Pharmaceutical composition for treating spinal cord injury and method for producing non-human primate animal model for spinal cord injury

Abstract

The present invention pertains to: a pharmaceutical composition for treating spinal cord injury, the pharmaceutical composition containing an mRNA encoding NeuroD1 protein; a method for producing a non-human primate animal model with which it is possible to assess spinal cord injury (in particular, chronic-stage spinal cord injury); and a method for assessing, by using the model, the effect of a test substance as a therapeutic agent for spinal cord injury.

Description

Pharmaceutical composition for treating spinal cord injury and method for producing a non-human primate animal model of spinal cord injury

[0001] The present invention relates to a method for treating spinal cord injury, a pharmaceutical composition for treating spinal cord injury, a non-human primate animal model of spinal cord injury and a method for producing the same, and a method for evaluating a therapeutic agent for spinal cord injury using the animal model.

[0002] Spinal cord injury is a condition in which the spinal cord is damaged by a heavy external load or trauma such as an accident. Spinal cord injury can cause motor dysfunction, respiratory dysfunction, urinary dysfunction, numbness, and pain. Symptoms vary depending on whether the injury occurs in the cervical, thoracic, or lumbar spinal cord. For example, cervical spinal cord injury, which accounts for the majority of spinal cord injuries, can cause quadriplegia, sensory impairment, respiratory dysfunction, urinary dysfunction, and visceral dysfunction.

[0003] In the treatment of spinal cord injury, rehabilitation has been the primary focus, with the aim of restoring motor function. However, the recovery effect is limited, and the development of more effective treatment methods has been desired. The development of drugs for treating spinal cord injury is particularly desirable, but at present, there are no drugs that have been approved and have demonstrated a clear effect on restoring motor function. Furthermore, in the chronic phase of spinal cord injury, where functional recovery through rehabilitation is difficult, the development of drugs that demonstrate therapeutic effects on spinal cord injury is even more urgent than in the acute phase.

[0004] One of the reasons for the lack of progress in the development of therapeutic drugs for spinal cord injury is the lack of sufficient animal models capable of predicting drug efficacy in humans. In rodents, such as mice and rats, the main corticospinal tract in the spinal cord is located in a single dorsal location, whereas in primates, including humans, the main corticospinal tract is separated into two lateral locations (Non-Patent Documents 1 and 2). Therefore, evaluation of drug efficacy using rodent spinal cord injury models is considered to have low accuracy in predicting drug efficacy in humans. Therefore, the use of non-human primate spinal cord injury animal models, which have a spinal cord structure similar to that of humans, is needed to develop therapeutic drugs for spinal cord injury (Non-Patent Document 3).

[0005] Spinal cord injury occurs either through transection or crushing of the spinal cord, but is primarily caused by spinal cord crushing. Known crush models that mimic crushing include the drop model, in which a weight is dropped, and the impactor model, which uses an impactor with controllable pressure. While these existing crush models for non-human primates use different crushing methods, they all crush the spinal cord by applying pressure to a single point on the spinal cord. With the drop model, it is technically difficult to precisely control the location within the spinal cord where the dropped weight strikes, making it technically difficult to align the injury site within the spinal cord. A known drop model crushes the cervical spinal cord by dropping a weight onto the center of the spinal cord, but motor function spontaneously recovers during the chronic phase, and therefore it cannot be said to adequately reflect the pathological condition in the chronic phase (Patent Document 1, Non-Patent Documents 4 and 5). A known impactor model crushes the cervical spinal cord is described in Non-Patent Document 6, in which the spinal cord is crushed on one side, and motor function spontaneously recovers.

[0006] As such, no primate spinal cord injury model suitable for evaluating drugs aimed at improving chronic motor dysfunction due to spinal cord injury has been reported to date, nor have there been any reports examining the recovery of motor function by administering drugs in the chronic phase.

[0007] In the field of treatment for subacute to chronic spinal cord injury, drugs that enable nerve regeneration have long been desired. To date, nerve regeneration approaches based on cell transplantation, activation of endogenous stem cells, and axonal regeneration have been pursued, but no clinical trials have been successful. A distinct approach from these nerve regeneration approaches is a method that utilizes in vivo direct reprogramming (DR). DR refers to inducing differentiation of somatic cells into target cells without the intervention of pluripotent stem cells such as iPS cells.

[0008] The transcription factor Neuronal Differentiation 1 (NeuroD1) is known to be one of the factors that achieve DR, which can convert astrocytes into neurons in the brain or spinal cord. It is known that ectopic expression of NeuroD1 in glial cells such as astrocytes and NG2 cells can convert glial cells into neurons (Patent Document 2). It has been reported that forced expression of NeuroD1 in astrocytes using a viral vector in mice with spinal cord crush injury resulted in DR from astrocytes to neurons (Patent Document 3, Non-Patent Document 7). It has also been reported that forced expression of NeuroD1 in spinal cord astrocytes using a viral vector in rats with spinal cord crush injury restored motor function (Non-Patent Document 8). However, existing spinal cord injury studies using NeuroD1 have been limited to rodents, and no reports have been published using primates. In addition, existing studies have used viral vectors to express NeuroD1 (Non-Patent Documents 7 and 8), and there have been no reports using mRNA. Furthermore, there have been no reports examining the recovery of motor function by expressing NeuroD1 in a spinal cord injury model in which motor impairment persists in the chronic phase.

[0009] International Publication No. 2003 / 045137 International Publication No. 2014 / 015261 International Publication No. 2021 / 076983

[0010] Goldman S. A. , Nat. Med. , 2018 Apr 10, 24(4):388-390Filipp M. E. et al. , Neural. Regen. Res. , 2019 Jan, 14(1):7-19Corre M. L. et al. , Neurotherapeutics, 2018 Jul, 15(3):751-769Iwanami A. et al. , J. Neurosci. Res. , 2005 Apr 15, 80(2):172-181 Kobayashi Y. et al. , PLoS One, 2012, 7(12): e52787 Salegio E. A. et al. , J. Neurotrauma, 2016 Mar 1, 33(5):439-459Puls B. et al. , Front. Cell Dev. Biol. , 2020, 8:591883Chen W. et al. , Brain Res. Bull. , 2017 Oct, 135:143-148

[0011] One object of the present invention is to provide a therapeutic agent or a method for treating spinal cord injury. Another object of the present invention is to provide a non-human primate animal model of spinal cord injury that can be used for screening or evaluating therapeutic agents for spinal cord injury, and a method for producing the animal model.

[0012] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have succeeded in constructing a non-human primate animal model of spinal cord injury that can evaluate spinal cord injury (particularly chronic spinal cord injury). Furthermore, using this non-human primate animal model, they have found that mRNA encoding NeuroD1 protein is effective as a therapeutic agent for spinal cord injury. The present invention was completed based on these findings.

[0013] That is, the present invention provides the following aspects.

[0014] [1] A pharmaceutical composition for treating spinal cord injury, comprising mRNA encoding NeuroD1 protein. [2] The pharmaceutical composition according to [1] above, wherein the NeuroD1 protein is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. [3] The pharmaceutical composition according to [1] or [2] above, wherein the mRNA comprises a nucleotide sequence having 60% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 5. [4] The pharmaceutical composition according to [3] above, wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 5. [5] The pharmaceutical composition according to any of [1] to [4] above, wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 7, or a partial sequence of said nucleotide sequence comprising positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7. [6] The pharmaceutical composition according to any of [1] to [5] above, wherein the mRNA comprises a 5'UTR and / or a 3'UTR. [7] The pharmaceutical composition according to [5] or [6] above, wherein the mRNA comprises the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7. [8] The pharmaceutical composition according to any of [1] to [7] above, wherein the mRNA comprises a polyA sequence. [9] The pharmaceutical composition according to [8] above, wherein the polyA sequence is 50 to 200 nucleotides in length.

[10] The pharmaceutical composition according to any of [1] to [9] above, wherein the mRNA has a 5'-cap structure.

[11] The pharmaceutical composition according to any of [1] to

[10] above, wherein the mRNA comprises a modified nucleoside.

[12] The pharmaceutical composition according to

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

[13] The pharmaceutical composition according to

[12] above, wherein the mRNA is mRNA in which some or all uridines have been substituted with N1-methylpseudouridine.

[14] The pharmaceutical composition according to any one of [1] to

[13] above, wherein the mRNA is encapsulated in a drug delivery carrier.

[15] The pharmaceutical composition according to

[14] above, wherein the drug delivery carrier is a lipid nanoparticle.

[16] The pharmaceutical composition according to any one of [1] to

[15] above, wherein the spinal cord injury is subacute to chronic spinal cord injury.

[17] A method for treating spinal cord injury, comprising the step of administering to a subject the pharmaceutical composition according to any one of [1] to

[16] above or mRNA encoding NeuroD1 protein.

[18] mRNA encoding NeuroD1 protein for use in treating spinal cord injury.

[19] Use of mRNA encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating spinal cord injury.

[20] A method for producing a non-human primate animal model of spinal cord injury, comprising crushing both the left and right spinal cords of a non-human primate by applying pressure to sites on the dorsal surface of the cervical spinal cord of the non-human primate, located on both the left and right sides of the spinal cord midline.

[21] A method for producing a non-human primate animal model of spinal cord injury, comprising crushing either the left or right spinal cord of a non-human primate by applying pressure to sites on the dorsal surface of the cervical spinal cord of the non-human primate, located on either the left or right side of the spinal cord midline.

[22] The method described in

[20] or

[21] above, wherein the non-human primate is a marmoset.

[23] The method described in any of

[20] to

[22] above, wherein the pressure is applied using an impactor.

[24] The method described in

[23] above, wherein the pressure is 255 to 295 kdyn.

[25] The method according to

[24] above, wherein the pressure is 280 kdyn.

[26] The method according to any one of

[20] to

[25] above, wherein the pressure is applied to a portion of the cervical spinal cord exposed by removing the vertebral arch of any cervical vertebra selected from the group consisting of the third to seventh cervical vertebrae.

[27] The method according to

[26] above, wherein the pressure is applied to a portion of the cervical spinal cord exposed by removing the vertebral arch of the fifth cervical vertebra.

[28] The method according to any one of

[20] to

[27] above, wherein the spinal cord injury is a spinal cord injury in the subacute to chronic phase.

[29] The method according to any one of

[20] to

[28] above, wherein the spinal cord injury is a spinal cord injury in the chronic phase.

[30] A non-human primate animal model of spinal cord injury produced by the method according to any one of

[20] to

[29] above.

[31] A method for evaluating the effect of a test substance as a therapeutic agent for spinal cord injury, comprising the steps of administering a test substance to the non-human primate animal model described in

[30] above, and determining the effect of the test substance on spinal cord injury in the non-human primate animal model.

[32] The method described in

[31] above, further comprising the step of performing rehabilitation on the non-human primate animal model.

[0015] The present invention further provides the following aspects.

[0016]

[33] The pharmaceutical composition according to the above-mentioned

[15] , wherein the lipid nanoparticles comprise di(heptadecan-9-yl)8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate) or a salt thereof.

[34] The pharmaceutical composition according to the above-mentioned

[15] , wherein the lipid nanoparticles comprise bis(2-nonylundecyl)7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) or a salt thereof.

[35] The pharmaceutical composition according to the above-mentioned

[15] , wherein the lipid nanoparticles comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof.

[36] The pharmaceutical composition according to any one of

[33] to

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

[37] The method according to

[17] above, comprising a step of administering to a subject mRNA encoding NeuroD1 protein, wherein the NeuroD1 protein is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1.

[38] The method according to

[17] or

[37] above, wherein the mRNA comprises a nucleotide sequence having 60% or more sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 5.

[39] The method according to

[38] above, wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 5.

[40] The method according to any one of

[17] and

[37] to

[39] above, wherein the mRNA comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 7, or a partial sequence of the nucleotide sequence, which comprises positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7.

[41] The method according to any one of

[17] and

[37] to

[40] above, wherein the mRNA comprises a 5'UTR and / or a 3'UTR.

[42] The method according to

[40] or

[41] above, wherein the mRNA comprises the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7.

[43] The method according to any one of

[17] and

[37] to

[42] above, wherein the mRNA comprises a polyA sequence.

[44] The method according to

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

[45] The method according to any one of

[17] and

[37] to

[44] above, wherein the mRNA has a 5'-cap structure.

[46] The method according to any one of

[17] and

[37] to

[45] above, wherein the mRNA comprises a modified nucleoside.

[47] The method according to

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

[48] The method according to

[47] above, wherein the mRNA is mRNA in which some or all uridines have been substituted with N1-methylpseudouridine.

[49] The method according to any one of

[17] and

[37] to

[48] above, wherein the mRNA is encapsulated in a drug delivery carrier.

[50] The method according to

[49] above, wherein the drug delivery carrier is a lipid nanoparticle.

[51] The method according to

[50] above, wherein the lipid nanoparticles comprise di(heptadecan-9-yl)8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate) or a salt thereof.

[52] The method according to

[50] above, wherein the lipid nanoparticles comprise bis(2-nonylundecyl)7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) or a salt thereof.

[53] The method according to

[50] above, wherein the lipid nanoparticles comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof.

[54] The method according to any one of

[51] to

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

[55] The method according to any one of

[17] and

[37] to

[54] above, wherein the spinal cord injury is subacute to chronic spinal cord injury.

[56] The mRNA for the above-mentioned use according to

[18] above, wherein the NeuroD1 protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[57] The mRNA for the above-mentioned use according to

[18] or

[56] above, wherein the mRNA comprises a base sequence having 60% or more sequence identity to the base sequence shown in SEQ ID NO: 4 or 5.

[58] The mRNA for the above-mentioned use according to

[57] above, wherein the mRNA comprises the base sequence shown in SEQ ID NO: 4 or 5.

[59] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[58] above, wherein the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 6 or 7, or a partial sequence of the nucleotide sequence, which comprises positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7.

[60] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[59] above, wherein the mRNA comprises a 5'UTR and / or a 3'UTR.

[61] The mRNA for the above-mentioned use according to

[59] or

[60] above, wherein the mRNA comprises the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7.

[62] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[61] above, wherein the mRNA comprises a polyA sequence.

[63] The mRNA for the above-mentioned use according to

[62] above, wherein the length of the polyA sequence is 50 to 200 nucleotides.

[64] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[63] above, wherein the mRNA has a 5'-cap structure.

[65] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[64] above, wherein the mRNA comprises a modified nucleoside.

[66] The mRNA for the above-mentioned use according to

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

[67] The mRNA for the above-mentioned use according to

[66] above, wherein some or all uridines have been substituted with N1-methylpseudouridine.

[68] The mRNA for the above-mentioned use according to any one of

[18] and

[56] to

[67] above, wherein the mRNA is encapsulated in a drug delivery carrier.

[69] The mRNA for the above-mentioned use according to

[68] above, wherein the drug delivery carrier is a lipid nanoparticle.

[70] The mRNA for the above use according to

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

[71] The mRNA for the above-mentioned use according to

[69] above, wherein the lipid nanoparticles comprise 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate)bis(2-nonylundecyl) or a salt thereof.

[72] The mRNA for the above-mentioned use according to

[69] above, wherein the lipid nanoparticles comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof.

