any one of the mRNAs (i) to (v) above and further comprises a 5'UTR and / or 3'UTR and a poly A sequence;

[0458] (xiii) mRNA which is the mRNA of (x) or (xii) above, and whose 5'UTR and / or 3'UTR is the 5'UTR and / or 3'UTR derived from the human α-globin gene;

[0459] (xiv) an mRNA which is any one of the mRNAs (i) to (v) above, and further comprises a 5'UTR consisting of the base sequence from positions 4 to 43 of SEQ ID NO: 11 or 13 and / or a 3'UTR consisting of the base sequence from positions 1121 to 1231 of SEQ ID NO: 11 or 13, and a poly A sequence;

[0460] (xv) an mRNA comprising a base sequence having a sequence identity of 70% or more (e.g., 80% or more, 90% or more, or 95% or more) with respect to the base sequence at positions 4 to 1231 of SEQ ID NO: 11 or 13 and encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 8 and a poly A sequence;

[0461] (xvi) an mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13 and a poly A sequence;

[0462] (xvii) an mRNA that is any one of the mRNAs (xi) to (xvi) above and has a poly A sequence with a length of 50 to 200 polynucleotides;

[0463] (xviii) an mRNA that is any one of the mRNAs (i) to (v) or (x) to (xvii) above and has a 5' cap structure, preferably Cap-1; and

[0464] (xix) an mRNA that is any one of the mRNAs (i) to (v) or (x) to (xviii) above and contains a modified nucleoside, preferably N1-methylpseudouridine;

[0465] (xx) An mRNA which is any one of the mRNAs (i) to (v) or (x) to (xix) above, and in which a part or all, preferably all, of the uridine is substituted with N1-methylpseudouridine.

[0466] Lipid nanoparticles are not particularly limited as long as they can deliver mRNA to target cells. For example, when mRNA is delivered to glial cells (such as astrocytes or microglia), lipid nanoparticles that can be delivered to glial cells (such as astrocytes) are selected. Whether this lipid nanoparticle can deliver mRNA to glial cells can be evaluated using CleanCap (registered trademark) eGFP mRNA (TriLink BioTechnologies company). Specifically, the above-mentioned mRNA is enclosed in the lipid nanoparticles as the evaluation object, and the mRNA enclosed in the above-mentioned lipid nanoparticles is imported to the glial cells. Then the expression intensity of the eGFP expressed in the glial cells is measured, and the cells that replace the mRNA enclosed in the lipid nanoparticles are compared with importing PBS, and the expression intensity of the eGFP is studied. Whether it changes (increase or decrease), it can be judged thus. When the expression intensity of eGFP increases when, for example, importing the mRNA enclosed in the lipid nanoparticles, it can be judged that the lipid nanoparticles can deliver mRNA to glial cells. For example, the number of cells expressing eGFP may be used as an indicator for evaluation instead of using the expression intensity of eGFP as an indicator.

[0467] In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention can be encapsulated in lipid nanoparticles that can be delivered to glial cells and delivered to glial cells. In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention can be encapsulated in lipid nanoparticles that can be delivered to astrocytes and delivered to astrocytes. In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention can be encapsulated in lipid nanoparticles that can be delivered to microglia and delivered to microglia.

[0468] As cationic lipids contained in lipid nanoparticles capable of encapsulating mRNA, for example, cationic lipids contained in lipid nanoparticles capable of delivering mRNA to astrocytes, for example, compound 1 (Ex1, Example 15) or a salt thereof or compound 2 (Ex2, Example 16) or a salt thereof, etc. can be cited. In one embodiment, lipid nanoparticles capable of delivering mRNA to astrocytes contain cationic lipids. In one embodiment, lipid nanoparticles capable of delivering mRNA to astrocytes contain compound 1 (Ex1) or a salt thereof and / or compound 2 (Ex2) or a salt thereof as cationic lipids.

[0469] In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles, the lipid nanoparticles contain cationic lipids. In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles, the lipid nanoparticles may contain compound 1 (Ex1) or a salt thereof and / or compound 2 (Ex2) or a salt thereof. In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles, the cationic lipid in the lipid nanoparticles may be compound 1 (Ex1) or a salt thereof. In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles, the cationic lipid in the lipid nanoparticles may be compound 2 (Ex2) or a salt thereof.

[0470] In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles, the lipid nanoparticles can contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof and / or 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azadiyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as cationic lipids.

[0471] In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and encapsulated in lipid nanoparticles, the lipid nanoparticles may contain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azadiyl}di(octanoic acid)di(heptadecan-9-yl) or a salt thereof as a cationic lipid.

[0472] In one embodiment, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein and the mRNA encapsulated in lipid nanoparticles, the lipid nanoparticles can contain 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azepinediyl]di(heptanoic acid)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0473] The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles. The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 1 (Ex1) or a salt thereof and / or compound 2 (Ex2) or a salt thereof. The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 1 (Ex1) or a salt thereof. The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 2 (Ex2) or a salt thereof. The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and compound 1 (Ex1) or a salt thereof. The present invention also includes a pharmaceutical composition, which contains mRNA encoding NeuroD1 protein and compound 2 (Ex2) or a salt thereof.

[0474] In one embodiment, the pharmaceutical composition of the present invention may be: containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 1 (Ex1) or a salt thereof, and the lipid nanoparticles further contain DMG-PEG2000, cholesterol and DSPC. In one embodiment, the pharmaceutical composition of the present invention may be: containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 1 (Ex1) or a salt thereof, and the lipid nanoparticles further contain DMG-PEG2000, cholesterol and DSPC, and contain cationic lipid, DSPC, cholesterol and DMG-PEG2000 in a ratio of 40:12:46.5:1.5.

[0475] In one embodiment, the pharmaceutical composition of the present invention may be: containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 2 (Ex2) or a salt thereof, and the lipid nanoparticles further contain DMG-PEG2000, cholesterol and DSPC. In one embodiment, the pharmaceutical composition of the present invention may be: containing mRNA encoding NeuroD1 protein and the mRNA is encapsulated in lipid nanoparticles containing compound 2 (Ex2) or a salt thereof, and the lipid nanoparticles further contain DMG-PEG2000, cholesterol and DSPC, and contain cationic lipid, DSPC, cholesterol and DMG-PEG200 in a ratio of 50:10:38.5:1.5.

[0476] The pharmaceutical composition of the present invention may contain two or more mRNAs encoding NeuroD1 proteins. For example, pharmaceutical compositions containing mRNAs in which a portion of uridine is replaced by N1-methylpseudouridine, mRNAs in which all uridine is replaced by N1-methylpseudouridine, mRNAs having a 5' cap structure, and / or mRNAs without a 5' cap structure are also included in the present invention.

[0477] For example, in the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein, a pharmaceutical composition containing two or more mRNA encoding NeuroD1 protein among the following (i) to (iv) is also included in the present invention.

[0478] (i) An mRNA comprising the base sequence shown in SEQ ID NO: 11, in which a part of uridine is substituted with N1-methylpseudouridine, and which has Cap-1 as a 5' cap structure.

[0479] (ii) mRNA containing the base sequence shown in SEQ ID NO: 11, in which all uridines are substituted with N1-methylpseudouridine, and has Cap-1 as a 5' cap structure (mRNA containing the base sequence shown in SEQ ID NO: 13, and has Cap-1 as a 5' cap structure).

[0480] (iii) mRNA consisting of the base sequence shown in SEQ ID NO: 11, in which a part of uridine is substituted with N1-methylpseudouridine.

[0481] (iv) mRNA consisting of the base sequence shown in SEQ ID NO: 11, in which all uridines are substituted with N1-methylpseudouridine (mRNA consisting of the base sequence shown in SEQ ID NO: 13).

[0482] In one embodiment, the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein is a pharmaceutical composition containing two or more mRNA encoding NeuroD1 protein among the following (i) to (iv).

[0483] (i) An mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein a portion of uridine is substituted with N1-methylpseudouridine, and has Cap-1 as a 5' cap structure.

[0484] (ii) mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, in which all uridines are substituted with N1-methylpseudouridine and which has Cap-1 as a 5' cap structure.

[0485] (iii) mRNA consisting of a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein a part of uridine is substituted with N1-methylpseudouridine.

[0486] (iv) An mRNA consisting of a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein all uridines are substituted with N1-methylpseudouridine.