[73] The mRNA for the above-mentioned use according to any of

[70] to

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

[74] The mRNA for the use described in any of

[18] and

[56] to

[73] above, wherein the spinal cord injury is subacute to chronic spinal cord injury.

[75] The use described in

[19] above, wherein the NeuroD1 protein is a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[76] The use described in

[19] or

[75] above, wherein the mRNA comprises a nucleotide sequence having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 or 5.

[77] The use described in

[76] above, wherein the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 4 or 5.

[78] The use described in any of

[19] and

[75] to

[77] above, wherein the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 6 or 7, or a partial sequence of the nucleotide sequence comprising positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7.

[79] The use according to any one of

[19] and

[75] to

[78] above, wherein the mRNA comprises a 5'UTR and / or a 3'UTR.

[80] The use according to

[78] or

[79] above, wherein the mRNA comprises the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7.

[81] The use according to any one of

[19] and

[75] to

[80] above, wherein the mRNA comprises a polyA sequence.

[82] The use according to

[81] above, wherein the length of the polyA sequence is 50 to 200 nucleotides.

[83] The use according to any one of

[19] and

[75] to

[82] above, wherein the mRNA has a 5' cap structure.

[84] The use according to any one of

[19] and

[75] to

[83] above, wherein the mRNA comprises a modified nucleoside.

[85] The use according to

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

[86] The use according to

[85] above, wherein the mRNA is mRNA in which some or all uridines have been substituted with N1-methylpseudouridine.

[87] The use according to any one of

[19] and

[75] to

[86] above, wherein the mRNA is encapsulated in a drug delivery carrier.

[88] The use according to

[87] above, wherein the drug delivery carrier is a lipid nanoparticle.

[89] The use according to

[88] above, wherein the lipid nanoparticle comprises 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate)di(heptadecan-9-yl)

[90] The use according to the above-mentioned

[88] , wherein the lipid nanoparticles comprise bis(2-nonylundecyl)7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) or a salt thereof.

[91] The use according to the above-mentioned

[88] , wherein the lipid nanoparticles comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof.

[92] The use according to any of the above-mentioned

[89] to

[91] , wherein the lipid nanoparticles further comprise DSPC, cholesterol, and DMG-PEG2000.

[93] The use according to any of the above-mentioned

[19] and

[75] to

[92] , wherein the spinal cord injury is subacute to chronic spinal cord injury.

[0017] The present invention provides a therapeutic agent, therapeutic pharmaceutical composition, or therapeutic method for spinal cord injury, which comprises mRNA encoding NeuroD1 protein. The present invention also provides a method for preparing a non-human primate model of spinal cord injury and for evaluating the efficacy of a test substance as a therapeutic agent for spinal cord injury. This non-human primate model of spinal cord injury is particularly useful as a non-human primate model of spinal cord injury in the subacute to chronic phases.

[0018] This specification includes the disclosures of Japanese Patent Application Nos. 2023-220683 and 2023-221885, from which the present application claims priority.

[0019] FIG. 1 shows the daily Field Rating Scale scores of common marmosets whose spinal cords were crushed under the crush conditions of Experiment 6 in Example 1. The black circles indicate the Field Rating Scale scores for each test day for three individuals that survived for approximately three months after the crush, and the line graph shows the change in the average values ​​for each test day. FIG. 2 shows the daily Field Rating Scale scores of common marmosets in "2. Confirmation of Reproducibility" in Example 1. The black circles indicate the Field Rating Scale scores for each test day for two individuals that survived for approximately three months after the crush, and the line graph shows the change in the average values ​​for each test day. The Field Rating Scale was evaluated every two weeks from day 14 after the crush, and the results for day 28 represent the results evaluated on day 29. FIG. 3 shows the results of "2. Observation of the damaged area in a spinal cord injury model" in Example 2. A is an image showing a bright-field image of a sagittal spinal cord tissue section containing the damaged area. The area indicated in brackets indicates the damaged area. B is a schematic diagram of a coronal spinal cord section showing the approximate position of the sagittal spinal cord tissue section in A. The dotted line in B indicates the approximate site of amputation. FIG. 4 shows the results of "2. Confirmation of NeuroD1 protein expression" in Example 7. A is an image showing the results of immunostaining for NeuroD1 protein in a sagittal spinal cord tissue section containing the damaged area. The white dotted line indicates the outline of the spinal cord tissue, and the area indicated in brackets indicates the damaged area. Fluorescence derived from NeuroD1 protein is shown as a white signal in the image. B is a schematic diagram of a coronal spinal cord section showing the approximate position of the sagittal spinal cord tissue section in A. The dotted line in B indicates the approximate site of amputation. FIG. 5 shows the results of evaluating the effect of NeuroD1 mRNA in a spinal cord injury model in Example 8. A shows the change in score based on the Field Rating Scale for each of the six control group (Control mRNA) individuals from before drug administration to 12 weeks after drug administration. B shows the change in score based on the Field Rating Scale for each of the six drug-administered group (ND1 mRNA) individuals from before drug administration to 12 weeks after drug administration. C shows the change in grip test results for each of the six control group (Control mRNA) individuals from before drug administration to 12 weeks after drug administration.Figure D shows the change in the grip test results from before drug administration to 12 weeks after drug administration for each of the six individuals in the drug-administered group (ND1 mRNA). In A and B, the Wilcoxon matched-pairs signed rank test was performed on the scores before drug administration and 12 weeks after drug administration, and in C and D, a paired t test was performed on the change in the grip test results before drug administration and 12 weeks after drug administration. A * in the figure indicates that P<0.05 in each significant difference test, and ns in the figure indicates that P≧0.05.

[0020] The present invention will be described in detail below.

[0021] Terms used herein are used in the sense commonly used by those skilled in the art unless otherwise defined below.

[0022] 1. Pharmaceutical Compositions and Treatment Methods of the Present Invention The present invention relates to the use of a polynucleotide that is an RNA encoding NeuroD1 protein for the treatment of spinal cord injury, particularly the use of mRNA encoding NeuroD1 protein for the treatment of spinal cord injury. The present invention provides a pharmaceutical composition for the treatment of spinal cord injury (also referred to as the "pharmaceutical composition of the present invention") comprising mRNA encoding NeuroD1 protein. Furthermore, the present invention provides a method for producing a pharmaceutical composition for the treatment of spinal cord injury, comprising producing the pharmaceutical composition using mRNA encoding NeuroD1 protein. The pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein as an active ingredient for the treatment of spinal cord injury. The pharmaceutical composition of the present invention comprises a therapeutically effective amount of mRNA encoding NeuroD1 protein.

[0023] "mRNA" refers to RNA comprising an RNA sequence encoding (at least one) polypeptide (e.g., a naturally occurring polypeptide, a polypeptide having amino acid insertions, deletions, substitutions, and / or additions relative to a naturally occurring polypeptide, or a non-naturally occurring polypeptide) under the control of sequences that enable or promote translation of the mRNA (e.g., a cap structure at the 5' end of the mRNA, a 5' untranslated region (UTR), a Kozak sequence, a 3' UTR, and / or a polyA 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. The terms "polypeptide" and "protein" are used interchangeably herein. In the present invention, "mRNA encoding a NeuroD1 protein" refers to an mRNA comprising a nucleotide sequence encoding a NeuroD1 protein under the control of sequences that enable or promote translation of the mRNA. As used herein, the term "nucleotide sequence encoding NeuroD1 protein" refers to a nucleotide sequence that can be translated into NeuroD1 protein, and the nucleotide sequence is an RNA sequence. The term "nucleotide sequence encoding NeuroD1 protein" may be, for example, RNA encoding a CDS (coding sequence; a sequence from the start codon to the stop codon (including the start codon and the stop codon)) that encodes NeuroD1 protein. In a typical embodiment, the mRNA encoding NeuroD1 protein encodes the NeuroD1 protein in an expressible manner. In the present invention, "encoding in an expressible manner" means that the nucleotide sequence encoding the NeuroD1 protein is operably (e.g., in-frame) placed under the control of a sequence that enables or promotes translation of the mRNA. In one embodiment, the mRNA encoding the NeuroD1 protein in the present invention is an mRNA comprising a base sequence encoding the NeuroD1 protein and functional sequences for expressing the NeuroD1 protein (e.g., including but not limited to, a CDS, a 5'UTR, a 3'UTR, a 5' cap structure, and a polyA sequence).In one embodiment, the mRNA encoding the NeuroD1 protein of the present invention is an mRNA comprising a 5' cap structure, a 5' UTR, an RNA sequence encoding the CDS of the NeuroD1 protein, a 3' UTR, and a polyA sequence.

[0024] "NeuroD1" (Neuronal differentiation 1) is a basic helix-loop-helix (bHLH) transcription factor belonging to the NeuroD family. NeuroD1 protein activates the transcription of genes containing specific DNA sequences known as E-boxes. In other words, NeuroD1 protein preferably has transcription factor activity. NeuroD1 has a central function in inducing differentiation of neural stem cells into neurons. It is known that ectopic expression of NeuroD1 in glial cells such as astrocytes and NG2 cells can convert glial cells into neurons (WO 2014 / 015261). Glial cells are classified into four types of cells: astrocytes, oligodendrocytes, ependymal cells, and microglia.

[0025] In one embodiment, the NeuroD1 is human NeuroD1. Human NeuroD1 proteins are typically proteins of (i) or (ii) below: (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, for example, 99.4% or more or 99.5% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, or (ii) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 with insertion, deletion, substitution, and / or addition of 1 to 50 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10, or 1 to 35).

[0026] SEQ ID NO: 1 is the amino acid sequence of human NeuroD1 protein, and is a polypeptide sequence encoded by the CDS of the human NeuroD1 gene (for example, having the base sequence shown in SEQ ID NO: 2 or 3).

[0027] In one embodiment, the human NeuroD1 protein is a protein of (i) or (ii) below: (i) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence having 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more sequence identity to the amino acid sequence set forth in SEQ ID NO: 1, and having transcription factor activity; (ii) a protein consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) in the amino acid sequence set forth in SEQ ID NO: 1, and having transcription factor activity.

[0028] Those skilled in the art can easily determine whether a protein has transcription factor activity by, for example, preparing a recombinant vector incorporating a polynucleotide encoding the protein into a vector having an arbitrary promoter, introducing the vector into cells, expressing the protein, measuring the expression level of a gene (herein also referred to as a controlled gene) whose expression (transcription) is regulated by the protein's function as a transcription factor, and evaluating whether the expression level of the controlled gene has changed (increased or decreased) compared to cells not introduced with the vector. For example, if a cell introduced with a vector containing a polynucleotide encoding NeuroD1 shows increased expression of a controlled gene whose expression is known to be enhanced by NeuroD1's function as a transcription factor, it can be determined that the NeuroD1 protein has transcription factor activity.

[0029] As used herein, "sequence identity" refers to the percentage (%) of residues that match between sequences when a reference biological sequence (such as a nucleotide sequence or an amino acid sequence) and a target biological sequence are aligned (usually the percentage of matching residues relative 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., 2015, 43: W580-W584) as the identity value obtained using default parameters. The parameters are as follows: Gap Open Penalty = 10 Gap Extend Penalty = 0.5 Matrix = EBLOSUM62 End Gap Penalty = false

[0030] The mRNA encoding the NeuroD1 protein may be a natural nucleic acid (a nucleic acid derived from a natural source) or an artificial nucleic acid, and the artificial nucleic acid may be, for example, a synthetic nucleic acid comprising natural and / or artificial nucleobases.

[0031] Unless otherwise specified in the sequence listing, the bases "adenine (A)," "thymine (T)," "guanine (G)," "cytosine (C)," and "uracil (U)" constituting the base sequences of the polynucleotides described herein (for example, the base sequences shown in SEQ ID NOS: 2 to 7), as well as the nucleosides and nucleotides containing them, may be in their natural form or a corresponding modified form, and / or may have other modifications (such as methylation). Note that the symbol t in the base sequences shown in the sequence listing represents thymine in DNA and uracil in RNA, unless otherwise specified, such as a modified base.

[0032] The mRNA may include a 5' cap structure, a 5' UTR, a CDS, a 3' UTR, and a polyA sequence. The RNA sequence of the CDS included 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 terminate translation from the mRNA to the NeuroD1 protein, and may be any of the sequences TAA, TGA, and TAG. Furthermore, multiple stop codons may be used consecutively to the stop codon included in the CDS (for example, TGATAG may be used consecutively to the stop codon TAA included in the CDS). The UTR contained in the mRNA encoding the NeuroD1 protein is not particularly limited as long as it enables expression of the NeuroD1 protein. For example, it may be a 5'UTR and / or 3'UTR derived from the NeuroD1 gene, or a 5'UTR and / or 3'UTR derived from a heterologous gene (e.g., the human α-globin gene or the human β-globin gene). The 5'UTR and 3'UTR may be derived from the same heterologous gene, or may be derived from different heterologous genes. The UTR may be a native UTR, or a modified UTR whose sequence has been altered by the insertion, deletion, substitution, and / or addition of one or several (e.g., 2, 3, 4, 5, or 6) nucleotides relative to the native UTR. As used herein, the term "gene-derived UTR" includes not only native UTRs but also such modified UTRs. The 5'UTR and / or 3'UTR may be a series of multiple UTRs, either directly or via a spacer sequence. The 5'UTR may include a portion of the Kozak sequence (e.g., the sequence 5' from the start codon in the Kozak sequence). Examples of 5' cap structures include Cap-0, Cap-1, and Cap-2 (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 7th position of the guanine base of the guanosine is methylated, and is also called a 7-methylguanylic acid cap.Cap-1 has a structure in which, in addition to Cap-0, the 2-position of the ribose of the first nucleotide at the 5' end of the mRNA (the nucleotide to which the guanosine of Cap-0 is linked in a 5'-5' bond) is methylated, and Cap-2 has a structure in which, in addition to Cap-0, the 2-positions of the ribose of the first and second nucleotides from the 5' end of the mRNA are methylated. The length of the polyA sequence is not particularly limited as long as it has a function in mRNA stabilization, nuclear export, translation, etc., and may be, for example, 20 to 1,000 nucleotides (e.g., 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). Multiple poly(A) sequences may be connected together with a spacer sequence consisting of nucleotides other than A (International Publication No. WO2016 / 005004, etc.).

[0033] In one embodiment, the mRNA encoding the NeuroD1 protein is either (i) or (ii) the following mRNA: (i) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having 90% or more, preferably 95% or more, more preferably 98% or more, and even more preferably 99% or more, for example, 99.4% or more or 99.5% or more, sequence identity to the amino acid sequence set forth in SEQ ID NO: 1; or (ii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 50 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, 1 to 7, 1 to 10, or 1 to 35) in the amino acid sequence set forth in SEQ ID NO: 1. The protein (NeuroD1 protein) encoded by the mRNA preferably has transcription factor activity.

[0034] In one embodiment, the mRNA encoding the NeuroD1 protein is the following mRNA (i) or (ii): (i) an mRNA consisting of the amino acid sequence shown in SEQ ID NO: 1 or an amino acid sequence having 98% or more, preferably 99% or more, more preferably 99.4% or more or 99.5% or more sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and comprising a nucleotide sequence encoding a protein having transcription factor activity (NeuroD1 protein); or (ii) an mRNA consisting of an amino acid sequence having an insertion, deletion, substitution, and / or addition of 1 to 10 amino acids (e.g., 1 to 2, 1 to 3, 1 to 5, or 1 to 7) in the amino acid sequence shown in SEQ ID NO: 1, and comprising a nucleotide sequence encoding a protein having transcription factor activity (NeuroD1 protein).