[0487] In one embodiment, the pharmaceutical composition of the present invention containing mRNA encoding NeuroD1 protein is a pharmaceutical composition containing two or more of the following (i) to (iv) encoding NeuroD1 protein and having a poly A sequence length of 40 to 200 polynucleotides (e.g., 50 to 200, 50 to 150, 50 to 100, 50 to 90, 60 to 150, 60 to 100, 60 to 90, 70 to 130, 70 to 120, 70 to 100, 70 to 90, 70 to 85, 70 to 80, 75 to 130, 75 to 120, 75 to 100, 75 to 90, preferably 74 to 84, 75 to 85, 75 to 83 or 76 to 82 nucleotides).

[0488] (i) An mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein a portion of uridine is substituted with N1-methylpseudouridine, and has Cap-1 as a 5' cap structure.

[0489] (ii) mRNA comprising a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, in which all uridines are substituted with N1-methylpseudouridine and which has Cap-1 as a 5' cap structure.

[0490] (iii) mRNA consisting of a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein a part of uridine is substituted with N1-methylpseudouridine.

[0491] (iv) An mRNA consisting of a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 and a poly A sequence, wherein all uridines are substituted with N1-methylpseudouridine.

[0492] The pharmaceutical composition of the present invention may also include a pharmaceutical composition containing two or more mRNAs encoding NeuroD1 proteins having different poly A sequence lengths.

[0493] The pharmaceutical composition of the present invention may also include a pharmaceutical composition formulated without encapsulating mRNA in DDS (eg, encapsulated in a drug delivery vehicle).

[0494] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable adjuvants, such as inactive carriers (solid, liquid carriers), excipients, surfactants, cosolvents, suspending agents, colorants, flavoring agents, preservatives, buffers, pH regulators, etc. The pharmaceutical composition of the present invention can be prepared or formulated using pharmaceutically acceptable adjuvants, such as commonly used pharmaceutical excipients, pharmaceutical carriers, etc. The "pharmaceutical composition" referred to in the present invention refers to a composition that can be used as a drug.

[0495] The pharmaceutical composition of the present invention can be used to treat cerebral infarction. In one embodiment, the pharmaceutical composition of the present invention can be used to treat perforator infarction (e.g., lacunar infarction and / or BAD). In one embodiment, the pharmaceutical composition of the present invention can be used to treat i) brain disorders in the basal ganglia-thalamus region of the brain, ii) brain disorders in the basal ganglia and / or thalamus of the brain, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule of the brain. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction with brain disorders in the perforator-maintained area. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction with brain disorders in the perforator-maintained area and the brain disorder is a brain disorder including i) brain disorders in the basal ganglia-thalamus region of the brain, ii) brain disorders in the basal ganglia and / or thalamus of the brain, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule of the brain. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction with localized brain disorders.

[0496] In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction in the subacute to chronic stage. In one embodiment, the pharmaceutical composition of the present invention can be used to treat perforator infarction in the subacute to chronic stage. In one embodiment, the pharmaceutical composition of the present invention can be used to treat lacunar infarction and / or BAD in the subacute to chronic stage. In one embodiment, the pharmaceutical composition of the present invention can be used to treat perforator infarction in the subacute to chronic stage, i) with brain disorders in the basal ganglia-thalamus region of the brain, ii) with brain disorders in the basal ganglia and / or thalamus of the brain, iii) with brain disorders in the basal ganglia-thalamus region of the brain and internal capsule, or iv) with brain disorders in the basal ganglia, thalamus and internal capsule of the brain. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction in the subacute to chronic stage with brain disorders in the perforator-derived area. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction with brain disorders in the perforator distribution area in the subacute to chronic stage, and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, the pharmaceutical composition of the present invention can be used to treat cerebral infarction with localized brain disorders in the subacute to chronic stage.

[0497] In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic cerebral infarction. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic perforator infarction. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic lacunar infarction and / or BAD. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic perforator infarction with i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic cerebral infarction with brain disorders in the perforator-derived area. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic cerebral infarction with brain disorders in the perforator distribution area, and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule. In one embodiment, the pharmaceutical composition of the present invention can be used to treat chronic cerebral infarction with localized brain disorders.

[0498] In a preferred embodiment, the pharmaceutical composition of the present invention can significantly improve the motor function of subjects suffering from cerebral infarction, such as subjects suffering from perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase), subjects suffering from cerebral infarction with brain disorders in the perforator distribution area (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase) and / or subjects suffering from cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase).

[0499] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 2 or more (e.g., 2 to 5, 3 to 5, or 3 or 4). In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 2 or more (e.g., 2 to 5, 3 to 5, or 3 or 4) in the subacute to chronic phase. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 2 or more (e.g., 2 to 5, 3 to 5, or 3 or 4) in the chronic phase. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 3 to 5 in the chronic phase. In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 3 or 4 in the chronic phase.

[0500] The NIHSS score, which is a human stroke neurological severity rating scale, ranges from 0 to 42, with higher scores indicating more severe conditions. In clinical trials of therapeutic agents for cerebral infarction, the NIHSS can also be used as an indicator for recruiting patients and for evaluating the effectiveness of drugs. In clinical trials of therapeutic agents for chronic cerebral infarction, for example, patients with NIHSS scores of 8 to 30 (https: / / clinicaltrials.gov / ct2 / show / NCT04088149), 6 to 20 (https: / / www.clinicaltrials.gov / ct2 / show / NCT04590118), or 5 to 14 (https: / / clinicaltrials.gov / ct2 / show / NCT03202147) are used as clinical trial subjects.

[0501] In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of cerebral infarction with an NIHSS of 5 or more (e.g., 8 or more) (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of cerebral infarction with an NIHSS of 8 or more in the chronic phase. In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 3 to 5 and an NIHSS of 8 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for the treatment of cerebral infarction with an mRS of 3 or 4 and an NIHSS of 8 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase).

[0502] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating perforator infarction with a chronic mRS of 2 to 5 (e.g., 3 to 5 or 3 or 4) (e.g., i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule; and / or, lacunar infarction and / or BAD). In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating perforator infarction with a chronic NIHSS of 5 or more (e.g., 8 or more) (e.g., i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule; and / or, lacunar infarction and / or BAD).

[0503] In one embodiment, the pharmaceutical composition of the present invention can be a pharmaceutical composition for treating cerebral infarction with a brain disorder in the perforator distribution area and having an mRS of 2 to 5 (e.g., 3 to 5 or 3 or 4) in the chronic phase (e.g., a cerebral infarction with a brain disorder in the perforator distribution area and the brain disorder is a brain disorder including i) a brain disorder in the basal ganglia-thalamus area, ii) a brain disorder in the basal ganglia and / or thalamus, iii) a brain disorder in the basal ganglia-thalamus area and internal capsule, or iv) a brain disorder in the basal ganglia, thalamus and internal capsule). In one embodiment, the pharmaceutical composition of the present invention can be a pharmaceutical composition for treating cerebral infarction with a NIHSS of 5 or more (e.g., 8 or more) in the chronic stage and having a brain disorder in the perforator-derived area (e.g., a cerebral infarction with a brain disorder in the perforator-derived area and the brain disorder is a brain disorder including i) a brain disorder in the basal ganglia-thalamus area, ii) a brain disorder in the basal ganglia and / or thalamus, iii) a brain disorder in the basal ganglia-thalamus area and internal capsule, or iv) a brain disorder in the basal ganglia, thalamus, and internal capsule).

[0504] In one embodiment, the pharmaceutical composition of the present invention is a pharmaceutical composition for treating cerebral infarction with localized brain disorders in which the mRS in the chronic phase is 2 to 5 (eg, 3 to 5 or 3 or 4).

[0505] In one embodiment, the pharmaceutical composition of the present invention can be a pharmaceutical composition for the treatment of brain disorders in the perforator-maintained area accompanied by cerebral infarction (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). As brain disorders in the perforator-maintained area, i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule can be listed, but are not limited to these. In one embodiment, the pharmaceutical composition of the present invention can be a pharmaceutical composition for the treatment of i) brain disorders in the basal ganglia-thalamus region accompanied by cerebral infarction (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase), ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule.