[0035] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0036] The base sequences shown in SEQ ID NO: 4 or 5 are base sequences in which the base sequences (DNA sequences) of SEQ ID NO: 2 or 3, which are the CDS of the human NeuroD1 gene, have been replaced with RNA sequences. The base sequence shown in SEQ ID NO: 3 is a base sequence that differs from the base sequence shown in SEQ ID NO: 2 only in the stop codon. Similarly, the base sequence shown in SEQ ID NO: 5 is a base sequence that differs from the base sequence shown in SEQ ID NO: 4 only in the stop codon.

[0037] The mRNA encoding the NeuroD1 protein may be codon-optimized. Codon optimization can be performed using methods known in the art.

[0038] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (vi): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) that has 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more (e.g., 98% or more, 99% or more, or 99.5% or more) sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, (ii) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) that has 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more (e.g., 98% or more, 99% or more, or 99.5% or more) sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, (iii) an mRNA containing a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid 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 (e.g., 98% or more, 99% or more, or 99.5% or more) to the base sequence shown in SEQ ID NO: 4 and 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 amino acid sequence shown in SEQ ID NO: 1; (iv) mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence that has 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more (e.g., 98% or more, 99% or more, or 99.5% or more) sequence identity to the base sequence shown in SEQ ID NO: 5 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1; (v) mRNA comprising a base sequence that has 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more (e.g., 98% or more, 99% or more, or 99.5% or more) sequence identity to the base sequence shown in SEQ ID NO: 4 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1;(vi) An mRNA having a base sequence that has 60% or more, preferably 70% or more, more preferably 80% or more, and even more preferably 90% or more or 95% or more (e.g., 98% or more, 99% or more, or 99.5% or more) sequence identity to the base sequence shown in SEQ ID NO: 5 and that encodes a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0039] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (vi): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, (ii) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, (iii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (iv) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (v) an mRNA comprising a nucleotide sequence having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1, and (vi) an mRNA comprising a nucleotide sequence having 60% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0040] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (vi): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, (ii) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, (iii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (iv) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (v) an mRNA comprising a nucleotide sequence having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1, and (vi) an mRNA comprising a nucleotide sequence having 70% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0041] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (vi): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, (ii) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, (iii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (iv) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (v) an mRNA comprising a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1, and (vi) an mRNA comprising a nucleotide sequence having 80% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0042] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (vi): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4, (ii) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5, (iii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (iv) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, (v) an mRNA comprising a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1, and (vi) an mRNA comprising a nucleotide sequence having 90% or more sequence identity to the nucleotide sequence shown in SEQ ID NO: 5 and encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0043] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of (i) to (iii) below: (i) an mRNA comprising the base sequence shown in SEQ ID NO: 4 or 5, (ii) an mRNA comprising the base sequence shown in SEQ ID NO: 4, and (iii) an mRNA comprising the base sequence shown in SEQ ID NO: 5.

[0044] The base sequence shown in SEQ ID NO: 4 or 5 encodes a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0045] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence set forth in SEQ ID NO: 1. Preferably, the protein (polypeptide) has transcription factor activity. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence shown at positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 6 or 7, and that contains a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains the nucleotide sequence shown in SEQ ID NO: 6 or 7, or a partial sequence of the nucleotide sequence that includes positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 6 or 7 (encoding the NeuroD1 protein). Herein, a partial sequence of SEQ ID NO: 6 or 7 that includes positions 44 to 1114 of SEQ ID NO: 6 or 7 may be, but is not limited to, 1071 to 1080 bases in length, 1071 to 1100 bases in length, 1071 to 1228 bases in length, 1071 to 1231 bases in length, or 1071 to 1310 bases in length. Herein, a partial sequence of SEQ ID NO: 6 or 7 that includes positions 44 to 1120 of SEQ ID NO: 6 or 7 may be, but is not limited to, 1077 to 1080 bases in length, 1077 to 1100 bases in length, 1077 to 1228 bases in length, 1077 to 1231 bases in length, or 1077 to 1310 bases in length. In one embodiment, the mRNA encoding NeuroD1 protein is an mRNA that includes the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7.The nucleotide sequence from positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7 encodes a protein (NeuroD1 protein) consisting of the amino acid sequence shown in SEQ ID NO: 1. The nucleotide sequence from positions 44 to 1114 of SEQ ID NO: 6 or 7 is the CDS of the human NeuroD1 gene, and the nucleotide sequence from positions 44 to 1120 of SEQ ID NO: 6 or 7 includes the CDS of the human NeuroD1 gene and multiple stop codons.

[0046] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA selected from the group consisting of the following (i) to (ix): (i) an mRNA comprising a nucleotide sequence (a nucleotide sequence encoding the NeuroD1 protein) that has 60% or more sequence identity (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) to the nucleotide sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7, (ii) an mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) that has 60% or more sequence identity to the nucleotide sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the amino acid sequence shown in SEQ ID NO: 1, (iii) mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 70% or more sequence identity to the base sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the amino acid sequence shown in SEQ ID NO: 1; (iv) mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 80% or more sequence identity to the base sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the amino acid sequence shown in SEQ ID NO: 1; (v) an mRNA containing a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 90% or more sequence identity to the base sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1;(vi) mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 95% or more sequence identity to the base sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the amino acid sequence shown in SEQ ID NO: 1; (vii) mRNA comprising a base sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence having 98% or more sequence identity to the base sequence of positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more sequence identity (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) to the amino acid sequence shown in SEQ ID NO: 1; (viii) mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence that has 99% or more sequence identity to the nucleotide sequence from positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1, and (ix) mRNA comprising a nucleotide sequence encoding a protein (NeuroD1 protein) consisting of an amino acid sequence that has 99.5% or more sequence identity to the nucleotide sequence from positions 44 to 1120 of SEQ ID NO: 6 or 7 and 90% or more (e.g., 95% or more, 98% or more, 99% or more, 99.4% or more, or 99.5% or more) sequence identity to the amino acid sequence shown in SEQ ID NO: 1. The NeuroD1 protein encoded by the mRNA preferably has transcription factor activity.

[0047] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and includes a 5'UTR and / or a 3'UTR. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a 5'UTR and / or a 3'UTR derived from a heterologous gene (e.g., a human alpha globin gene or a human beta globin gene). In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding the NeuroD1 protein and including a 5'UTR and / or a 3'UTR derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further including a 5'UTR and / or a 3'UTR derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising a 5'UTR and / or a 3'UTR derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprising a 5'UTR and / or a 3'UTR derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein, and comprises a 5'UTR consisting of the nucleotide sequence of positions 4 to 43 of SEQ ID NO: 6 or 7 and / or a 3'UTR consisting of the nucleotide sequence of positions 1121 to 1231 of SEQ ID NO: 6 or 7. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence of positions 1 to 1231 or 4 to 1231 of SEQ ID NO: 6 or 7.In one embodiment, the mRNA encoding the NeuroD1 protein has a sequence identity of 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) to the base sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and is an mRNA containing a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0048] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein and also comprises a poly-A sequence. A "poly-A sequence" is a region of an mRNA downstream, e.g., directly downstream (i.e., 3') from the 3'UTR, that contains multiple (typically, but not limited to, 10 or more) consecutive adenosine monophosphates. 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 50 to 200 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 mRNA encoding the NeuroD1 protein has a polyA sequence of 75 to 85 nucleotides in length.

[0049] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and comprises a 5'UTR and / or a 3'UTR and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) an mRNA having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence.

[0050] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that includes a nucleotide sequence encoding the NeuroD1 protein and also includes a 5'UTR and / or a 3'UTR and a polyA sequence derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that includes a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further includes a 5'UTR and / or a 3'UTR and a polyA sequence derived from the human alpha globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to (i) the base sequence shown in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) the base sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and further comprises a 5'UTR and / or 3'UTR derived from the human α-globin gene and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and that contains a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and that further contains a 5'UTR and / or a 3'UTR derived from the human α-globin gene and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and that further contains a 5'UTR and / or a 3'UTR derived from the human α-globin gene and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein, and also comprises a 5'UTR consisting of the base sequence of positions 4 to 43 of SEQ ID NO: 6 or 7 and / or a 3'UTR consisting of the base sequence of positions 1121 to 1231 of SEQ ID NO: 6 or 7, and a polyA sequence.In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a polyA sequence.

[0051] In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence set forth in SEQ ID NO: 4 or 5, and further comprises a 5'UTR consisting of the nucleotide sequence of positions 4 to 43 of SEQ ID NO: 6 or 7 and / or a 3'UTR consisting of the nucleotide sequence of positions 1121 to 1231 of SEQ ID NO: 6 or 7, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a polyA sequence.

[0052] mRNA can be produced using methods known in the art. One example of a method for producing mRNA involves inserting any UTR (e.g., the 5'UTR and 3'UTR of human α-globin) and the base sequence of any nucleic acid molecule (e.g., the base sequence encoding the NeuroD1 protein) into a commercially available plasmid (e.g., a high copy plasmid for E. coli (e.g., pUC18, pCU18, etc.)) to produce a plasmid. The produced plasmid can be amplified, for example, using E. coli and purified using methods known in the art. The purified plasmid can be linearized using methods known in the art, such as, but not limited to, linearization methods using restriction enzymes and buffers. The linearized reaction product can be purified, for example, using PureLink® (Thermo Fisher Scientific). The purification method can be varied depending on the size of the linearized reaction product. The purified linearized reaction product may be used as a DNA template directly, or may be subjected to polymerase chain reaction (PCR) to generate a DNA template. The generated DNA template may be transcribed using an in vitro transcription (IVT) system. This system typically includes a transcription buffer, nucleoside triphosphates (NTPs), an RNase inhibitor, and a polymerase (e.g., T7 RNA polymerase). In the present invention, the NTPs may be natural or non-natural (modified). The DNA template may be removed using methods known in the art, such as, but not limited to, treatment with deoxyribonuclease I (DNase I). The transcribed RNA may be purified using, for example, a silica column or tangential flow filtration (TFF). If the RNA contains a poly(A) sequence, it may be purified using, for example, an oligo dT column. The mRNA may be produced through a capping reaction and / or a poly(A) addition reaction. The capping reaction can be performed using a method known in the art for adding a 5' cap structure to the 5' end of a sequence transcribed by IVT or for adding a 5' cap structure to the 5' end during IVT.The capping reaction may be carried out using a method known in the art, such as, but not limited to, a method using vaccinia capping enzyme and 2'O-methyltransferase or a method using CleanCap (registered trademark) (TriLink BioTechnologies). If the sequence transcribed by IVT does not contain a polyA sequence, a polyA addition reaction may be carried out. The constructed mRNA may be purified using a method known in the art.

[0053] In one embodiment, the mRNA encoding the NeuroD1 protein may have a cap structure or may include a 5'-terminal sequence suitable for the capping method. For example, when using CleanCap® Reagent AG (TriLink BioTechnologies), the mRNA may include the nucleotide sequence AGG at the 5'-end. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR, a nucleotide sequence encoding the NeuroD1 protein (e.g., a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1), a 3'UTR, and a poly(A) sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR, a nucleotide sequence encoding a protein having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 or 5 and consisting of the amino acid sequence shown in SEQ ID NO: 1, a 3'UTR, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR, the nucleotide sequence shown in SEQ ID NO: 4 or 5, a 3'UTR, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR, a nucleotide sequence encoding a protein having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7 and consisting of the amino acid sequence set forth in SEQ ID NO: 1, a 3'UTR, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR, the nucleotide sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, a 3'UTR, and a polyA sequence.

[0054] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR derived from the human α globin gene, a nucleotide sequence encoding the NeuroD1 protein (e.g., a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1), a 3'UTR derived from the human α globin gene, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR derived from the human α globin gene, a nucleotide sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence set forth in SEQ ID NO: 4 or 5 and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, a 3'UTR derived from the human α globin gene, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR derived from the human α globin gene, the nucleotide sequence shown in SEQ ID NO: 4 or 5, a 3'UTR derived from the human α globin gene, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7, the 5'UTR derived from the human α globin gene, a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence at positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7 and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, a 3'UTR derived from the human α globin gene, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' side, the base sequence (AGG) from positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR derived from the human α-globin gene, the base sequence from positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, a 3'UTR derived from the human α-globin gene, and a polyA sequence.In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising, from the 5' end, the nucleotide sequence (AGG) from positions 1 to 3 of SEQ ID NO: 6 or 7, a 5'UTR consisting of the nucleotide sequence from positions 4 to 43 of SEQ ID NO: 6 or 7, a 3'UTR consisting of the nucleotide sequence from positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and a 3'UTR consisting of the nucleotide sequence from positions 1121 to 1231 of SEQ ID NO: 6 or 7, and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein has, from the 5' side, 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence (AGG) at positions 1 to 3 of SEQ ID NO: 6 or 7 and the nucleotide sequence at positions 4 to 1231 of SEQ ID NO: 6 or 7, and includes a nucleotide sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a polyA sequence.

[0055] In one embodiment, the mRNA encoding the NeuroD1 protein has a 5' cap structure (e.g., Cap-0, Cap-1, or Cap-2). In one embodiment, the 5' cap structure contained in the mRNA encoding the NeuroD1 protein is Cap-1.

[0056] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) an mRNA having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that includes a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, further comprising 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 includes (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii), and encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, further comprising a 5'UTR and / or a 3'UTR, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and further comprises a 5' UTR and / or a 3' UTR and a polyA sequence, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has a 5' cap structure and comprises (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii), and that encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5' UTR and / or a 3' UTR and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has a 5' cap structure and comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5' UTR and / or a 3' UTR derived from the human α-globin gene. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) the nucleotide sequence of (i) or (ii), and encodes a protein consisting of the amino acid sequence of SEQ ID NO: 1, and further comprises a 5' UTR and / or a 3' UTR derived from the human alpha globin gene, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has a nucleotide sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, and further comprises a 5' UTR and / or a 3' UTR derived from the human alpha globin gene and a polyA sequence, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) the nucleotide sequence of (i) or (ii), and comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5' UTR and / or 3' UTR derived from the human α-globin gene and a polyA sequence, and is an mRNA having a 5' cap structure. In one embodiment, the 5' cap structure contained in these mRNAs encoding the NeuroD1 protein is Cap-1.

[0057] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which is a nucleotide sequence including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and that comprises a 5'UTR and / or a 3'UTR. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which is a nucleotide sequence including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and that comprises a 5'UTR and / or a 3'UTR and a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which is a nucleotide sequence including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and that comprises a polyA sequence. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes a nucleotide sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes a nucleotide sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and has 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 comprising a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes a nucleotide sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and has a 5' UTR and / or a 3' UTR and a polyA sequence, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes a nucleotide sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes a nucleotide sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene and a polyA sequence, and has a 5' cap structure. In one embodiment, the 5' cap structure contained in these mRNAs encoding the NeuroD1 protein is Cap-1.

[0058] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 4 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, and having Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or 4 to 1231 of SEQ ID NO: 6 or 7, and comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, and has Cap-1 as a 5' cap structure.

[0059] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6 or 7.

[0060] In the present invention, the mRNA encoding the NeuroD1 protein may be modified. For example, modified mRNA can be produced by using modified NTPs in the production of mRNA. Modified mRNA may have modifications (e.g., chemical modifications) located on the nucleic acid base, sugar moiety, and / or phosphate of the nucleotide. Modified mRNA includes, for example, modified nucleosides. A "modified nucleoside" is a nucleoside that has been modified, for example, by methylation, atom exchange, double bond saturation, deamination, or substitution of an oxygen atom or the like with a sulfur atom. Modified nucleosides may include, but are not limited to, pseudouridine, N1-methylpseudouridine (also referred to as 1-methylpseudouridine), 5-methylcytidine, 7-methylguanosine, N6-methyladenosine, 1-methyladenosine, dihydrouridine, inosine, 4-thiouridine, and the like. A polynucleotide in which "some or all" of a particular type of nucleoside is substituted with a modified nucleoside includes a polynucleotide in which some of the nucleosides of that type contained in the original polynucleotide sequence (specifically, the mRNA encoding the NeuroD1 protein) are substituted with modified nucleosides and others are not, and a polynucleotide in which all of the nucleosides of that type contained in the original polynucleotide sequence are substituted with modified nucleosides. In a polynucleotide sequence containing modified nucleosides, the term "part" with respect to the proportion of nucleosides of a particular type substituted with a modified nucleoside encompasses one or more nucleosides, and the proportion of nucleosides of a particular type substituted with a modified nucleoside relative to the total number of residues may be, but is not limited to, 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%. In one embodiment, the mRNA encoding the NeuroD1 protein of 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, some or all of the uridines may be substituted with N1-methylpseudouridine. In one embodiment, in the mRNA encoding the NeuroD1 protein of the present invention, all of the uridines may be substituted with N1-methylpseudouridine.

[0061] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a base sequence encoding the NeuroD1 protein, and comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which includes a base sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and which contains a modified nucleoside, preferably N1-methylpseudouridine.

[0062] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding the NeuroD1 protein, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) an mRNA having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which includes a base sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine.

[0063] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein, further comprising a 5'UTR and / or a 3'UTR, and is an mRNA comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, further comprising a 5'UTR and / or a 3'UTR, and is an mRNA comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains (i) the base sequence shown in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and further contains a 5'UTR and / or a 3'UTR, and further contains a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and further comprises a 5'UTR and / or a 3'UTR, wherein the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which comprises a base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR, wherein the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine.

[0064] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further comprises a 5'UTR and / or a 3'UTR, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further contains a 5'UTR and / or a 3'UTR, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 and further comprises a 5'UTR and / or a 3'UTR, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence set forth in SEQ ID NO: 6 or 7, which includes a nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine.

[0065] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein, further comprising a 5'UTR and / or a 3'UTR and a polyA sequence, and is an mRNA containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, further comprising a 5'UTR and / or a 3'UTR and a polyA sequence, and is an mRNA containing a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains (i) the base sequence shown in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and further contains a 5'UTR and / or a 3'UTR and a polyA sequence, and further contains a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which comprises a base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine.

[0066] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that contains (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further contains a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence set forth in SEQ ID NO: 6 or 7, which comprises a nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine.

[0067] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein, and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, and is an mRNA comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, and is an mRNA comprising a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is (i) the base sequence shown in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and further comprises 5'UTR and / or 3'UTR derived from the human α-globin gene, and further comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which includes a base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine.

[0068] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, and further includes 5'UTR and / or 3'UTR derived from the human α-globin gene, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which includes a base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine.

[0069] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein, and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a nucleotide sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (i) or (ii) and encodes a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprises a 5'UTR and / or 3'UTR derived from the human α-globin gene and a polyA sequence, and further comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence set forth in SEQ ID NO: 6 or 7, which comprises a base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, and the mRNA comprises a modified nucleoside, preferably N1-methylpseudouridine.

[0070] In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and further comprises a 5'UTR and / or a 3'UTR derived from a human α-globin gene and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii) of the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1, and further comprises a 5'UTR and / or 3'UTR derived from the human α-globin gene and a poly(A) sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein comprises the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6, and further comprises a 5'UTR and / or a 3'UTR derived from the human α-globin gene and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine.In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the base sequence shown in SEQ ID NO: 6 or 7, which comprises a base sequence including positions 44 to 1114 or positions 44 to 1120 of SEQ ID NO: 6 or 7, and which also comprises a 5'UTR and / or 3'UTR and a polyA sequence derived from the human α-globin gene, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine.

[0071] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and that contains a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, and that contains a modified nucleoside. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and that contains a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, and that contains N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6, and is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, in which some or all uridines are substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6, and is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, in which all uridines are substituted with N1-methylpseudouridine.

[0072] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, which comprises a modified nucleoside. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, which comprises N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, in which some or all uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridine. The mRNA encoding the NeuroD1 protein in which all uridines have been substituted with N1-methylpseudouridine may be, for example, a polynucleotide comprising the nucleotide sequence set forth in SEQ ID NO: 7.

[0073] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding the NeuroD1 protein, which comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example, (i) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (ii) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) an mRNA having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to (i) or (ii) and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, which comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR, which comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA having a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example, (i) the base sequence set forth in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii), and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising a 5'UTR and / or a 3'UTR, which contains a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, and comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA having a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example, (i) the base sequence set forth in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) a base sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii), and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising a 5'UTR and / or a 3'UTR and a polyA sequence, and further comprising a modified nucleoside, preferably N1-methylpseudouridine, and having a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein and further comprises a 5'UTR and / or a 3'UTR derived from the human alpha globin gene, and comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA having a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example, (i) the base sequence set forth in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii), and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising a 5'UTR and / or a 3'UTR derived from the human α-globin gene, which contains a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure.In one embodiment, the mRNA encoding the NeuroD1 protein comprises a base sequence encoding the NeuroD1 protein, and further comprises a 5'UTR and / or a 3'UTR derived from the human alpha globin gene and a polyA sequence, and comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA having a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, for example, (i) the base sequence set forth in SEQ ID NO: 4 or 5, (ii) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (iii) 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (i) or (ii), and encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and further comprising a 5'UTR and / or 3'UTR derived from the human α-globin gene and a polyA sequence, and further comprising a modified nucleoside, preferably N1-methylpseudouridine, and having a 5' cap structure.

[0074] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the base sequence shown in SEQ ID NO: 6 or 7, which includes a base sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, a modified nucleoside, preferably N1-methylpseudouridine, and a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the base sequence shown in SEQ ID NO: 6 or 7, which includes a base sequence from positions 44 to 1114 or from positions 44 to 1120 of SEQ ID NO: 6 or 7, and which includes a 5' UTR and / or a 3' UTR, a modified nucleoside, preferably N1-methylpseudouridine, and a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a 5'UTR and / or a 3'UTR and a polyA sequence, and further comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and further comprises a polyA sequence, further comprises a modified nucleoside, preferably N1-methylpseudouridine, and has a 5' cap structure. In one embodiment, the 5' cap structure contained in these mRNAs encoding the NeuroD1 protein is Cap-1.

[0075] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine, and the mRNA has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, which includes positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and which includes a 5'-UTR and / or a 3'-UTR, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine, and the mRNA has a 5'-cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and also comprises a 5'UTR and / or a 3'UTR and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine, and the mRNA has a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein comprises a partial sequence of the nucleotide sequence shown in SEQ ID NO: 6 or 7, including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, and also comprises a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine, and the mRNA has a 5' cap structure. In one embodiment, the 5' cap structure contained in these mRNAs encoding the NeuroD1 protein is Cap-1.

[0076] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, which comprises a modified nucleoside, preferably N1-methylpseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 4 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, which comprises a modified nucleoside, preferably N1-methylpseudouridine, and has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein has a sequence identity of 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) to the base sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and is an mRNA that includes a base sequence encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 and a polyA sequence, and that includes a modified nucleoside, preferably N1-methylpseudouridine, and has Cap-1 as a 5' cap structure.

[0077] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine, and which has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 4 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine, and which has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein has a sequence identity of 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) to the base sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and comprises a base sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 and a polyA sequence, in which some or all, preferably all, uridines are substituted with N1-methylpseudouridine, and the mRNA has Cap-1 as the 5' cap structure.

[0078] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 6 and a polyA sequence, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of the polynucleotide consisting of the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 6; (ii) adenosine at position 1 of SEQ ID NO: 6 is 2'-O-methyladenosine; and (iii) some or all of the uridines are substituted with N1-methylpseudouridine.

[0079] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 7 and a polyA sequence, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of the polynucleotide consisting of the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 7; and (ii) adenosine at position 1 of SEQ ID NO: 7 is 2'-O-methyladenosine.

[0080] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence from positions 4 to 1231 of SEQ ID NO: 6 and a polyA sequence, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of the polynucleotide consisting of the nucleotide sequence from positions 4 to 1231 of SEQ ID NO: 6; (ii) adenosine at position 4 of SEQ ID NO: 6 is 2'-O-methyladenosine; and (iii) some or all of the uridines are substituted with N1-methylpseudouridine.

[0081] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence of positions 4 to 1231 of SEQ ID NO: 7 and a polyA sequence, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of the polynucleotide consisting of the nucleotide sequence of positions 4 to 1231 of SEQ ID NO: 7; and (ii) adenosine at position 4 of SEQ ID NO: 7 is 2'-O-methyladenosine.

[0082] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, in which some or all uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 7, in which which has Cap-1 as a 5' cap structure.

[0083] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 6, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 6; (ii) adenosine at position 1 of SEQ ID NO: 6 is 2'-O-methyladenosine; and (iii) some or all of the uridines are substituted with N1-methylpseudouridine.

[0084] In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA comprising the nucleotide sequence set forth in SEQ ID NO: 7, in which: (i) 7-methylguanosine is bound via a triphosphate bond (5'-5' bond) to the 5' end of a polynucleotide consisting of the nucleotide sequence set forth in SEQ ID NO: 7; and (ii) adenosine at position 1 of SEQ ID NO: 7 is 2'-O-methyladenosine.

[0085] 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 nucleotide sequence set forth in SEQ ID NO: 6, which contains a modified nucleoside. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6, which contains N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6, in which some or all uridines have been substituted with N1-methylpseudouridine. In one embodiment, the mRNA encoding the NeuroD1 protein is an mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridine. The mRNA encoding the NeuroD1 protein in which all uridines have been substituted with N1-methylpseudouridine may be, for example, an mRNA consisting of the nucleotide sequence set forth in SEQ ID NO: 7.

[0086] In one embodiment, the pharmaceutical composition of the present invention comprising mRNA encoding NeuroD1 protein may be such that the mRNA is encapsulated in a DDS. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA may be encapsulated in a drug delivery system (DDS).

[0087] The DDS that can be used in the present invention preferably controls the distribution of mRNA encoding NeuroD1 protein in the body, enhances the effects of the mRNA, and suppresses side effects. In one embodiment, the DDS may be a drug delivery carrier. As used herein, the term "drug delivery carrier" is not particularly limited as long as it encapsulates mRNA and can control the distribution of the mRNA in the body, but examples include nanoparticles such as lipid nanoparticles and polymer particles (e.g., nanomicelle carriers composed of a block copolymer of polyethylene glycol and a biocompatible cationic polymer). In one embodiment, the pharmaceutical composition of the present invention contains mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in a drug delivery carrier. In one embodiment, the pharmaceutical composition of the present invention contains mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in a nanoparticle.

[0088] "Lipid nanoparticles" (LNPs) are nanoparticles composed of lipids with a diameter in the range of 10 nm to 1000 nm (10 nm or more and less than 1000 nm), and are used as DDSs that encapsulate nucleic acid drugs and the like. Known pharmaceutical compositions containing mRNA encoding a protein and encapsulating the mRNA in lipid nanoparticles include, for example, LNP-encapsulated RNA vaccines (e.g., L.R. Baden 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 a cationic lipid, a PEGylated lipid, a sterol, or a neutral phospholipid. In one embodiment, the lipid nanoparticles contain a cationic lipid. In one embodiment, the lipid nanoparticles comprise a neutral phospholipid, a sterol, and a PEGylated lipid. In one embodiment, the lipid nanoparticles comprise a cationic lipid, a neutral phospholipid, a sterol, and a PEGylated lipid. Examples of PEGylated lipids include DSPE-mPEG, ALC-0159, DMG-mPEG, DMG-PEG2000, and the like. Examples of sterols include cholesterol. Examples of neutral phospholipids include distearoylphosphatidylcholine (DSPC), dipalmitoylphosphatidylcholine (DPPC), 1,2-dioleoyl-3-sn-phosphatidylethanolamine (DOPE), and the like. Examples of cationic lipids (or ionizable lipids) include DLin-MC3-DMA, which is used in lipid nanoparticles encapsulating siRNA. In one embodiment, the lipid nanoparticles comprise DSPC, cholesterol, and DMG-PEG2000.In one example, lipid nanoparticles containing RNA can be constructed with a cationic lipid core surrounding the RNA, surrounded by an outer lipid layer 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)), 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 produced by rapidly mixing lipid-containing ethanol with RNA-containing low-pH buffer in a mixer (e.g., microfluidic mixing) to promote RNA encapsulation, then gradually adjusting the pH to near neutral (e.g., pH 7.4), and removing the ethanol to form lipid nanoparticles. The PEGylated lipids, sterols, neutral phospholipids, and cationic lipids that make up the lipid nanoparticles can be produced using methods known in the art.

[0089] 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]azanediyl}di(octanoic acid)di(heptadecan-9-yl)) (see Example 9) or a salt thereof, compound 2 (Ex2) represented by formula (II) (i.e., 7,7'-[({[1-(N,N-dimethylglycyl)piperidine-4- [2-nonylundecyl]-8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (see Example 11) or a salt thereof.

[0090]

[0091]

[0092]

[0093] In the present invention, the salt of a compound refers to a pharmaceutically acceptable salt, which may form an acid addition salt depending on the type of substituent. Specific examples include acid addition salts with inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid, and acid addition salts with 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.

[0094] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles.