[0506] In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for treating brain disorders in perforator-derived areas associated with cerebral infarction with a chronic mRS of 2 to 5 (eg, 3 to 5 or 3 or 4).

[0507] In one embodiment, the pharmaceutical composition of the present invention may be a pharmaceutical composition for treating brain disorders in the perforator-derived area associated with chronic cerebral infarction with a NIHSS of 5 or more (eg, 8 or more).

[0508] The present invention further provides a method for treating cerebral infarction (hereinafter also referred to as "the method for treating the present invention"), which comprises the step of administering the pharmaceutical composition of the present invention, NeuroD1 protein, or a polynucleotide encoding NeuroD1 protein to a subject. In one embodiment, the method for treating the present invention may be a method for treating cerebral infarction comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of the present invention, NeuroD1 protein, or a polynucleotide encoding NeuroD1 protein to a subject. In one embodiment, the method for treating the present invention may be a method for treating cerebral infarction comprising the step of administering a therapeutically effective amount of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein to a subject. In one embodiment, the method for treating the present invention may be a method for treating cerebral infarction comprising the step of administering a therapeutically effective amount of a polynucleotide encoding NeuroD1 protein to a subject. In a typical embodiment, the polynucleotide encoding NeuroD1 protein encodes NeuroD1 protein in an expressible state. In one embodiment, the method for treating the present invention comprises the step of administering a DNA encoding NeuroD1 protein to a subject. In one embodiment, the method for treating the present invention comprises the step of administering a DNA encoding NeuroD1 protein to a subject. In one embodiment, the method for treating the present invention comprises the step of administering an expression vector containing a polynucleotide encoding NeuroD1 protein to a subject. In one embodiment, the treatment method of the present invention comprises the step of administering a therapeutically effective amount of an expression vector containing a polynucleotide encoding NeuroD1 protein to a subject. In one embodiment, the expression vector used in the treatment method of the present invention is an AAV vector, a retroviral vector, a Sendai virus vector or a lentiviral vector. In one embodiment, the expression vector used in the treatment method of the present invention is an AAV vector. In one embodiment, the treatment method of the present invention comprises the step of administering mRNA encoding NeuroD1 protein to a subject. In one embodiment, the treatment method of the present invention comprises the step of administering a therapeutically effective amount of mRNA encoding NeuroD1 protein to a subject. In one embodiment, the mRNA used in the treatment method of the present invention is encapsulated in DDS. In one embodiment, the mRNA used in the treatment method of the present invention is encapsulated in a drug delivery carrier. In one embodiment, the mRNA used in the treatment method of the present invention is encapsulated in nanoparticles. In one embodiment, the mRNA used in the treatment method of the present invention is encapsulated in lipid nanoparticles.

[0509] In one embodiment, the treatment method of the present invention may be a treatment method for perforator infarction (e.g., i) brain disorders in the basal ganglia-thalamus region of the brain, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule; and / or, lacunar infarction and / or BAD). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction in the subacute to chronic phase. In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction in the chronic phase. In one embodiment, the treatment method of the present invention may be a treatment method for perforator infarction in the subacute to chronic phase (e.g., i) brain disorders in the basal ganglia-thalamus region of the brain, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule; and / or, lacunar infarction and / or BAD). In one embodiment, the treatment method of the present invention may be a treatment method for lacunar infarction and / or BAD in the subacute to chronic phase. In one embodiment, the treatment method of the present invention may be a treatment method for perforator infarction in the chronic phase (e.g., i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule; and / or, lacunar infarction and / or BAD). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with brain disorders in the perforator-derived area (e.g., brain disorders in the perforator-derived area and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule). In one embodiment, the treatment method of the present invention can be a treatment method for cerebral infarction with brain disorders in the perforator distribution area in the subacute to chronic stage (for example, cerebral infarction with brain disorders in the perforator distribution area and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule).In one embodiment, the treatment method of the present invention can be a treatment method for chronic cerebral infarction with brain disorders in the perforator distribution area (for example, cerebral infarction with brain disorders in the perforator distribution area and the brain disorders are brain disorders including i) brain disorders in the basal ganglia-thalamus area, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus area and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule).

[0510] In one embodiment, the treatment method of the present invention can be a treatment method for cerebral infarction with localized brain disorders. In one embodiment, the treatment method of the present invention can be a treatment method for cerebral infarction with localized brain disorders in the subacute to chronic stages. In one embodiment, the treatment method of the present invention can be a treatment method for cerebral infarction with localized brain disorders in the chronic stage.

[0511] In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with an mRS of 2 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with an mRS of 3 to 5 (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with an NIHSS of 5 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with an NIHSS of 8 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). In one embodiment, the treatment method of the present invention may be a treatment method for cerebral infarction with an mRS of 3 to 5 and an NIHSS of 8 or more (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase).

[0512] Cerebral infarction with an mRS of 2 or more that is a subject of the use of the pharmaceutical composition of the present invention or a subject of treatment of the present invention (hereinafter also referred to as "subject of treatment of the present invention") includes cerebral infarction evaluated as an mRS of 2 or more at any period after the onset of cerebral infarction. Cerebral infarction with an mRS of 3 to 5 that is a subject of treatment of the present invention includes cerebral infarction evaluated as an mRS of 3 to 5 at any period after the onset of cerebral infarction. For example, a case where the mRS is evaluated as 3 from the onset of cerebral infarction to the time when the mRS score reaches stability, and then the symptoms recover and the mRS improves to 2, is also included in the cerebral infarction with an mRS of 3 to 5 that is a subject of treatment of the present invention. Similarly, cerebral infarction with an NIHSS of 5 or more that is a subject of treatment of the present invention includes cerebral infarction evaluated as an NIHSS of 5 or more at any period after the onset of cerebral infarction. Cerebral infarction with an NIHSS of 8 or more that is a subject of treatment of the present invention includes cerebral infarction evaluated as an NIHSS of 8 or more at any period after the onset of cerebral infarction. For example, the case where the NIHSS is evaluated as 8 or more during the period from the onset of cerebral infarction to the NIHSS score reaching stability, and then the symptoms recover and the NIHSS score improves, is also included in the cerebral infarction with an NIHSS of 8 or more that is the treatment target of the present invention. The cerebral infarction that is the treatment target of the present invention may be a perforator infarction (e.g., lacunar cerebral infarction and / or BAD), for example, a perforator infarction in the subacute phase to the chronic phase or the chronic phase. Alternatively, the cerebral infarction that is the treatment target of the present invention may be a cerebral infarction other than a perforator infarction, for example, a cerebral infarction other than a perforator infarction in the subacute phase to the chronic phase.

[0513] The subject to which the treatment method and pharmaceutical composition of the present invention are applied can be any mammal (subject) including primates (humans and non-human primates), livestock (horses, cattle, sheep, goats, pigs, etc.), ornamental animals (dogs, cats, rabbits, etc.), experimental (test) animals (mice, rats, etc.), etc. The preferred subject is primates, and the particularly preferred subject is humans. The subject to which the treatment method and pharmaceutical composition of the present invention are applied is preferably a subject to which the treatment method and pharmaceutical composition are applied, for example, a subject having cerebral infarction, such as perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase) or cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase), or a subject suspected of having cerebral infarction, such as perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase) or cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase). As perforator infarction, i) perforator infarction with brain disorders in the basal ganglia-thalamus region, ii) perforator infarction with brain disorders in the basal ganglia and / or thalamus, iii) perforator infarction with brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) perforator infarction with brain disorders in the basal ganglia, thalamus and internal capsule, but not limited to these. The subject of the treatment method and pharmaceutical composition of the present invention can also be a subject with or suspected of having cerebral infarction with brain disorders in the perforator distribution area (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase). As cerebral infarction with brain disorders in the perforator distribution area, the brain disorders can be listed as cerebral infarctions including i) brain disorders in the basal ganglia-thalamus region, ii) brain disorders in the basal ganglia and / or thalamus, iii) brain disorders in the basal ganglia-thalamus region and internal capsule, or iv) brain disorders in the basal ganglia, thalamus and internal capsule, but not limited to these.