[0095] In one embodiment, the pharmaceutical composition of the present invention comprises an mRNA selected from the group consisting of the following (i) to (xix), and the mRNA is encapsulated in a lipid nanoparticle: (i) an mRNA comprising a nucleotide sequence encoding a NeuroD1 protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, (ii) an mRNA comprising (a) the nucleotide sequence set forth in SEQ ID NO: 4 or 5, (b) the nucleotide sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (c) a nucleotide sequence having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of (a) or (b), (iii) (a) the base sequence shown in SEQ ID NO: 4 or 5, (b) the base sequence of positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, or (c) an mRNA comprising a base sequence that has 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the base sequence of (a) or (b) and encodes a NeuroD1 protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (iv) an mRNA comprising a partial sequence of the base sequence shown in SEQ ID NO: 6 or 7, which is a base sequence including positions 44 to 1114 or 44 to 1120 of SEQ ID NO: 6 or 7, (v) any of the mRNAs (i) to (iv) above, which comprises a 5'UTR and / or a 3'UTR, (vi) any of the mRNAs (i) to (iv) above, which comprises a polyA sequence, (vii) any of the mRNAs (i) to (iv) above, comprising a 5'UTR and / or a 3'UTR and a polyA sequence; (viii) any of the mRNAs (v) or (vii) above, wherein the 5'UTR and / or the 3'UTR are derived from the human α-globin gene; (ix) any of the mRNAs (i) to (iv) above, comprising a 5'UTR consisting of the nucleotide sequence of positions 4 to 43 of SEQ ID NO: 6 or 7 and / or a 3'UTR consisting of the nucleotide sequence of positions 1121 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence;(x) an mRNA having 60% or more (e.g., 70% or more, 80% or more, 90% or more, or 95% or more) sequence identity to the nucleotide sequence of positions 1 to 1231 or positions 4 to 1231 of SEQ ID NO: 6 or 7, and comprising a nucleotide sequence encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1, and a polyA sequence; (xi) an mRNA comprising the nucleotide sequence of positions 1 to 1231 of SEQ ID NO: 6 or 7 and a polyA sequence; (xii) an mRNA of any of (vi) to (xi) above, wherein the length of the polyA sequence is 50 to 200 polynucleotides; (xiii) an mRNA of any of (i) to (xii) above, wherein the mRNA has a 5' cap structure, preferably Cap-1; (xiv) an mRNA of any of (i) to (xiii) above, wherein the mRNA contains a modified nucleoside, preferably N1-methylpseudouridine; (xv) mRNA of any of (i) to (xiv) above, in which some or all, preferably all, uridines have been substituted with N1-methylpseudouridine; (xvi) mRNA comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6 or 7; (xvii) mRNA comprising or consisting of the nucleotide sequence shown in SEQ ID NO: 6, in which some or all uridines have been substituted with N1-methylpseudouridine; (xviii) mRNA comprising the nucleotide sequence shown in SEQ ID NO: 6, in which some or all uridines have been substituted with N1-methylpseudouridine and which has Cap-1 as the 5' cap structure; and (xix) mRNA comprising the nucleotide sequence shown in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridine and which has Cap-1 as the 5' cap structure (mRNA comprising the nucleotide sequence shown in SEQ ID NO: 7, in which it has Cap-1 as the 5' cap structure).

[0096] The lipid nanoparticles are not particularly limited as long as they are capable of delivering mRNA to the target cells. For example, when delivering mRNA to glial cells (e.g., astrocytes or microglia), lipid nanoparticles capable of delivering mRNA to glial cells (e.g., astrocytes) can be selected. Whether a lipid nanoparticle can deliver mRNA to glial cells can be evaluated using, for example, CleanCap® eGFP mRNA (TriLink BioTechnologies). Specifically, the mRNA is encapsulated in the lipid nanoparticles to be evaluated, and the mRNA encapsulated in the lipid nanoparticles is introduced into glial cells. Subsequently, the expression intensity of eGFP expressed in the glial cells is measured, and whether the expression intensity of eGFP has changed (increased or decreased) can be determined by comparing it with cells to which PBS has been introduced instead of the mRNA encapsulated in the lipid nanoparticles. For example, if the expression intensity of eGFP increases when mRNA encapsulated in the lipid nanoparticles is introduced, it can be determined that the lipid nanoparticles are capable of delivering mRNA to glial cells. Instead of using the expression intensity of eGFP as an index, evaluation can also be performed using, for example, the number of cells expressing eGFP as an index.

[0097] In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention is encapsulated in lipid nanoparticles that can be delivered to glial cells and can be delivered to glial cells. In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention is encapsulated in lipid nanoparticles that can be delivered to astrocytes and can be delivered to astrocytes. In one embodiment, the mRNA contained in the pharmaceutical composition of the present invention is encapsulated in lipid nanoparticles that can be delivered to microglia and can be delivered to microglia.

[0098] Examples of cationic lipids contained in lipid nanoparticles encapsulating mRNA, for example, cationic lipids contained in lipid nanoparticles capable of delivering mRNA to astrocytes, include compound 1 (Ex1, Example 9) or a salt thereof, compound 2 (Ex2, Example 10) or a salt thereof, or compound 3 (Ex3, Example 11) or a salt thereof. In one embodiment, the lipid nanoparticles capable of delivering mRNA to astrocytes contain a cationic lipid. In one embodiment, the lipid nanoparticles capable of delivering mRNA to astrocytes contain compound 1 (Ex1) or a salt thereof, compound 2 (Ex2) or a salt thereof, and / or compound 3 (Ex3) or a salt thereof as the cationic lipid.

[0099] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles, wherein the lipid nanoparticles comprise a cationic lipid. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles, wherein the lipid nanoparticles may comprise Compound 1 (Ex1) or a salt thereof, Compound 2 (Ex2) or a salt thereof, and / or Compound 3 (Ex3) or a salt thereof. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles, wherein the cationic lipid in the lipid nanoparticles may be Compound 1 (Ex1) or a salt thereof. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles, wherein the cationic lipid in the lipid nanoparticles may be Compound 2 (Ex2) or a salt thereof. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles, and the cationic lipid in the lipid nanoparticles may be compound 3 (Ex3) or a salt thereof.

[0100] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles. The lipid nanoparticles may comprise, as a cationic lipid, di(heptadecan-9-yl)8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate) or a salt thereof, bis(2-nonylundecyl)7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) or a salt thereof, and / or 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof.

[0101] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles. The lipid nanoparticles may comprise 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate)di(heptadecan-9-yl) or a salt thereof as a cationic lipid.

[0102] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles. The lipid nanoparticles may comprise 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate)bis(2-nonylundecyl) or a salt thereof as a cationic lipid.

[0103] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles. The lipid nanoparticles may comprise 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate or a salt thereof as a cationic lipid.

[0104] The present invention also includes pharmaceutical compositions comprising mRNA encoding NeuroD1 protein, wherein the mRNA is encapsulated in lipid nanoparticles. The present invention also includes pharmaceutical compositions comprising mRNA encoding NeuroD1 protein, wherein the mRNA is encapsulated in lipid nanoparticles comprising Compound 1 (Ex1) or a salt thereof, Compound 2 (Ex2) or a salt thereof, and / or Compound 3 (Ex3) or a salt thereof. The present invention also includes pharmaceutical compositions comprising mRNA encoding NeuroD1 protein, wherein the mRNA is encapsulated in lipid nanoparticles comprising Compound 1 (Ex1) or a salt thereof. The present invention also includes pharmaceutical compositions comprising mRNA encoding NeuroD1 protein, wherein the mRNA is encapsulated in lipid nanoparticles comprising Compound 2 (Ex2) or a salt thereof. The present invention also includes pharmaceutical compositions comprising mRNA encoding NeuroD1 protein, wherein the mRNA is encapsulated in lipid nanoparticles comprising Compound 3 (Ex3) or a salt thereof. The present invention also includes a pharmaceutical composition comprising mRNA encoding NeuroD1 protein and Compound 1 (Ex1) or a salt thereof. The present invention also includes a pharmaceutical composition comprising mRNA encoding NeuroD1 protein and Compound 2 (Ex2) or a salt thereof. The present invention also includes a pharmaceutical composition comprising mRNA encoding NeuroD1 protein and Compound 3 (Ex3) or a salt thereof.

[0105] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 1 (Ex1) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 1 (Ex1) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC, and the cationic lipid, DSPC, cholesterol, and DMG-PEG2000 may be contained in a ratio of 40:12:46.5:1.5.

[0106] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 2 (Ex2) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 2 (Ex2) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC, and the cationic lipid, DSPC, cholesterol, and DMG-PEG2000 may be contained in a ratio of 50:10:38.5:1.5.

[0107] In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 3 (Ex3) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC. In one embodiment, the pharmaceutical composition of the present invention comprises mRNA encoding NeuroD1 protein, and the mRNA is encapsulated in lipid nanoparticles comprising compound 3 (Ex3) or a salt thereof, and the lipid nanoparticles may further comprise DMG-PEG2000, cholesterol, and DSPC, and the cationic lipid, DSPC, cholesterol, and DMG-PEG2000 may be contained in a ratio of 50:10:38.5:1.5.

[0108] The pharmaceutical composition of the present invention may contain mRNA encoding multiple types of NeuroD1 proteins. For example, the present invention also includes pharmaceutical compositions containing mRNA in which some uridines are substituted with N1-methylpseudouridine, mRNA in which all uridines are substituted with N1-methylpseudouridine, mRNA with a 5' cap structure, and / or mRNA without a 5' cap structure.

[0109] For example, the pharmaceutical composition of the present invention containing an mRNA encoding a NeuroD1 protein also includes pharmaceutical compositions containing mRNA encoding two or more of the following (i) to (iv): (i) an mRNA comprising the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which some uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure; (ii) an mRNA comprising the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which all uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure; (iii) an mRNA consisting of the nucleotide sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which some uridines have been substituted with N1-methylpseudouridines; and (iv) An mRNA consisting of the base sequence from positions 1 to 1231 of SEQ ID NO: 6 and a polyA sequence, in which all uridines are substituted with N1-methylpseudouridine.

[0110] In one embodiment, the pharmaceutical composition of the present invention containing an mRNA encoding a NeuroD1 protein is a pharmaceutical composition containing mRNA encoding two or more of the following (i) to (iv): (i) an mRNA comprising the nucleotide sequence shown in SEQ ID NO: 6, in which some uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure; (ii) an mRNA comprising the nucleotide sequence shown in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridines and which has Cap-1 as a 5' cap structure (an mRNA comprising the nucleotide sequence shown in SEQ ID NO: 7, which has Cap-1 as a 5' cap structure); (iii) an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 6, in which some uridines have been substituted with N1-methylpseudouridines; and (iv) an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 6, in which all uridines have been substituted with N1-methylpseudouridines (i.e., an mRNA consisting of the nucleotide sequence shown in SEQ ID NO: 7).

[0111] In one embodiment, the pharmaceutical composition of the present invention containing an mRNA encoding a NeuroD1 protein is a pharmaceutical composition containing an mRNA encoding two or more of the following NeuroD1 proteins (i) to (iv), wherein the length of the polyA sequence is 40 to 200 nucleotides (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): (i) mRNA comprising the base sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which some uridines have been substituted with N1-methylpseudouridine and which has Cap-1 as the 5' cap structure; (ii) mRNA comprising the base sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which all uridines have been substituted with N1-methylpseudouridine and which has Cap-1 as the 5' cap structure; (iii) mRNA consisting of the base sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which some uridines have been substituted with N1-methylpseudouridine; and (iv) mRNA consisting of the base sequence from positions 1 to 1231 of SEQ ID NO: 6 and a poly-A sequence, in which all uridines have been substituted with N1-methylpseudouridine.

[0112] The pharmaceutical composition of the present invention may also include a pharmaceutical composition containing mRNA encoding multiple types of NeuroD1 proteins with different lengths of polyA sequences.

[0113] The pharmaceutical compositions of the present invention may also include pharmaceutical compositions formulated without encapsulating mRNA in a DDS (for example, encapsulating it in a drug delivery carrier).

[0114] The pharmaceutical composition of the present invention may contain pharmaceutically acceptable adjuvants, such as inert carriers (solid or liquid carriers), excipients, surfactants, solubilizing agents, suspending agents, colorants, flavoring agents, preservatives, buffers, pH adjusters, etc. The pharmaceutical composition of the present invention can be prepared or formulated by commonly used methods using pharmaceutically acceptable adjuvants, such as commonly used pharmaceutical excipients and pharmaceutical carriers. The term "pharmaceutical composition" as used herein means a composition that can be used as a medicament.

[0115] The pharmaceutical composition of the present invention can be used for treating spinal cord injuries.

[0116] Spinal cord injury (SCI) is a condition in which the spinal cord is damaged by severe external stress or trauma, such as in an accident. Spinal cord injury disrupts the nerve pathways in the spinal cord, resulting in motor dysfunction in the limbs and impaired function of internal organs such as the lungs. While some spinal cord injuries are caused by non-traumatic factors such as tumors and circulatory disorders, most are caused by traumatic factors such as traffic accidents, falls from heights, or tripping. Symptoms of spinal cord injury vary depending on the location and severity of the injury. Damage to the cervical spinal cord can affect both the upper and lower body, while damage to the lumbar spinal cord can affect the lower body. Disabilities associated with spinal cord injury include motor paralysis (motor dysfunction) and sensory paralysis. Motor dysfunction can include walking difficulties, balance problems, limited range of motion, difficulty with flexion, extension, adduction, and abduction, and decreased arm and grip strength. In particular, the action of grasping or holding "objects" is frequently used in daily life and is extremely important for maintaining one's life, but spinal cord injury makes it difficult to flex one's fingers and significantly reduces grip strength, which becomes a major obstacle to daily life. Spinal cord injury resulting in impairment of both limbs is also referred to as "spinal cord injury associated with quadriplegia," and spinal cord injury resulting in impairment of motor function of the upper arm is also referred to as "spinal cord injury associated with impairment of motor function of the upper arm." The spinal cord injury in the present invention is preferably or includes cervical spinal cord injury.

[0117] Examination or diagnosis of spinal cord injury in humans can be performed using X-ray (roentgen) imaging, MRI, or computed tomography (CT) of the injured area. Known assessment scales for human spinal cord injury include the American Spinal Injury Association (ASIA) Score Scale and the Quality of Life Scale.

[0118] Many patients with spinal cord injury experience spasticity and / or contracture as a pathological condition. Spasticity is characterized by abnormal muscle tension, resulting in stiffness and difficulty moving the limbs, involuntary movements, clenched fingers that are difficult to open, bending of the elbows, and bending of the toes toward the soles of the feet. Contracture is a condition in which muscles atrophy due to paralysis or loss of muscle flexibility due to immobility, resulting in loss of normal joint movement. Spasticity, in particular, is observed in almost all patients with chronic spinal cord injury. In clinical practice, spinal cord injury patients typically undergo rehabilitation aimed at maintaining joint movement to prevent and / or prevent the progression of spasticity and / or contracture. Examples of such rehabilitation include stretching, range of motion exercises, massage, and heat therapy.

[0119] In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat subacute to chronic spinal cord injury. In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat chronic spinal cord injury. In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat spinal cord injury accompanied by upper arm dysfunction (e.g., subacute to chronic or chronic spinal cord injury). In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat spinal cord injury accompanied by limb paralysis (e.g., subacute to chronic or chronic spinal cord injury). In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat spinal cord injury, including cervical spinal cord injury (e.g., subacute to chronic or chronic spinal cord injury). In one embodiment, the pharmaceutical composition or mRNA encoding a NeuroD1 protein of the present invention can be used to treat spinal cord injury caused by crush injury (e.g., subacute to chronic or chronic spinal cord injury).

[0120] The pharmaceutical composition of the present invention or mRNA encoding the NeuroD1 protein can improve the motor function of a subject who has suffered a spinal cord injury, for example, a subject who has suffered a spinal cord injury in the subacute to chronic phase or a spinal cord injury in the chronic phase.

[0121] The present invention also provides a method for treating spinal cord injury (hereinafter also referred to as the "therapeutic method of the present invention"), comprising the step of administering to a subject the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein. In one embodiment, the therapeutic method of the present invention may be a method for treating spinal cord injury, comprising the step of administering to a subject a therapeutically effective amount of the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein. In one embodiment, the therapeutic method of the present invention may be a method for treating spinal cord injury, comprising the step of administering to a subject a therapeutically effective amount of mRNA encoding NeuroD1 protein. In one embodiment, the therapeutic method of the present invention may be a method for treating spinal cord injury, comprising the step of administering to a subject a therapeutically effective amount of mRNA encoding NeuroD1 protein, and the step of rehabilitation to alleviate spasticity and contracture. In a typical embodiment, the mRNA encoding NeuroD1 protein encodes NeuroD1 protein in an expressible state. In one embodiment, the mRNA used in the therapeutic method of the present invention is encapsulated in a drug delivery carrier, in one embodiment, the mRNA used in the therapeutic method of the present invention is encapsulated in a nanoparticle, and in one embodiment, the mRNA used in the therapeutic method of the present invention is encapsulated in a lipid nanoparticle.

[0122] The subject to which the therapeutic methods and pharmaceutical compositions of the present invention are applied may be any mammal (subject), including primates (humans and non-human primates), livestock (horses, cows, sheep, goats, pigs, etc.), pets (dogs, cats, rabbits, etc.), laboratory (test) animals (mice, rats, etc.), etc. Primates are preferred, and humans are particularly preferred. The subject to which the therapeutic methods and pharmaceutical compositions of the present invention are applied is preferably one in need of the therapeutic method and pharmaceutical composition, etc., and may be, for example, a subject with spinal cord injury, for example, a spinal cord injury in the subacute to chronic phase (e.g., chronic phase), or a subject suspected of having a spinal cord injury, for example, a spinal cord injury in the subacute to chronic phase (e.g., chronic phase).

[0123] In humans, the period from immediately after the onset of spinal cord injury to less than two weeks, during which symptoms may worsen, is called the "acute phase." In humans, the period from two weeks to less than four weeks after the onset of spinal cord injury is called the "subacute phase" (also called the recovery phase), and the period from four weeks after onset is called the "chronic phase." The subacute phase is a period during which a higher degree of recovery from paralysis can be expected compared to the chronic phase. In humans, the subacute to chronic phases of spinal cord injury refer to the period from two weeks after the onset of spinal cord injury, and may include, for example, the period from four weeks after the onset of spinal cord injury, or may include the period from three months after the onset of spinal cord injury, for example, six months after the onset of spinal cord injury. The "acute phase," "subacute phase," and "chronic phase" in non-human animal subjects can be appropriately defined by those skilled in the art as periods corresponding to the "acute phase," "subacute phase," and "chronic phase" in humans, respectively.

[0124] The dosage and frequency of administration of the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to a subject can be adjusted appropriately depending on the disease to be treated, its severity, the age, body weight, condition, etc. The dosage can be, for example, 0.00001 mg / kg body weight to 1 mg / kg body weight of mRNA encoding NeuroD1 protein per administration.

[0125] The method of administering the pharmaceutical composition of the present invention or mRNA encoding the NeuroD1 protein to a subject is not particularly limited, and examples include local administration by surgical means (e.g., intraspinal administration, intrathecal administration, intraspinal parenchymal administration, etc.), cardiac catheter administration, administration using an ultrasound contrast agent (microbubbles), intravenous administration, lower limb puncture administration, local injection administration, subcutaneous administration, intradermal administration, intramuscular administration, etc.

[0126] In one embodiment, the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein is administered to a subject by local administration via surgical means (e.g., intraspinal administration, intrathecal administration, intraspinal parenchymal administration, etc.). In one embodiment, the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein is administered to a subject by intraspinal administration. In one embodiment, the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein is administered to a subject by intrathecal administration. In one embodiment, the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein is administered to a subject by intraspinal parenchymal administration.

[0127] As used herein, the term "damaged region" refers to the region of the spinal cord where damage has occurred.

[0128] As used herein, the term "administration site" refers to the location where the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein is administered to a subject.

[0129] The method for administering the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to a subject comprises administering the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to at least one administration site in a damaged area of ​​the subject. When multiple damaged areas are present, the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein may be administered to at least one administration site in each damaged area. In one embodiment, the method may involve administering the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to one or more damaged areas of the subject, at one administration site per damaged area.

[0130] The number of times the pharmaceutical composition of the present invention or mRNA encoding the NeuroD1 protein is administered to a subject is not particularly limited and may be a single administration or multiple administrations. The timing of the single administration may be any time after the onset of spinal cord injury, for example, during the subacute to chronic phase or the chronic phase, and is not particularly limited, and may be, for example, 2 weeks, 1 month (4 weeks), 3 months, 6 months, 1 year, 1.5 years, 2 years, 3 years, 5 years, 7 years, 10 years, etc. after the onset of spinal cord injury. In the case of multiple administrations, for example, the first administration may be any time after the onset of spinal cord injury, for example, during the subacute to chronic phase or the chronic phase, and subsequent administrations may be at regular intervals, for example, every six months.

[0131] The device used for local administration of the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to a subject is not particularly limited as long as it allows administration to the target site.

[0132] The therapeutic method of the present invention can effectively treat spinal cord injury. In one embodiment, the therapeutic method of the present invention can effectively treat spinal cord injury in the subacute to chronic phase. In one embodiment, the therapeutic method of the present invention can effectively treat spinal cord injury in the chronic phase. In a preferred embodiment, the therapeutic method of the present invention can significantly improve the motor function of a subject suffering from spinal cord injury (e.g., spinal cord injury in the subacute to chronic phase, particularly spinal cord injury in the chronic phase). In the present invention, "treatment" of spinal cord injury includes, but is not limited to, recovery from nerve fiber transection and neuronal loss in the damaged area, and also includes elimination, alleviation, improvement, or prevention or delay of progression of symptoms of spinal cord injury (e.g., motor dysfunction). Rehabilitation, including rehabilitation to alleviate spasticity and contracture, may also be performed as part of the therapeutic method. In one embodiment, the treatment of spinal cord injury in the present invention may be treatment of symptoms of spinal cord injury (e.g., motor dysfunction). In one embodiment, the therapeutic method of the present invention is a method for treating spinal cord injury comprising the step of administering to a subject the pharmaceutical composition of the present invention or mRNA encoding the NeuroD1 protein. The therapeutic method of the present invention may include a step of administering the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to a subject, as well as a step of providing rehabilitation to the subject. Rehabilitation may be performed before the start of administration of the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein, during the administration period, and / or after completion of administration. In one embodiment, the therapeutic method of the present invention is a method for treating spinal cord injury, comprising a step of administering the pharmaceutical composition of the present invention or mRNA encoding NeuroD1 protein to a subject, and a step of providing continuous rehabilitation during the treatment period.

[0133] The present invention also provides the pharmaceutical composition or mRNA encoding the NeuroD1 protein of the present invention for use in treating spinal cord injury (eg, subacute to chronic spinal cord injury, or chronic spinal cord injury).

[0134] The present invention also provides the use of mRNA encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for treating spinal cord injury (eg, subacute to chronic spinal cord injury, or chronic spinal cord injury).

[0135] 2. Preparation of a Non-Human Primate Animal Model The present invention also provides a method for preparing a non-human primate animal model of spinal cord injury (hereinafter also referred to as the "method for preparing the animal model of the present invention"), which comprises crushing the spinal cord by applying pressure (compression) to the cervical spinal cord of a non-human primate.

[0136] a) Non-human Primates As used herein, "non-human primates" refers to any primate (order Primates) animal other than humans. The non-human primates (monkeys) used in the present invention are preferably non-human primates that can be evaluated by scoring motor dysfunction using the Original Open Field Rating Scale (Kitamura K. et al., PLoS One, 2011, 6(11):e27706) or a modified Field Rating Scale (see Table 1 below). Examples of non-human primates used in the present invention include apes, monkeys belonging to the Cercopithecidae family, and monkeys belonging to the Capuchin family. "Ape" refers to monkeys belonging to the Hominoidea of ​​the Order Primates (Primates). Examples of apes include chimpanzees, gorillas, orangutans, bonobos, and gibbons. Examples of monkeys belonging to the Capuchin family include, but are not limited to, monkeys such as the tufted capuchin monkey (capuchin monkey) belonging to the Capuchin genus, monkeys such as the common squirrel monkey (squirrel monkey) belonging to the Sciuridae genus, and monkeys such as the common marmoset (marmoset) belonging to the Marmoset genus. Examples of monkeys belonging to the Cercopithecidae family include, but are not limited to, monkeys such as the Japanese macaque, rhesus macaque, cynomolgus macaque, Taiwan macaque, pig-tailed macaque, and bonnet monkey belonging to the Macaca genus; monkeys such as the hamadryas baboon and gelada baboon belonging to the Baboon genus; and monkeys such as the mandrill and drill belonging to the Mandrill genus. In one embodiment, the non-human primate used in the animal model production method of the present invention is a marmoset. In one embodiment, the non-human primate used in the animal model production method of the present invention is a common marmoset. In one embodiment, the non-human primate used in the method for producing an animal model of the present invention is a marmoset (e.g., a common marmoset) that is approximately 1 year of age or older. The non-human primate used in the method for producing an animal model of the present invention may be male or female.

[0137] The common marmoset (Callithrix jacchus) is a primate classified in the genus Callithrix, family Marmosets, order Primates. Common marmosets are widely used in animal experiments because they share many similarities with humans in terms of the brain, nervous system, circulatory system, digestive system, endocrine system, immune system, behavior, and so on. Furthermore, the brain of a common marmoset has a well-developed prefrontal cortex, motor cortex, and premotor cortex, and contains association fibers such as the arcuate fasciculus, uncinate fasciculus, inferior longitudinal fasciculus, superior longitudinal fasciculus, and inferior fronto-occipital fasciculus, which are not found in rodent brains. Thus, the common marmoset shares many structures and functions with the human brain. Marmosets are highly active and exhibit a wide variety of behaviors, making them an ideal animal species for behavioral observation.

[0138] b) Crush of the Spinal Cord in Non-Human Primates The "spinal cord" is a cylindrical bundle of nerves extending from the brain and is part of the central nervous system. It is divided into four regions: cervical, thoracic, lumbar, and sacral. The "cervical spinal cord" is the highest part of the spinal cord, continuing cranially to the medulla oblongata and caudally to the thoracic spinal cord. The cervical spinal cord has eight nerve segments, each from which a pair of spinal nerves emerges. The cervical spinal cord primarily controls superficial sensation in the neck, respiratory muscles, and muscles and superficial sensation in the upper limbs. The sympathetic nerves that control the head and neck also emerge from the cervical spine. The spine surrounds and protects the spinal cord. The spine is made up of bones called vertebrae, and consists of the cervical, thoracic, lumbar, sacral, and caudal vertebrae, from the head to the tail. The cervical spine consists of seven vertebrae, referred to as the first through seventh cervical vertebrae, numbered C1 through C7. The vertebrae have a vertebral body that serves as a support column on the ventral side, and the spinal canal through which the spinal cord passes on the dorsal side. The dorsal side of the spinal canal is covered by the vertebral arch, and the vertebral arch and vertebral body are connected by the pedicles. Spinous processes are present behind the vertebral arch. The spinal cord has a central canal that connects to the ventricles, and the "H"-shaped area surrounding it is gray matter, where neuronal cell bodies are collected. The gray matter is surrounded by white matter, through which countless nerve fibers run. One example of this bundle of nerve fibers is the corticospinal tract. The corticospinal tract is a bundle of axons (nerve fibers) that run from the motor cortex of the cerebral cortex through the spinal cord to lower motor neurons, and most of the axons that make up the corticospinal tract are upper motor neuron axons. In primates, including humans, the main corticospinal tract exists in two lateral locations, one on the left and right, and one on the left and right ventromedial locations.

[0139] In the method for producing an animal model of the present invention, the spinal cord of a non-human primate is crushed by applying pressure to a site on the dorsal surface of the cervical spinal cord located on at least one of the left and right sides of the spinal midline.

[0140] As used herein, "crush" refers to the destruction of internal tissues due to external impact or pressure. "Crushing the spinal cord" refers to destroying and damaging the spinal cord by applying external pressure to the spinal cord. Whether the spinal cord is damaged can be evaluated, for example, by confirming the presence or absence of nerve fiber rupture and / or the presence or absence of neuronal loss. The presence or absence of nerve fiber rupture can be evaluated, for example, by magnetic resonance imaging (MRI), electrophysiological techniques, immunohistochemical staining using a cytoskeleton marker, or the like. The presence or absence of neuronal loss can be evaluated, for example, by immunohistochemical staining using a neuronal marker, or the like.

[0141] In the method for producing an animal model of the present invention, both the left and right spinal cords of a non-human primate may be crushed by applying pressure to at least two sites on the dorsal surface of the cervical spinal cord located on both the left and right sides of the spinal cord midline. The sites (at least two sites) on the dorsal surface of the cervical spinal cord located on both the left and right sides of the spinal cord midline to which pressure is applied are preferably sites that crush the lateral corticospinal tracts located on the two outer sides of the spinal cord. In one embodiment, the sites (at least two sites) on the dorsal surface of the cervical spinal cord located on both the left and right sides of the spinal cord midline can be defined by exposing the spinal cord (cervical spinal cord) by resecting (removing) the vertebral arches, including the spinous processes of the cervical vertebrae, to a position where the edges of the spinal cord are visible, and then defining the sites from the position on the dorsal surface where a tip (described below) does not touch the remaining portions of the left and right vertebral arches to at least the spinal cord midline (e.g., beyond the spinal cord midline to the region opposite the side where the center of the pressure site is located). In one embodiment, the method for producing an animal model of the present invention may be a method for producing a non-human primate animal model of spinal cord injury, comprising crushing both the left and right spinal cords of a non-human primate by applying pressure to two sites on the dorsal surface of the cervical spinal cord of the non-human primate, one on the left and one on the right side of the spinal cord midline. Compression may be applied sequentially to the pressure sites on the left and right sides, or may be applied simultaneously. In one embodiment, the non-human primate used in the animal model of the present invention is a marmoset, for example, a common marmoset.

[0142] In the method for producing an animal model of the present invention, pressure may be applied to a site on the dorsal surface of the cervical spinal cord of a non-human primate, either on the left or right side of the spinal midline, to crush either the left or right side of the spinal cord. The site on the dorsal surface of the cervical spinal cord, either on the left or right side of the spinal midline, to which pressure is applied is preferably a site that crushes either of the two lateral corticospinal tracts located on the left and right sides of the spinal cord. In one embodiment, the site on the dorsal surface of the cervical spinal cord, either on the left or right side of the spinal midline, can be determined by excising the vertebral arch, including the spinous processes of the cervical vertebrae, to a position where the edges of the spinal cord are visible, thereby exposing the spinal cord (cervical spinal cord), and then defining the site as a region extending from the point where a tip (described below) does not touch the remaining part of the resected (removed) vertebral arch to at least the spinal cord midline (e.g., beyond the spinal midline to the region opposite the side where the center of the pressure site is located). In one embodiment, the method for producing an animal model of the present invention may be a method for producing a non-human primate model of spinal cord injury, comprising crushing either the left or right side of the cervical spinal cord of a non-human primate by applying pressure to a site on the dorsal surface located on either the left or right side of the spinal cord midline. In one embodiment, the non-human primate used in the animal model of the present invention is a marmoset.