[0514] For humans, the period from less than 72 hours after the onset of cerebral infarction, when there is a possibility of worsening symptoms, is called the "acute phase". For humans, the period from 72 hours to less than 1 month after the onset of the disease is called the "subacute phase" (also called the recovery phase), and the period from 1 month after the onset of the disease is called the "chronic phase". The subacute phase is a period when a higher degree of recovery from paralysis can be expected compared to the chronic phase. For humans, the subacute phase to the chronic phase of cerebral infarction refers to the period from 72 hours after the onset of cerebral infarction, for example, from 1 month after the onset of cerebral infarction. In addition, it can also be the period from 3 months after the onset of cerebral infarction, for example, from 6 months after the onset of cerebral infarction. The "acute phase", "subacute phase" and "chronic phase" of non-human animal subjects can be appropriately determined by a technician in this field in the form of periods equivalent to the "acute phase", "subacute phase" and "chronic phase" of humans, respectively.

[0515] The dosage and frequency of administration of the pharmaceutical composition of the present invention, NeuroD1 protein or polynucleotide encoding NeuroD1 protein to a subject can be appropriately adjusted according to the disease, severity, age, weight and condition of the subject to be treated. The dosage can be, for example, 0.0001 mg to 15 mg / kg body weight per administration as NeuroD1 protein.

[0516] The method of administering the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention to a subject is not particularly limited, and examples include local administration by surgical means, cardiac catheter administration, administration using an ultrasonic contrast agent (microballoon), intravenous administration, lower limb puncture administration, local injection administration, subcutaneous administration, intradermal administration, intramuscular administration, etc., and preferably, local administration to the infarct foci or obstruction foci of the subject by surgical means (e.g., intracranial administration, intraparenchymal administration, intraventricular administration, etc.).

[0517] In one embodiment, the pharmaceutical composition, NeuroD1 protein, or polynucleotide encoding NeuroD1 protein of the present invention is administered by topical administration using surgical means. In one embodiment, the pharmaceutical composition, NeuroD1 protein, or polynucleotide encoding NeuroD1 protein of the present invention is administered to the subject by intracranial administration. In one embodiment, the pharmaceutical composition, NeuroD1 protein, or polynucleotide encoding NeuroD1 protein of the present invention is administered to the subject by intracerebral administration. In one embodiment, the pharmaceutical composition, NeuroD1 protein, or polynucleotide encoding NeuroD1 protein of the present invention is administered to the subject by intracerebral administration.

[0518] The method of administering the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention to a subject comprises the step of administering the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention to at least one administration site in the infarct or disorder of the subject. When there are two or more infarcts or disorder foci, the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention may be administered to at least one administration site in each infarct or disorder foci. In one embodiment, the present method may administer the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention to the administration site of each infarct or disorder foci in one or more infarcts or disorder foci of the subject.

[0519] The number of administrations of the pharmaceutical composition of the present invention, NeuroD1 protein, or polynucleotide encoding NeuroD1 protein to a subject is not particularly limited, and may be either a single administration or multiple administrations. The period of single administration may be any period after the onset of cerebral infarction, such as the subacute to chronic phase or the chronic phase, and is not particularly limited, and may be, for example, 1 month after the onset of the disease, 3 months after the onset of the disease, 6 months after the onset of the disease, 1 year after the onset of the disease, 2 years after the onset of the disease, 3 years after the onset of the disease, 5 years after the onset of the disease, 7 years after the onset of the disease, 10 years after the onset of the disease, etc. In the case of multiple administrations, for example, the first administration may be any period after the onset of cerebral infarction, such as the subacute to chronic phase or the chronic phase, and after the first administration, the administration may be performed at regular intervals, such as every six months.

[0520] The instrument used when administering the pharmaceutical composition, NeuroD1 protein or polynucleotide encoding NeuroD1 protein of the present invention into the brain of a subject is not particularly limited as long as it can be administered to the target site, and for example, a biopsy / injection needle kit (MIZUHO, MES-CG07-200-01), a puncture needle (International Publication WO2020 / 241833), etc. can be used.

[0521] The treatment method of the present invention can effectively treat cerebral infarction. In one embodiment, the treatment method of the present invention can effectively treat cerebral infarction in the subacute to chronic stage. In one embodiment, the treatment method of the present invention can effectively treat cerebral infarction in the chronic stage. In one embodiment, the treatment method of the present invention can effectively treat perforator infarction (e.g., lacunar infarction and / or BAD). In one embodiment, the treatment method of the present invention can effectively treat perforator infarction in the subacute to chronic stage (especially the chronic stage). In one embodiment, the treatment method of the present invention can effectively treat lacunar infarction and / or BAD in the subacute to chronic stage (especially the chronic stage). In one embodiment, the treatment method of the present invention can effectively treat cerebral infarction with brain disorders in the perforator distribution area. In one embodiment, the treatment method of the present invention can effectively treat cerebral infarction with brain disorders in the perforator distribution area in the subacute to chronic stage (especially the chronic stage). In a preferred embodiment, the treatment method of the present invention can significantly improve the motor function of subjects suffering from perforator infarction (e.g., perforator infarction in the subacute to chronic phase, especially perforator infarction in the chronic phase), cerebral infarction with brain disorders in the perforator distribution area (e.g., cerebral infarction in the subacute to chronic phase, especially cerebral infarction in the chronic phase), and / or subjects suffering from subacute to chronic phase cerebral infarction (e.g., cerebral infarction in the chronic phase). In the present invention, "treatment" of cerebral infarction is not limited to eliminating or alleviating the cause of cerebral infarction, i.e., thrombus, or the interruption of cerebral blood flow caused thereby, but includes eliminating, alleviating, improving, or preventing or delaying the progression of symptoms of cerebral infarction (e.g., motor dysfunction). In one embodiment, "treatment" of cerebral infarction in the present invention can be treatment of symptoms of cerebral infarction (e.g., motor dysfunction).

[0522] The treatment method and pharmaceutical composition of the present invention are preferably able to bring about the recovery of both the disorder of the basal ganglia-thalamus region of the brain and the disorder of the internal capsule. The disorder of the basal ganglia-thalamus region of the brain and its recovery can be shown, for example, by the expression level of the dopamine transporter as an indicator, as shown in the examples described below. The disorder of the internal capsule and its recovery can be shown, for example, by DTI analysis, etc., using the FA value representing the corticospinal tract as an indicator, as shown in the examples described below. The treatment method and pharmaceutical composition of the present invention are also preferably able to bring about the inhibition of the volume increase of the lateral ventricles associated with brain disorders. The inhibition of the increase in the volume of the lateral ventricles indicates the inhibition of brain parenchymal atrophy or the recovery of brain parenchymal tissue damage. Regarding the volume of the lateral ventricles, it can be determined by any brain image analysis, such as the analysis of T1-weighted images and / or T2-weighted images obtained by MRI as shown in the examples described below. The treatment method and pharmaceutical composition of the present invention are preferably also able to promote the recovery of motor dysfunction.

[0523] The present invention also provides the pharmaceutical composition of the present invention, NeuroD1 protein or polynucleotide encoding NeuroD1 protein, which is used to treat cerebral infarction, preferably perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase), cerebral infarction with brain disorders in the perforator distribution area (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase), cerebral infarction with localized brain disorders (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase) or cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase). The present invention also provides the pharmaceutical composition of the present invention, NeuroD1 protein or polynucleotide encoding NeuroD1 protein, which is used to treat cerebral infarction with mRS of 2 or more (e.g., 3 to 5) and / or NIHSS of 5 or more (e.g., 8 or more) (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase).

[0524] The present invention also provides the use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating cerebral infarction, preferably perforator infarction (e.g., perforator infarction in the subacute to chronic phase or perforator infarction in the chronic phase), cerebral infarction with brain disorders in the perforator distribution area (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase), cerebral infarction with localized brain disorders (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase), or cerebral infarction in the subacute to chronic phase (e.g., cerebral infarction in the chronic phase). The present invention also provides the use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating cerebral infarction with an mRS of 2 or more (e.g., 3 to 5) and / or an NIHSS of 5 or more (e.g., 8 or more) (e.g., cerebral infarction in the subacute to chronic phase or cerebral infarction in the chronic phase).

[0525] As described above, the present invention also includes the use of NeuroD1 protein or polynucleotides encoding NeuroD1 protein in the manufacture of NeuroD1 protein or polynucleotides encoding NeuroD1 protein for the treatment method of the present invention, the treatment of cerebral infarction, and a pharmaceutical composition for the treatment of cerebral infarction. Cerebral infarctions that are the treatment targets of the pharmaceutical use of these NeuroD1 proteins or polynucleotides encoding NeuroD1 proteins include cerebral infarctions described in the above-mentioned "pharmaceutical composition of the present invention". Those skilled in the art can easily implement the pharmaceutical use of NeuroD1 protein or polynucleotides encoding NeuroD1 protein of the present invention with reference to the relevant description of the "pharmaceutical composition of the present invention".

[0526] Example

[0527] The present invention will be described in more detail below using examples, but the technical scope of the present invention is not limited to these examples.

[0528] Unless otherwise stated, the operations of each step described in the following examples can be carried out using known techniques. In addition, regarding the use of commercially available kits or reagents, the experiments were carried out according to the attached protocols unless otherwise stated.

[0529] In addition, the naming of the compounds described in this specification may use naming software such as ACD / Name (registered trademark, Advanced Chemistry Development, Inc.).

[0530] For convenience, the concentration unit mol / L is expressed as M. For example, 1M sodium hydroxide aqueous solution refers to 1 mol / L sodium hydroxide aqueous solution.

[0531] In the development of therapeutic agents for cerebral infarction, rodent cerebral infarction models have been used in various studies of acute cerebral infarction therapeutic agents in the past. On the other hand, the guidelines of the International Stroke Association recommend the use of non-human primates, whose cerebrovascular and tissue structures are closer to humans, for evaluation in order to reduce the deviation between non-clinical and clinical results. Regarding cerebral infarction models using non-human primates, MCAO models and internal capsule disorder models have been reported, but these are not models that can maintain highly severe motor dysfunction.

[0532] The present inventors believe that the rodent cerebral infarction model cannot be used for comprehensive motor function evaluation by motor dysfunction scores such as NHPSS, and is difficult to be used in the study of cerebral infarction therapeutic agents that restore motor dysfunction. In addition, in the cerebral infarction models using rodents, non-human primates, etc. reported in the past, there has been no report of cases in which comprehensive motor skills evaluation using NHPSS, etc. can be performed with high reproducibility until the subacute to chronic phases. The present inventors believe that the existing cerebral infarction model cannot fully evaluate the cerebral infarction therapeutic agents in the subacute to chronic phases. Therefore, it was decided to use non-human primates to construct a cerebral infarction model that can be evaluated by motor dysfunction scores and can maintain motor dysfunction for a long time. The present inventors believe that infarction in the basal ganglia of the brain may be important for motor dysfunction based on the fact that cerebral cortical infarction is prone to symptoms of decreased consciousness, and BAD, one of the perforator infarction methods, is prone to infarction in the cerebral basal ganglia, and has a tendency to continue motor paralysis after cerebral infarction. Furthermore, according to the previous insights of the present inventors, the present inventors believe that thalamic disorders may be important for the long-term maintenance of motor dysfunction. In addition, the present inventors believe that the internal capsule is a collection of nerve fibers, so the internal capsule disorder is also important for the target cerebral infarction model. Based on these insights and investigations, the present inventors speculated that by causing disorders in the areas (basal ganglia, thalamus and internal capsule) innervated by perforators in non-human primates, it is possible to construct a cerebral infarction model (particularly a perforator infarction model or a model of cerebral infarction with brain disorders in the perforator innervated area) that can maintain long-term motor dysfunction, and thus implemented the following research.

[0533] It should be noted that when making the non-human primate model of cerebral infarction, a model of brain disorder caused in the right brain or left brain of non-human primates was studied. Most of the patients with cerebral infarction have infarction in the right brain or left brain, so it is believed that the animal model of cerebral infarction that induces brain disorder in the right brain or left brain is closer to the clinical animal model of cerebral infarction. In the present embodiment, brain disorder is induced in the left brain by injecting endothelin into the left brain, thus causing right hemi-body motor dysfunction.

[0534] (Example 1) Study on the construction of cerebral infarction model in cynomolgus monkeys

[0535] The study used male cynomolgus monkeys (over 3 years old, New Japan Science) to create a cerebral infarction model (non-human primate model of cerebral infarction) with motor dysfunction lasting for more than 3 months and with basal ganglia disorders, thalamic disorders, and internal capsule disorders. It is believed that by injecting endothelin-1, which has a vasoconstrictor effect, into the basal ganglia region and thalamic region of the brain, it is possible to induce vasoconstriction at the injection site, cause local cerebral ischemia, and cause neuronal cell death. Therefore, endothelin-1 (human origin; Peptide Research Institute, product number: 4198-v, Cys-Ser-Cys-Ser-Ser-Leu-Met-Asp-Lys-Glu-Cys-Val-Tyr-Phe-Cys-His-Le u-Asp-Ile-Ile-Trp (in Cys 1 With Cys 15 Between and Cys 3 With Cys 11 There is a disulfide bond between them; sequence number 9)) was injected into the brain of cynomolgus monkeys according to the following various injection sites, injection amounts and injection rates to investigate the extent of symptoms associated with neuronal cell death and induction of motor dysfunction.

[0536] First, ketamine hydrochloride (ketalar for intramuscular injection 500 mg; Daiichi Sankyo Propharma, license number: 21600AMZ00252; hereinafter referred to as "ketamine hydrochloride") was introduced as a general anesthetic into male cynomolgus monkeys at a dose of about 10 mg / kg body weight by intramuscular injection (hereinafter referred to as "im"). The cynomolgus monkeys under anesthesia were intubated with a tracheal tube (Comedy Angiography, 301-40G), and anesthesia was maintained by inhalation of isoflurane (isoflurane inhalation anesthetic solution "VTRS"; Mailan Pharmaceutical Co., Ltd., license number: 22700AMX00134; hereinafter referred to as "isoflurane") under artificial respiration or spontaneous respiration. After shaving the hair on the head of the anesthetized cynomolgus monkey with a clipper, the cynomolgus monkey was fixed in a brain positioning fixture (NARISHIGE, SN-3N). Then, the skin of the cynomolgus monkey's head was incised while taking into account the bleeding. The steps up to this point were the same in all the following research experiments.

[0537] The intracerebral injection site of endothelin-1 was studied with reference to the well-known brain map of cynomolgus monkeys (J. Szabo et al., J. Comp. Neurol., vol. 222 (2), pp. 265-300, 1984). Regarding the brain map of cynomolgus monkeys, the Anterior-Posterior (AP) coordinates are used for the anterior-posterior axis, and the Midline (ML) coordinates are used for the left-right axis. Therefore, as the coordinates of the intracerebral injection site of endothelin-1, the AP coordinates are used for the anterior-posterior axis, and the ML coordinates are used for the left-right axis. First, the intersection of the line connecting the two external auditory canals (interaural line) and the sagittal suture of the skull is used as the base point of the injection site (AP coordinate: 0 mm, ML coordinate: 0 mm (center)). The injection site is shown as follows.

[0538] A: The distance from the base point of the AP coordinate to the front

[0539] L: The distance to the left from the base point of the ML coordinate

[0540] D: The depth of the brain from the dura mater in the vertical downward direction

[0541] (a) Research Experiment 1

[0542] The site and amount of endothelin-1 injected into the brain were investigated. Using a drill (Muromachi Machine Industry, HSD-1000; hereinafter referred to as "drill"), holes with a diameter of about 1 mm were made in the skull of one of the two cynomolgus monkeys at positions (i) A: 20.0 mm, L: 7.0 mm, (ii) A: 20.0 mm, L: 9.0 mm, and (iii) A: 20.0 mm, L: 11.0 mm, and in the skull of the other monkey, holes were made at positions (iv) A: 15.0 mm, L: 7.0 mm, (v) A: 15.0 mm, L: 9.0 mm, and (vi) A: 15.0 mm, L: 11.0 mm in addition to the above positions (i) to (iii). (Total 3 or 6 holes.) A cannula (TOP spinal needle; Top Co., Ltd., 02036 (25G×89mm); hereinafter referred to as "cannula") and a tube (Plastics One, C312VT; hereinafter referred to as "tube") were inserted from each hole and indwelled in such a way that the tip of the cannula reached a depth (D) of 19.0 mm. Using the indwelling cannula, endothelin-1 diluted with 0.1% acetic acid (prepared by diluting acetic acid (Wako, 017-00256) with Milli-Q (registered trademark) water) was injected into a total of 3 or 6 sites corresponding to the above-mentioned positions of each individual. Endothelin-1 was injected at a concentration of 2.0 μg / μL, 2 μL / site, and an injection rate of 1 μL / min. The 3-site injection and 6-site injection were each carried out with N=1.