[0143] As used herein, with respect to the dorsal surface of the cervical spinal cord, "spinal midline" means the line along the dorsal median groove of the spinal cord that bisects the spinal cord into left and right sides.

[0144] As used herein, "bilateral spinal cord" refers to the left and right sides of the spinal cord when the spinal cord is bisected along the median sulcus of the spinal cord. As used herein, "one of the left or right spinal cord" refers to either the left or right side of the spinal cord when the spinal cord is bisected along the median sulcus of the spinal cord.

[0145] As used herein, "left side" with respect to the body of a subject or non-human primate means the left-hand side of the subject or non-human primate, and "right side" means the right-hand side of the subject or non-human primate.

[0146] As used herein, "sites on the dorsal surface of the cervical spinal cord, located on both the left and right sides of the spinal cord midline" refers to both the site (area) on the dorsal surface of the cervical spinal cord to be compressed, located to the left of the spinal cord midline, and the site (area) to be compressed, located to the right of the spinal cord midline. As used herein, "site located to the left of the spinal cord midline" refers to a site to be compressed that has its center on the left side of the spinal cord midline. A site to be compressed "located to the left of the spinal cord midline" may be a site consisting only of the area to the left of the spinal cord midline, or a site whose center is offset to the left of the spinal cord midline but also includes a region near the center of the spinal cord on the right side of the spinal cord midline. Similarly, "site located to the right of the spinal cord midline" refers to a site to be compressed that has its center on the right side of the spinal cord midline. The pressure area "located on the right side of the spinal cord midline" may be a pressure area consisting only of the area to the right of the spinal cord midline, or a pressure area whose center is offset to the right of the spinal cord midline but also includes the area near the center of the spinal cord on the left side beyond the spinal cord midline.

[0147] As used herein, the term "pressure site" refers to the area on the surface of the cervical spinal cord to which pressure is applied when applying pressure to the cervical spinal cord. As used herein, the "center" of the pressure site refers to the geometric center of the area to which pressure is applied. For example, when applying pressure using a round impactor tip, the "center" of the pressure site is the position where the center (geometric center) of the circle on the surface shape of the pressure-applying part of the tip is applied, and when applying pressure using a square impactor tip, the "center" of the pressure site is the position where the intersection (geometric center) of the medians of the rectangle on the surface shape of the pressure-applying part of the tip is applied. The position of the pressure-applying part can be determined by the position of the center of the pressure-applying part.

[0148] As used herein, the term "crushing conditions" refers to "conditions for inducing crushing by applying pressure."

[0149] The means for crushing in the method for producing an animal model of the present invention is not particularly limited as long as it can crush the cervical spinal cord with an arbitrarily set pressure, and for example, an impactor can be used. In one embodiment, the method for producing an animal model of the present invention is a method for producing a non-human primate animal model of spinal cord injury, which comprises crushing the spinal cord by applying pressure to the cervical spinal cord of a non-human primate using an impactor.

[0150] As used herein, an "impactor" refers to a machine capable of applying pressure to a desired area. The impactor of the present invention is preferably an impactor that can inflict force damage on a spinal cord, such as the cervical spinal cord, at an arbitrarily set pressure. Use of such an impactor makes it possible to apply a desired pressure to the cervical spinal cord with good reproducibility. The impactor is not particularly limited as long as it can apply a desired pressure to the cervical spinal cord with good reproducibility, but for example, an IH impactor (Precision Systems and Instrumentation, Inc., IH-0400) can be used.

[0151] In one embodiment, the method for producing an animal model of the present invention is a method for producing a non-human primate model of spinal cord injury, comprising crushing both the left and right spinal cords of a non-human primate by applying pressure using an impactor to sites on the dorsal surface of the cervical spinal cord of the non-human primate, located on both the left and right sides of the spinal cord midline.

[0152] In one embodiment, the method for producing an animal model of the present invention is a method for producing a non-human primate model of spinal cord injury, comprising crushing either the left or right side of the cervical spinal cord of a non-human primate by applying pressure with an impactor to a site on the dorsal surface of the spinal cord, which is located on either the left or right side of the spinal cord midline.

[0153] In the method for producing an animal model of the present invention, the pressure applied to the cervical spinal cord, for example, the pressure of the impactor (pressure exerted by the impactor), may be, for example, more than 200 kdyn, more than 250 kdyn, less than 300 kdyn, more than 200 kdyn and less than 300 kdyn, more than 250 kdyn and less than 300 kdyn, 255 to 295 kdyn, 260 to 290 kdyn, 265 to 285 kdyn, or 280 kdyn. In one embodiment, the pressure is preferably 255 to 295 kdyn or 270 to 290 kdyn, and more preferably 280 kdyn. Here, 1 kdyn (kilodyne) = 0.01 N (Newton).

[0154] The impactor pressure described above is particularly suitable for application to monkeys, preferably belonging to the genus Marmoset, such as the common marmoset, but is not limited to application to such species. The impactor pressure can be appropriately determined by those skilled in the art depending on the species used (e.g., the body weight of the species).

[0155] In the animal model preparation method of the present invention, pressure may be applied by attaching a tip (impactor tip) to the impactor. The size of the tip is not particularly limited as long as it can crush the left or right spinal cord. Taking marmosets as an example, for example, a tip with a diameter of 2.5 mm may be used. The size of the tip can be appropriately determined by those skilled in the art depending on the species of organism used (e.g., the body weight of the species). The impactor tip may have any shape, and may have, for example, a round (circular) or angular (rectangular) pressure-applying surface shape.

[0156] In the method for producing an animal model of the present invention, the time for which pressure is applied using an impactor (hereinafter also referred to as "crush time") can be appropriately selected as long as it is possible to crush the spinal cord, and the crush time may be, for example, 1 second or more, 3 seconds or more, 5 seconds or more, 1 second to 1 minute, 3 seconds to 1 minute, 5 seconds to 1 minute, 1 second to 30 seconds, 3 seconds to 30 seconds, 5 seconds to 30 seconds, 1 second to 10 seconds, 3 seconds to 10 seconds, or 5 seconds to 10 seconds. In one embodiment, the crush time in the method for producing an animal model of the present invention is 5 seconds.

[0157] In one embodiment, the method for producing an animal model of the present invention may involve applying pressure to a portion of the cervical spinal cord exposed by removal of the vertebral arch of any one of the cervical vertebrae C1 to C7 levels (e.g., C3 to C7, C4 to C6, C4 or C5, C5 or C6, or C5). In one embodiment, the method for producing an animal model of the present invention comprises applying pressure to a portion of the cervical spinal cord exposed by removal of the vertebral arch of at least one cervical vertebra selected from the group consisting of the third to seventh cervical vertebrae (cervical vertebrae C3, C4, C5, C6, and C7). In one embodiment, the method for producing an animal model of the present invention comprises applying pressure to a portion of the cervical spinal cord exposed by removal of the vertebral arch of cervical vertebra C5.

[0158] When crush injury is performed two or more times, pressure may be applied to the same region of the cervical spinal cord, or pressure may be applied to different regions of the cervical spinal cord.

[0159] In the method for producing an animal model of the present invention, the site to which pressure is applied (pressure site) is not particularly limited as long as it can crush the target spinal cord (cervical spinal cord), but to induce quadriplegia, it is preferable to apply pressure to sites on the dorsal surface of the cervical spinal cord located on both the left and right sides of the spinal midline. Alternatively, to induce motor dysfunction in either the left or right half of the body, it is preferable to apply pressure to sites on the dorsal surface located on either the left or right side of the spinal midline.

[0160] In the method for producing an animal model of the present invention, for example, when crushing the left side of the spinal cord, pressure may be applied only to the region to the left of the spinal cord midline without crossing the midline, or pressure may be applied to a region where the center of the pressure site is offset to the left of the midline but also includes a region near the center of the spinal cord on the right side crossing the midline. Furthermore, in the method for producing an animal model of the present invention, for example, when crushing the right side of the spinal cord, pressure may be applied only to the region to the right of the midline without crossing the midline, or pressure may be applied to a region where the center of the pressure site is offset to the right of the midline but also includes a region near the center of the spinal cord on the left side crossing the midline. In the method for producing an animal model of the present invention, when crushing both the left and right spinal cords, the above-mentioned crushing of the left spinal cord and the above-mentioned crushing of the right spinal cord may be combined.

[0161] In the method for producing an animal model of the present invention, the number of times pressure is applied to each site can be appropriately selected. In one embodiment, the method for producing an animal model of the present invention is a method for producing a non-human primate animal model of spinal cord injury, which comprises crushing the cervical spinal cord of a non-human primate by applying pressure once to each site on the dorsal surface located on both the left and right sides of the spinal cord midline (a total of two times).

[0162] In the method for producing an animal model of the present invention, motor dysfunction can be induced in the left half of the body by crushing the left side of the spinal cord, and motor dysfunction can be induced in the right half of the body by crushing the right side of the spinal cord.

[0163] The present invention also provides a non-human primate animal model of spinal cord injury (hereinafter also referred to as "the non-human primate animal model of the present invention") prepared by the above-mentioned method for preparing an animal model of the present invention.

[0164] The non-human primate animal model of the present invention has its spinal cord crushed in the animal model production method of the present invention, and is therefore useful as a spinal cord injury model (animal model). In one embodiment, the non-human primate animal model of the present invention can also be used as an acute spinal cord injury model. In one embodiment, the non-human primate animal model of the present invention can maintain injury for a relatively long period (e.g., 2 weeks to 3 months or more), and therefore can be used as a model of subacute to chronic spinal cord injury. In one embodiment, the non-human primate animal model of the present invention is useful as a model of subacute to chronic spinal cord injury. In one embodiment, the non-human primate animal model of the present invention is particularly useful as a model of chronic spinal cord injury. In one embodiment, the animal model production method of the present invention can induce acute to chronic spinal cord injury by crushing the spinal cord of a non-human primate. In one embodiment, the animal model production method of the present invention can induce subacute to chronic spinal cord injury by crushing the spinal cord of a non-human primate. In one embodiment, the animal model production method of the present invention can induce chronic spinal cord injury by crushing the spinal cord of a non-human primate.

[0165] In non-human primates, including marmosets, the acute phase refers to the period from immediately after spinal cord injury to less than two weeks after the onset of spinal cord injury, during which symptoms may worsen. The subacute phase refers to the period from two weeks (14 days after spinal cord injury) to less than four weeks after the onset of spinal cord injury, and the chronic phase refers to the period from four weeks after the onset of spinal cord injury. In the present invention, the subacute to chronic phase during which spinal cord injury in a non-human primate animal model is evaluated or non-human primates are treated may be the period from two weeks after the onset of spinal cord injury, for example, from three weeks after the onset of spinal cord injury, or from three weeks after the onset of spinal cord injury or one month after the onset of spinal cord injury, for example, from three weeks to six months or one month to six months after the onset of spinal cord injury, or from three weeks to three months or one month to three months after the onset of spinal cord injury. In this specification, "one week after" means seven days after the onset of spinal cord injury, "two weeks after" means 14 days after the onset of spinal cord injury, and "three weeks after" means 21 days after the onset of spinal cord injury.

[0166] One of the reasons why severe sequelae remain in the chronic phase of spinal cord injury is that in mammalian central nervous systems, astrocytes react around the injury site to form a scab-like tissue called a glial scar, which inhibits nerve regeneration. A chronic spinal cord injury model in which glial scars are formed is very useful for developing therapeutic agents for chronic spinal cord injury. In one embodiment, the non-human primate animal model of the present invention exhibits neuronal loss, nerve fiber transection, and astrocyte accumulation in the chronic phase of spinal cord injury.

[0167] In one embodiment, the method for producing an animal model of the present invention can induce spinal cord injury accompanied by quadriplegia by crushing the bilateral spinal cord of a non-human primate. In one embodiment, the non-human primate animal model of the present invention can also be used as a spinal cord injury model accompanied by quadriplegia. In one embodiment, the method for producing an animal model of the present invention can induce spinal cord injury accompanied by upper arm motor dysfunction by crushing the cervical spinal cord of a non-human primate. In one embodiment, the non-human primate animal model of the present invention can also be used as a spinal cord injury model accompanied by upper arm motor dysfunction. The upper arm motor dysfunction may include a decrease in grip strength. In one embodiment, the non-human primate animal model of the present invention can also be used as a spinal cord injury model accompanied by spasticity and / or contracture.

[0168] c) Methods for Confirming and Evaluating Spinal Cord Injury Whether the non-human primate animal model of the present invention is a non-human primate animal model of spinal cord injury can be determined by, for example, histological techniques such as TTC staining or hematoxylin-eosin (HE) staining, or by confirming that the non-human primate animal model has an injured area using X-ray (radiography), MRI, or computed tomography (CT), similar to diagnostic methods for human spinal cord injury. In addition to the above methods, whether the non-human primate animal model is a subacute to chronic spinal cord injury model can be confirmed based on the persistence of the impairment. For example, if motor dysfunction persists for at least two weeks, preferably four weeks, after the onset of spinal cord injury, the non-human primate animal model of spinal cord injury can be determined to be useful as a non-human primate animal model of subacute to chronic spinal cord injury. However, the method for confirming the persistence of the impairment is not limited thereto and can be changed depending on the evaluation method.

[0169] The indicators used to evaluate the state of motor dysfunction (symptoms, severity, duration of dysfunction, etc.) of the non-human primate animal model of the present invention are not particularly limited as long as they can evaluate the state of motor dysfunction of the animal, and can be used, for example, the Field Rating Scale described in this specification.

[0170] Evaluation using the Field Rating Scale can be performed by scoring based on Table 1 below. The Field Rating Scale described in Table 1 is an index modified from the original open field rating scale, and can be used to evaluate the state of motor dysfunction (symptoms, severity, duration of the disorder, etc.) in the non-human primate animal model of the present invention.

[0171] When the Field Rating Scale is used to assess motor dysfunction, for example, a non-human primate animal model of the present invention can be assessed as having quadriplegia if it cannot maintain a sitting position and the total score on the Field Rating Scale does not exceed 10. When the Field Rating Scale is used to assess the persistence of quadriplegia, for example, a non-human primate animal model of the present invention assessed as having quadriplegia can be assessed as not having spontaneous recovery (quadriplegia persisting) if the total score on the Field Rating Scale does not exceed 10, and can be assessed as having spontaneous recovery (quadriplegia not persisting) if the total score on the Field Rating Scale exceeds 10.

[0172] In one embodiment, the non-human primate animal model of the present invention is useful as an animal model of spinal cord injury accompanied by quadriplegia. In one embodiment, the non-human primate animal model of the present invention is useful as an animal model of spinal cord injury accompanied by forelimb motor dysfunction.

[0173]

[0174] The non-human primate animal model of the present invention preferably involves spasticity and / or contracture, which reflects the clinical picture. Therefore, the non-human primate animal model of the present invention is useful as a model of spinal cord injury accompanied by spasticity and / or contracture. In the method for producing an animal model of the present invention, spinal cord injury accompanied by spasticity and / or contracture can be induced by crushing the spinal cord of a non-human primate.