[0543] In Experiment 1 of this study, mild motor dysfunction was observed in the 2 cynomolgus monkeys treated as above in the 3rd injection site, and disappeared 1 week after treatment. In the 6th injection site, motor dysfunction of the right front and back limbs was clearly observed until 2 weeks after treatment, but recovered naturally to the extent that mild paralysis was observed 4 weeks later.

[0544] (b) Research Experiment 2

[0545] The injection site of endothelin-1 and the concentration of endothelin-1 injected were changed according to the conditions used in the study 1. In the skull of cynomolgus monkeys, holes with a diameter of about 1 mm were drilled at the positions of (i) A: 22.0 mm, L: 11.0 mm, (ii) A: 19.0 mm, L: 11.0 mm, (iii) A: 16.0 mm, L: 11.0 mm, (iv) A: 22.0 mm, L: 8.0 mm, (v) A: 19.0 mm, L: 8.0 mm, and (vi) A: 16.0 mm, L: 8.0 mm (a total of 6 holes). A microsyringe (HAMILTON, 1705RN; hereinafter referred to as "microsyringe") connected to a microsyringe (NARISHIGE, IMS-20; hereinafter referred to as "microsyringe") was inserted through each hole and indwelled so that the tip of the needle reached a depth (D) of 19.0 mm. Endothelin-1 diluted with 0.1% acetic acid was injected into a total of 6 sites corresponding to the above-mentioned sites of each individual using a microsyringe. Endothelin-1 was injected at a concentration of 4.0 μg / μL, 2 μL / site, and an injection rate of 1 μL / min. This was performed with N=2.

[0546] Regarding the symptoms of motor dysfunction shown by the cynomolgus monkeys treated as above, one individual had mild paralysis of the right front and rear limbs, which was the dysfunctional side (dysfunctional side), while the other individuals showed no symptoms.

[0547] In Research Experiment 2, precipitation was observed in the remaining sample of the endothelin-1 solution, and therefore, the concentration of endothelin-1 was reduced to 2.0 μg / μL from Research Experiment 3 onwards to prevent precipitation of endothelin-1 in the solution.

[0548] (c) Research Experiment 3

[0549] According to the results of research experiments 1 and 2, when the line connecting the two external auditory canals is used as the base point of the AP coordinate, endothelin-1 may not be injected into the target area due to individual differences. Therefore, based on the previous insights of the inventors, it was thought of using the anterior fontanelle (Bregma), the intersection of the sagittal suture and the coronal suture in the front of the skull surface, as the base point. After research experiment 3, the anterior fontanelle was used as the base point of the injection site (B: 0mm, ML coordinates: 0mm (center)). The injection site is shown as follows.

[0550] B: The distance in the anterior-posterior axis direction with the anterior fontanelle as the base point (a positive value indicates the distance in the front.)

[0551] L: The distance to the left from the base point of the ML coordinates

[0552] D: The depth of the brain from the dura mater in the vertical downward direction

[0553] Since there is no brain map of cynomolgus monkeys with bregma as the base point, the injection site was studied based on the inventors' previous knowledge.

[0554] In addition, in Research Experiment 3, the amount of endothelin-1 injected per injection site was increased compared to the conditions of Research Experiment 2. In Research Experiment 3, holes with a diameter of about 1 mm were drilled at the positions of (i) B: 2.0 mm, L: 8.0 mm, (ii) B: 5.0 mm, L: 8.0 mm, and (iii) B: 9.0 mm, L: 8.0 mm in the skull of cynomolgus monkeys (total 3 sites). A microsyringe connected to a microinjector was inserted into each hole and indwelled in such a way that the tip of the needle reached a depth (D) of 19.0 mm. Using a microsyringe, endothelin-1 diluted with 0.1% acetic acid was injected into a total of 3 sites corresponding to the above-mentioned positions of each individual. Endothelin-1 was injected at a concentration of 2.0 μg / μL, an amount of 10 μL / site, and an injection rate of 1 μL / min. This was implemented with N=2.

[0555] In one of the treated cynomolgus monkeys, the evaluation scale mRS (Table 1) was 5 on the day after treatment, and supine posture, decreased consciousness level, decreased voluntary movement, and complete paralysis of the right front and back limbs on the impaired side were confirmed. The mRS of this individual was also 4 one week after surgery. Another cynomolgus monkey individual that underwent the same treatment had an mRS of 3 on the day after treatment. Although the unconsciousness level was decreased, decreased voluntary movement and complete paralysis of the right front and back limbs on the impaired side were confirmed, and decreased voluntary movement and complete paralysis of the right front and back limbs on the impaired side were continuously observed in this individual one week after treatment. The above results indicate that by injecting endothelin according to the conditions of Research Experiment 3, motor dysfunction of the individual can be induced and the induced symptoms are more stable than those of Research Experiments 1 and 2. It can be seen that the motor dysfunction of the two treated cynomolgus monkeys in Research Experiment 3 lasted at least until the subacute stage.

[0556] (d) Research Experiment 4

[0557] The following study was conducted based on the conditions of Study 3. In order to more reliably induce brain disorders, the injection amount of endothelin-1 was increased to 2.0 μg / μL and 30 μL / site. In the skulls of two cynomolgus monkeys, holes with a diameter of about 1 mm were drilled with a drill at the positions of (i) B: 2.0 mm, L: 8.0 mm, (ii) B: 5.0 mm, L: 8.0 mm, and (iii) B: 9.0 mm or 11.0 mm, L: 8.0 mm (a total of 3 locations. Only the injection site of (iii) was different for each individual.). A microsyringe connected to a microinjector was inserted through each hole and indwelled in such a way that the tip of the needle reached a depth (D) of 19.0 mm (from the dura mater). Endothelin-1 was injected into a total of 3 sites corresponding to the above-mentioned positions in each individual using a microsyringe. Endothelin-1 was injected at a concentration of 2.0 μg / μL, a volume of 30 μL / site, and an injection rate of 1 μL / min. The experiment was performed with N=1 except that only the injection site in (iii) was different.

[0558] In the two treated cynomolgus monkeys, the mRS (Table 1) was 4 about one month after the treatm...

Claims

1. A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of the non-human primate brain to thereby induce basal ganglia disorders, thalamus disorders and internal capsule disorders.

2. The method according to claim 1, wherein: Endothelin is administered to one or more nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain.

3. The method according to claim 1 or 2, wherein: Endothelin is administered to at least three nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain.

4. The method according to any one of claims 1 to 3, wherein: Endothelin is administered to at least four nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain.

5. The method according to any one of claims 1 to 4, wherein: Endothelin is administered to all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain.

6. The method according to any one of claims 1 to 5, wherein: Cerebral infarction is a subacute to chronic stage of cerebral infarction.

7. The method according to any one of claims 1 to 6, wherein: Cerebral infarction is a chronic stage of cerebral infarction.

8. The method according to any one of claims 1 to 7, wherein: The cerebral infarction was perforator infarction.

9. The method according to any one of claims 1 to 7, wherein: Cerebral infarction is a cerebral infarction with brain disorders in the perforator distribution area.

10. The method according to claim 8 or 9, wherein: Perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

11. The method according to claim 8 or 9, wherein: Perforator infarction or cerebral infarction with brain damage in the perforator distribution area is a chronic cerebral infarction.

12. The method according to any one of claims 2 to 11, wherein: The method comprises the steps of determining an administration site in such a manner as to target the nerve nucleus and administering endothelin to the administration site.

13. A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of a non-human primate to thereby induce basal ganglia disorders, thalamus disorders and internal capsule disorders, comprising: (a) determining the administration site in such a manner as to target at least four nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and (b) a step of administering endothelin to the administration site.

14. A method for preparing a non-human primate model of cerebral infarction, comprising the steps of administering endothelin to the basal ganglia-thalamus region of a non-human primate to thereby induce basal ganglia disorders, thalamus disorders and internal capsule disorders, comprising: (a) determining the administration site in such a manner as to target all five nerve nuclei selected from the group consisting of the putamen, caudate nucleus, globus pallidus, ventrolateral thalamic nucleus, and ventroposterior thalamic nucleus in the basal ganglia-thalamus region of the brain; and (b) a step of administering endothelin to the administration site.

15. The method according to claim 13 or 14, wherein: The number of administration sites is at least 3.

16. The method according to any one of claims 1 to 15, wherein: The non-human primate was a cynomolgus macaque.

17. The method according to any one of claims 1 to 16, wherein: The amount of endothelin administered per administration site is at least 10 μg.

18. The method according to any one of claims 1 to 17, wherein: The endothelin is endothelin-1.

19. A non-human primate model of cerebral infarction having disorders of the basal ganglia, thalamus and internal capsule, which is prepared by the method according to any one of claims 1 to 18.

20. A method for preparing a non-human primate model of cerebral infarction in the subacute to chronic stages, comprising the step of administering endothelin to cynomolgus monkeys to induce cerebral basal ganglia disorders, thalamus disorders and internal capsule disorders, wherein the endothelin is administered to the following sites: Based on the bregma, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 8-12 mm, L: 4-13 mm, D: 12-20 mm.

21. The method according to claim 20, wherein: The administration site of endothelin includes any one of the following (1) to (3): (1) With bregma as the base point, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, (iii) B: 8-10 mm, L: 4-6 mm, D: 12-14 mm, and (iv) B: 8-10 mm, L: 11-13 mm, D: 14-16 mm; (2) based on the bregma, the administration sites are located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 8-10 mm, L: 7-9 mm, D: 18-20 mm; and (3) Based on the bregma, the administration sites were located at (i) B: 1-3 mm, L: 7-9 mm, D: 18-20 mm, (ii) B: 4-6 mm, L: 7-9 mm, D: 18-20 mm, and (iii) B: 10-12 mm, L: 7-9 mm, D: 18-20 mm.

22. The method according to claim 20 or 21, wherein: The administration site of endothelin includes any one of the following (1) to (3): (1) With bregma as the base point, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm; (2) With bregma as the base point, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, and (iii) B: 9 mm, L: 8 mm, D: 19 mm; as well as (3) Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, and (iii) B: 11 mm, L: 8 mm, D: 19 mm.

23. The method according to any one of claims 20 to 22, wherein: The administration sites of endothelin include the following: Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm.

24. The method according to any one of claims 20 to 23, wherein: The administration sites of endothelin are the following administration sites: Based on the bregma, the administration sites were located at (i) B: 2 mm, L: 8 mm, D: 19 mm, (ii) B: 5 mm, L: 8 mm, D: 19 mm, (iii) B: 9 mm, L: 5 mm, D: 13 mm, and (iv) B: 9 mm, L: 12 mm, D: 15 mm.

25. The method according to any one of claims 20 to 24, wherein: Cerebral infarction is a chronic stage of cerebral infarction.

26. The method according to any one of claims 20 to 25, wherein: Cerebral infarction is perforator infarction or cerebral infarction with brain disorders in the perforator distribution area.

27. The method according to any one of claims 20 to 26, wherein: The amount of endothelin administered per administration site is at least 10 μg.

28. The method according to any one of claims 20 to 27, wherein: The endothelin is endothelin-1.

29. A cynomolgus monkey model of cerebral infarction in the subacute to chronic stages, which is a model prepared by the method according to any one of claims 20 to 28.

30. A method for screening a therapeutic agent for cerebral infarction, comprising: The step of administering a test substance to the non-human primate model of claim 19 or 29; and a step of determining the effect of the test substance on cerebral infarction in the non-human primate model.

31. A pharmaceutical composition for treating perforator infarction or subacute to chronic cerebral infarction, comprising NeuroD1 protein or a polynucleotide encoding NeuroD1 protein.

32. A pharmaceutical composition for treating cerebral infarction with brain disorders in the perforator-derived area, comprising NeuroD1 protein or a polynucleotide encoding NeuroD1 protein.

33. The pharmaceutical composition according to claim 31 or 32, wherein Perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

34. The pharmaceutical composition according to any one of claims 31 to 33, wherein Perforator infarction or cerebral infarction with brain damage in the perforator distribution area is a chronic cerebral infarction.

35. The pharmaceutical composition according to claim 31, wherein Cerebral infarction from acute stage to chronic stage is called chronic stage cerebral infarction.

36. The pharmaceutical composition according to any one of claims 31 to 35, wherein A polynucleotide encoding NeuroD1 protein is contained, wherein the polynucleotide is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

8.

37. The pharmaceutical composition according to any one of claims 31 to 36, wherein The polynucleotide encoding NeuroD1 protein includes a polynucleotide having a sequence identity of 70% or more to the base sequence shown in SEQ ID NO: 7 or 10.

38. The pharmaceutical composition according to any one of claims 31 to 37, wherein The polynucleotide encoding NeuroD1 protein is a polynucleotide containing the base sequence shown in SEQ ID NO: 7 or 10.

39. The pharmaceutical composition according to any one of claims 31 to 38, wherein The polynucleotide encoding NeuroD1 protein is mRNA encoding NeuroD1 protein.

40. The pharmaceutical composition according to claim 39, wherein The mRNA encoding the NeuroD1 protein contains the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, encoding the NeuroD1 protein.

41. The pharmaceutical composition according to claim 39 or 40, wherein The mRNA encoding NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

42. The pharmaceutical composition according to any one of claims 39 to 41, wherein The mRNA encoding NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

43. The pharmaceutical composition according to any one of claims 39 to 42, wherein The mRNA encoding NeuroD1 protein contains a poly A sequence.

44. The pharmaceutical composition according to claim 43, wherein The length of the poly A sequence is 50 to 200 nucleotides.

45. The pharmaceutical composition according to any one of claims 39 to 44, wherein The mRNA encoding NeuroD1 protein has a 5' cap structure.

46. ​​The pharmaceutical composition according to any one of claims 39 to 45, wherein The mRNA encoding NeuroD1 protein contains modified nucleosides.

47. The pharmaceutical composition according to claim 46, wherein The modified nucleoside is N1-methylpseudouridine.

48. The pharmaceutical composition according to claim 47, wherein The mRNA encoding the NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

49. The pharmaceutical composition according to any one of claims 39 to 48, wherein The mRNA encoding NeuroD1 protein was encapsulated in a drug delivery system (DDS).

50. The pharmaceutical composition according to any one of claims 39 to 49, wherein The mRNA encoding NeuroD1 protein was encapsulated in lipid nanoparticles.

51. The pharmaceutical composition according to any one of claims 31 to 38, wherein The invention also contains a polynucleotide encoding NeuroD1 protein, wherein the polynucleotide is DNA.

52. The pharmaceutical composition according to any one of claims 31 to 38 and 51, wherein The invention comprises an expression vector containing a polynucleotide encoding NeuroD1 protein.

53. The pharmaceutical composition according to claim 52, wherein The expression vector is any one of an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

54. The pharmaceutical composition according to claim 53, wherein The expression vector is an adeno-associated virus vector.

55. A method for treating perforator infarction or subacute to chronic cerebral infarction, comprising the step of administering the pharmaceutical composition according to any one of claims 31 to 54, or NeuroD1 protein or a polynucleotide encoding NeuroD1 protein to a subject.

56. A method for treating cerebral infarction with brain disorders in the perforator distribution area, comprising the step of administering the pharmaceutical composition according to any one of claims 31 to 54, or the NeuroD1 protein or a polynucleotide encoding the NeuroD1 protein to a subject.

57. The method of claim 55 or 56, wherein: Perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

58. The method according to any one of claims 55 to 57, wherein: Perforator infarction or cerebral infarction with brain damage in the perforator distribution area is a chronic cerebral infarction.

59. The method of claim 55, wherein: Cerebral infarction in the subacute to chronic stage is called chronic cerebral infarction.

60. The method according to any one of claims 55 to 59, wherein: The method comprises the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide comprises a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

8.

61. The method according to any one of claims 55 to 60, wherein: The polynucleotide encoding NeuroD1 protein includes a polynucleotide having a sequence identity of 70% or more to the base sequence shown in SEQ ID NO: 7 or 10.

62. The method according to any one of claims 55 to 61, wherein: The polynucleotide encoding NeuroD1 protein is a polynucleotide containing the base sequence shown in SEQ ID NO: 7 or 10.

63. The method according to any one of claims 55 to 62, wherein: The method comprises the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide is mRNA encoding NeuroD1 protein.

64. The method of claim 63, wherein: The mRNA encoding the NeuroD1 protein contains the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, encoding the NeuroD1 protein.

65. The method of claim 63 or 64, wherein: The mRNA encoding NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

66. The method according to any one of claims 63 to 65, wherein: The mRNA encoding NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

67. The method according to any one of claims 63 to 66, wherein: The mRNA encoding NeuroD1 protein contains a poly A sequence.

68. The method of claim 67, wherein: The length of the poly A sequence is 50 to 200 nucleotides.

69. The method according to any one of claims 63 to 68, wherein: The mRNA encoding NeuroD1 protein has a 5' cap structure.

70. The method according to any one of claims 63 to 69, wherein: The mRNA encoding NeuroD1 protein contains modified nucleosides.

71. The method of claim 70, wherein: The modified nucleoside is N1-methylpseudouridine.

72. The method of claim 71, wherein: The mRNA encoding the NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

73. The method according to any one of claims 63 to 72, wherein: The mRNA encoding NeuroD1 protein was encapsulated in a drug delivery system (DDS).

74. The method according to any one of claims 63 to 73, wherein: The mRNA encoding NeuroD1 protein was encapsulated in lipid nanoparticles.

75. The method according to any one of claims 55 to 62, wherein: The method comprises the step of administering a polynucleotide encoding NeuroD1 protein to a subject, wherein the polynucleotide is DNA.

76. The method according to any one of claims 55 to 62 and 75, wherein The method comprises the step of administering an expression vector containing a polynucleotide encoding NeuroD1 protein to a subject.

77. The method of claim 76, wherein: The expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

78. The method of claim 77, wherein: The expression vector is an adeno-associated virus vector.

79. A NeuroD1 protein or a polynucleotide encoding the NeuroD1 protein, for use in treating perforator infarction or subacute to chronic cerebral infarction.

80. A NeuroD1 protein or a polynucleotide encoding the NeuroD1 protein, for use in treating cerebral infarction with brain disorders in perforator-derived areas.

81. The protein or polynucleotide for use according to claim 79 or 80, wherein: Perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

82. The protein or polynucleotide for use according to any one of claims 79 to 81, wherein: Perforator infarction or cerebral infarction with brain damage in the perforator distribution area is a chronic cerebral infarction.

83. The protein or polynucleotide for use according to claim 79, wherein: Cerebral infarction in the subacute to chronic stage is called chronic cerebral infarction.

84. The polynucleotide for use according to any one of claims 79 to 83, wherein The polynucleotide encoding NeuroD1 protein is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

8.

85. The polynucleotide for use according to any one of claims 79 to 84, wherein The polynucleotide encoding NeuroD1 protein includes a polynucleotide having a sequence identity of 70% or more to the base sequence shown in SEQ ID NO: 7 or 10.

86. The polynucleotide for use according to any one of claims 79 to 85, wherein The polynucleotide encoding NeuroD1 protein is a polynucleotide containing the base sequence shown in SEQ ID NO: 7 or 10.

87. The polynucleotide for use according to any one of claims 79 to 86, wherein The polynucleotide is mRNA encoding NeuroD1 protein.

88. The polynucleotide for use according to claim 87, wherein The mRNA encoding the NeuroD1 protein contains the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, encoding the NeuroD1 protein.

89. The polynucleotide for use according to claim 87 or 88, wherein The mRNA encoding NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

90. The polynucleotide for use according to any one of claims 87 to 89, wherein The mRNA encoding NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

91. The polynucleotide for use according to any one of claims 87 to 90, wherein The mRNA encoding NeuroD1 protein contains a poly A sequence.

92. The polynucleotide for use according to claim 91, wherein The length of the poly A sequence is 50 to 200 nucleotides.

93. The polynucleotide for use according to any one of claims 87 to 92, wherein The mRNA encoding NeuroD1 protein has a 5' cap structure.

94. The polynucleotide for use according to any one of claims 87 to 93, wherein The mRNA encoding NeuroD1 protein is a polynucleotide containing modified nucleosides.

95. The polynucleotide for use according to claim 94, wherein The modified nucleoside is N1-methylpseudouridine.

96. The polynucleotide for use according to claim 95, wherein The mRNA encoding the NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

97. The polynucleotide for use according to any one of claims 87 to 96, wherein The mRNA encoding NeuroD1 protein was encapsulated in a drug delivery system (DDS).

98. The polynucleotide for use according to any one of claims 87 to 97, wherein The mRNA encoding NeuroD1 protein was encapsulated in lipid nanoparticles.

99. The polynucleotide for use according to any one of claims 79 to 86, wherein The polynucleotide is DNA.

100. The polynucleotide for use according to any one of claims 79 to 86 and 99, wherein The polynucleotide is an expression vector containing the polynucleotide encoding NeuroD1 protein.

101. The polynucleotide for use according to claim 100, wherein The expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

102. The polynucleotide for use according to claim 101, wherein The expression vector is an adeno-associated virus vector.

103. Use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating perforator infarction or subacute to chronic cerebral infarction.

104. Use of NeuroD1 protein or a polynucleotide encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating cerebral infarction with brain disorders in perforator-derived areas.

105. The use according to claim 103 or 104, wherein: Perforator infarction or cerebral infarction with brain disorders in the perforator distribution area is a subacute to chronic cerebral infarction.

106. The use according to any one of claims 103 to 105, wherein: Perforator infarction or cerebral infarction with brain damage in the perforator distribution area is a chronic cerebral infarction.

107. The use according to claim 103, wherein: Cerebral infarction in the subacute to chronic stage is called chronic cerebral infarction.

108. The use according to any one of claims 103 to 107, wherein: The polynucleotide encoding NeuroD1 protein is a polynucleotide containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO:

8.

109. The use according to any one of claims 103 to 108, wherein: The polynucleotide encoding NeuroD1 protein includes a polynucleotide having a sequence identity of 70% or more to the base sequence shown in SEQ ID NO: 7 or 10.

110. The use according to any one of claims 103 to 109, wherein: The polynucleotide encoding NeuroD1 protein is a polynucleotide containing the base sequence shown in SEQ ID NO: 7 or 10.

111. The use according to any one of claims 103 to 110, wherein: The polynucleotide is mRNA encoding NeuroD1 protein.

112. The use according to claim 111, wherein: The mRNA encoding the NeuroD1 protein contains the base sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 11 or 13, encoding the NeuroD1 protein.

113. The use according to claim 111 or 112, wherein: The mRNA encoding NeuroD1 protein contains a 5'UTR and / or a 3'UTR.

114. The use according to any one of claims 111 to 113, wherein: The mRNA encoding NeuroD1 protein is an mRNA containing a polynucleotide consisting of the base sequence from base 1 to base 1231 of SEQ ID NO: 11 or 13.

115. The use according to any one of claims 111 to 114, wherein: The mRNA encoding NeuroD1 protein contains a poly A sequence.

116. The use according to claim 115, wherein: The length of the poly A sequence is 50 to 200 nucleotides.

117. The use according to any one of claims 111 to 116, wherein: The mRNA encoding NeuroD1 protein has a 5' cap structure.

118. The use according to any one of claims 111 to 117, wherein: The mRNA encoding NeuroD1 protein contains modified nucleosides.

119. The use according to claim 118, wherein: The modified nucleoside is N1-methylpseudouridine.

120. The use according to claim 119, wherein: The mRNA encoding the NeuroD1 protein is a polynucleotide in which a part or all of the uridine is replaced by N1-methylpseudouridine.

121. The use according to any one of claims 111 to 120, wherein: The mRNA encoding NeuroD1 protein was encapsulated in a drug delivery system (DDS).

122. The use according to any one of claims 111 to 121, wherein The mRNA encoding NeuroD1 protein was encapsulated in lipid nanoparticles.

123. The use according to any one of claims 103 to 110, wherein: The polynucleotide is DNA.

124. The use according to any one of claims 103 to 110 and 123, wherein The polynucleotide is an expression vector containing the polynucleotide encoding NeuroD1 protein.

125. The use according to claim 124, wherein: The expression vector is an adeno-associated virus vector, a retrovirus vector, a Sendai virus vector or a lentivirus vector.

126. The use according to claim 125, wherein The expression vector is an adeno-associated virus vector.

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