[0175] 3. Method for Evaluating a Test Substance Using a Non-Human Primate Model The present invention also provides a method for evaluating the effect of a test substance as a therapeutic agent for spinal cord injury (therapeutic effect on spinal cord injury) in a non-human primate model of the present invention (hereinafter also referred to as a "method for evaluating a test substance of the present invention"). More specifically, the method for evaluating a test substance of the present invention may be a method for evaluating the effect of a test substance as a therapeutic agent for spinal cord injury, comprising the steps of administering the test substance to a non-human primate model of the present invention and determining the effect of the test substance on spinal cord injury in the non-human primate model.

[0176] In one embodiment, the method for evaluating a test substance of the present invention is a method for evaluating the therapeutic effect of a test substance on spinal cord injury, preferably on acute, subacute to chronic, or chronic spinal cord injury. The method for evaluating a test substance of the present invention may be a method for evaluating the therapeutic effect of a test substance on spinal cord injury in the same subject as the subject to which the above-mentioned treatment method of the present invention is applied.

[0177] In one embodiment, the method for evaluating a test substance of the present invention is a method for evaluating the therapeutic effect of a test substance on spinal cord injury accompanied by upper arm motor dysfunction. The upper arm motor dysfunction may include a decrease in grip strength. In one embodiment, the method for evaluating a test substance of the present invention is a method for evaluating the therapeutic effect of a test substance on spinal cord injury accompanied by quadriplegia. In one embodiment, the method for evaluating a test substance of the present invention is a method for evaluating the therapeutic effect of a test substance on spinal cord injury including cervical spinal cord injury. In one embodiment, the method for evaluating a therapeutic agent for spinal cord injury of the present invention is a method for evaluating the therapeutic effect of a test substance on spinal cord injury accompanied by spasticity and / or contracture.

[0178] In the test substance evaluation method of the present invention, the test substance administered to the non-human primate animal model of the present invention may be any substance, such as a peptide, protein, cell, low molecular weight compound, medium molecular weight compound, antibody, cell, or nucleic acid. The nucleic acid may be DNA or RNA (e.g., messenger RNA (mRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), antisense oligonucleotide, etc.), and may be a natural nucleic acid or an artificial nucleic acid. The artificial nucleic acid may be, for example, a synthetic nucleic acid containing natural and / or artificial nucleic acid bases. Examples of peptides, proteins, cells, low molecular weight compounds, and medium molecular weight compounds include substances with nerve 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, and mesenchymal stem cells. The test substance may be a substance known as a therapeutic agent for spinal cord injury, a substance not known as a therapeutic agent for spinal cord injury, or a novel substance. The test substance may be a candidate substance for a therapeutic agent for spinal cord injury.

[0179] In one embodiment, the method for evaluating a test substance of the present invention may be a method for screening for a therapeutic agent for spinal cord injury using a non-human primate animal model of the present invention. The method for screening for a therapeutic agent for spinal cord injury using a non-human primate animal model of the present invention comprises screening a test substance for its effectiveness as a therapeutic agent for spinal cord injury. In another embodiment, the method for evaluating a test substance of the present invention may be a method for evaluating the effectiveness of a test substance known as a therapeutic agent for spinal cord injury as a therapeutic agent for spinal cord injury using a non-human primate animal model of the present invention. This method for evaluating the effectiveness as a therapeutic agent for spinal cord injury comprises determining the effectiveness of the test substance (therapeutic agent for spinal cord injury) based on its effect as a therapeutic agent for spinal cord injury.

[0180] In the method for evaluating a test substance of the present invention, means for administering the test substance include, but are not limited to, oral administration, injection (intravenous injection, subcutaneous injection, intramuscular injection, etc.), administration to the spinal cord (intraspinal administration, intrathecal administration, intraspinal parenchymal administration), transplantation, etc. The test substance can be administered to the non-human primate animal model of the present invention at any time. However, for the purpose of evaluating the effect of the test substance as a spinal cord injury therapeutic agent on spinal cord injury in the subacute to chronic phases, it is preferable to administer the test substance to the non-human primate animal model of the present invention in the subacute to chronic phase (e.g., 2 weeks or 4 weeks after the onset of spinal cord injury). Similarly, for the purpose of evaluating a spinal cord injury therapeutic agent on spinal cord injury in the chronic phase, it is preferable to administer the test substance to the non-human primate animal model of the present invention in the chronic phase (e.g., 4 weeks after the onset of spinal cord injury). Alternatively, for the purpose of evaluating a spinal cord injury therapeutic agent on spinal cord injury in the acute phase, it is preferable to administer the test substance to the non-human primate animal model of the present invention in the acute phase (e.g., from immediately after the onset of spinal cord injury to less than 2 weeks after the onset of spinal cord injury).

[0181] The method for evaluating a test substance of the present invention includes a step of determining the effect of the test substance administered to a non-human primate animal model of the present invention. The effect of the administered test substance on spinal cord injury in a non-human primate animal model of the present invention can be determined, for example, by observing motor functions such as grip strength, mobility, sitting, standing, and jumping ability in individual animal models, diagnosing with CT or MRI, performing electromyography, or evaluation based on the Field Rating Scale, and determining whether or not spinal cord injury or disorders associated with spinal cord injury (motor dysfunction, sensory paralysis, visceral damage, etc.) have improved compared to before administration of the test substance. A test substance that has been administered to a non-human primate animal model of the present invention and has been determined to have an effect as a therapeutic agent for spinal cord injury is determined to have an effect as a therapeutic agent for spinal cord injury. Furthermore, when a test substance is administered to a non-human primate animal model of the present invention in the subacute to chronic phase (e.g., chronic phase), and the test substance is determined to have an improvement in spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be determined to have an effect as a therapeutic agent for spinal cord injury in the subacute to chronic phase (e.g., chronic phase). Alternatively, when a test substance is administered to a non-human primate animal model of the present invention in the acute phase, and the test substance is determined to have an improvement in spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be determined to have an effect as a therapeutic agent for spinal cord injury in the acute phase. In the method for screening a therapeutic agent for spinal cord injury, when a test substance is administered to a non-human primate animal model of the present invention, and the test substance is determined to have an improvement in spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be identified as a therapeutic agent for spinal cord injury. Furthermore, in the method for screening therapeutic agents for spinal cord injury, a test substance is administered to a non-human primate animal model of the present invention in the subacute to chronic phase (e.g., chronic phase), and the test substance is determined to have improved spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, and the test substance can be identified as a therapeutic agent for spinal cord injury in the subacute to chronic phase (e.g., chronic phase).Alternatively, in a method for screening therapeutic agents for spinal cord injury, a test substance is administered to a non-human primate animal model of the present invention in the acute phase, and the test substance is determined to have improved spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, and the test substance can be identified as a therapeutic agent for spinal cord injury in the acute phase. In a method for evaluating the effectiveness of a test substance known as a therapeutic agent for spinal cord injury as a therapeutic agent for spinal cord injury, if a test substance is administered to a non-human primate animal model of the present invention and the result is determined to have improved spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be confirmed to be effective as a therapeutic agent for spinal cord injury, and the level of the therapeutic effect of the test substance for spinal cord injury can be determined based on the level of improvement in spinal cord injury or a disorder associated with spinal cord injury. In addition, in a method for evaluating the efficacy of a test substance known as a therapeutic agent for spinal cord injury as a therapeutic agent for spinal cord injury, if the test substance is administered to a non-human primate animal model of the present invention in the subacute to chronic phase (e.g., chronic phase) and the results are determined to show an improvement in spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be confirmed to be effective as a therapeutic agent for spinal cord injury in the subacute to chronic phase (e.g., chronic phase), and the level of therapeutic effect of the test substance for spinal cord injury in the subacute to chronic phase (e.g., chronic phase) can be determined based on the level of improvement in spinal cord injury or a disorder associated with spinal cord injury. Alternatively, in a method for evaluating the efficacy of a test substance known as a therapeutic agent for spinal cord injury as a therapeutic agent for spinal cord injury, if the test substance is administered to a non-human primate animal model of the present invention in the acute phase and the results are determined to show an improvement in spinal cord injury or a disorder associated with spinal cord injury compared to before administration of the test substance, the test substance can be confirmed to be effective as a therapeutic agent for acute spinal cord injury, and the level of therapeutic effect of the test substance for acute spinal cord injury can be determined based on the level of improvement in spinal cord injury or a disorder associated with spinal cord injury.

[0182] In the method for evaluating a test substance of the present invention, when evaluation is performed using grip strength as an index to determine the effect of a test substance administered to a non-human primate animal model of the present invention, for example, the grip test described in Example 8 can be used.

[0183] The method for evaluating a test substance of the present invention may include a step of performing rehabilitation, for example, before, during, and / or after administration of the test substance. The rehabilitation is not particularly limited and may be, for example, range of motion training (e.g., joint flexion and extension exercises) for the part of the body suffering from motor dysfunction.

[0184] In one embodiment, the method for evaluating a test substance of the present invention comprises the steps of performing rehabilitation on a non-human primate animal model of the present invention, administering a test substance, and determining the effect of the test substance on spinal cord injury in the non-human primate animal model.

[0185] The present invention will be described in more detail below using examples, although the technical scope of the present invention is not limited to these examples.

[0186] Unless otherwise specified, the procedures in each step described in the following examples could be performed using known techniques. Furthermore, for the parts in which commercially available kits or reagents were used, experiments were performed according to the attached protocols unless otherwise specified.

[0187] In some cases, naming software such as ACD / Name (registered trademark, Advanced Chemistry Development, Inc.) is used to name the compounds described in this specification.

[0188] For convenience, the unit of concentration, mol / L, is represented as M. For example, a 1M aqueous solution of sodium hydroxide means a 1 mol / L aqueous solution of sodium hydroxide.

[0189] Example 1 Construction of a Spinal Cord Injury Model 1. Investigation of Conditions for Construction of a Spinal Cord Injury Model A method for constructing a spinal cord injury model in which quadriplegia persists for 3 months or more was investigated using common marmosets (purchased from CLEA Japan, Inc., Japan SLC, Inc., or Shin Nippon Scientific Research, Inc., or bred in-house at Astellas Pharma Inc., aged approximately 1 year or older).

[0190] Common marmosets were induced with anesthesia using a 50 mg / mL solution of ketamine hydrochloride (Ketalar 500 mg for intramuscular injection; Daiichi Sankyo Propharma, Approval Number: 21600AMZ00252; hereafter referred to as "ketamine hydrochloride") at a dose of 0.12 to 0.35 mL / animal, and a 20 mg / mL solution of xylazine (Selactal 2% injection; Bayer Yakuhin, Approval Order Number: 27 Animal Drug No. 2679; hereafter referred to as "xylazine") at a dose of 0.02 to 0.08 mL / animal. The common marmoset under anesthesia was intubated with an endotracheal intubation tube, an Atom Nutrition Catheter (6 Fr) (Atom Medical Co., Ltd., 2103111C), and maintenance anesthesia was performed by inhalation of isoflurane (isoflurane inhalation anesthetic solution "VTRS"; Mylan Pharmaceuticals Co., Ltd., Approval Number: 22700AMX00134; hereinafter referred to as "isoflurane"). The hair on the back of the anesthetized common marmoset was shaved and the marmoset was placed face down on a 37°C heating mat. The neck skin was incised, and the connective tissue was removed to expose the vertebrae. The vertebral arch, including the spinous process of cervical vertebra C5, was resected to expose the spinal cord (cervical spinal cord).

[0191] The common marmosets prepared in this manner were subjected to spinal cord (cervical) crush injury using an IH impactor (Precision Systems and Instrumentation, IH-0400; in the following examples, this device will be referred to as the "impactor") under the crush conditions described in Table 2. Crush injury was induced by applying pressure to each pressure site on the dorsal portion of the spinal cord (cervical spinal cord) exposed by resection of the vertebral arch of cervical vertebra C5 for 5 seconds. After crush injury, the muscles and skin surrounding the injured site were sutured and kept warm until the animal woke up. The common marmosets were then returned to their home cages (covered with a warming mat and soft bedding). The state of motor dysfunction after crush injury was evaluated using the Field Rating Scale (Table 1). As an evaluation criterion, an animal was judged to have quadriplegia if it could not maintain a sitting position and the total score for the forelimbs and hindlimbs based on the Field Rating Scale (Table 1) did not exceed 10 points. In subsequent daily evaluations of animals judged to have quadriplegia, if the total score for the forelimbs and hindlimbs based on the Field Rating Scale (Table 1) did not exceed 10 points, it was judged to have no spontaneous recovery, and if it exceeded 10 points, it was judged to have spontaneous recovery.

[0192]

[0193] Considering the thickness of the cervical spinal cord in common marmosets, the inventors first hypothesized that it would be possible to crush both the left and right spinal cords simultaneously by applying pressure with an impactor using a 3.5 mm diameter tip with the center of the tip positioned on the midline of the spinal cord. Therefore, in Experiments 1 to 3, we investigated whether it would be possible to induce motor dysfunction equally on both sides of the spinal cord in an individual by applying pressure once to the above-mentioned exposed area using a 3.5 mm diameter round tip or a 3.5 mm long square tip (both manufactured by Precision Systems and Instrumentation) with the center of the pressure applied positioned on the midline of the spinal cord on the dorsal surface of the spinal cord.

[0194] In Experiment 1, crushing was performed at a pressure of 200 kdyn. As a result, the motor dysfunction induced by a pressure of 200 kdyn was quadriplegia on the first day after crushing, but this quadriplegia was mild and recovered spontaneously within 1 to 2 weeks after crushing.

[0195] In Experiments 2 and 3, the tip shape and number of compressions were varied and the impactor pressure was set at 300 kdyn, the upper limit of the recommended pressure range. In Experiments 2 and 3, all animals showed quadriplegia on the first day after crushing. However, most animals recovered spontaneously from quadriplegia within four weeks after crushing. Therefore, it was concluded that the method of crushing both the left and right spinal cords simultaneously by applying pressure with the center of the pressure applied on the midline of the spinal cord cannot induce long-term motor dysfunction, even at the maximum crushing pressure of the impactor.

[0196] In primates, the main corticospinal tract is located on both the left and right dorsal sides. In Experiments 1-3, the center of the pressure application site was placed on the spinal cord midline, which may have resulted in inadequate damage to the corticospinal tract present on both the left and right sides of the spinal cord. Therefore, in Experiments 4-6, a method was investigated in which the left and right spinal cords were crushed separately by applying pressure from the dorsal side using a standard impact tip (Precision Systems and Instrumentation, Inc.), a round tip with a diameter of 2.5 mm. The spinal cord (cervical spinal cord) was exposed by resecting the vertebral arch, including the spinous process of cervical vertebra C5, to a position where the spinal cord edge could be visualized. The impactor pressure application sites were placed on the dorsal surface of the cervical spinal cord on the left and right sides of the spinal cord midline in the exposed area, respectively, and pressure was applied once for each pressure site (total of two times). Pressure was applied sequentially to the left and right pressure sites. The impactor was positioned on both sides to ensure that the tip did not touch the remaining portions of the left and right vertebral arches. Under crushing conditions of 300 kdyn (Experiment 4), one of three animals sustained tetraplegia approximately three months after the crush, but the remaining two animals died or were euthanized due to failure to regain spontaneous breathing due to the crush. Crushing conditions with a maximum impactor pressure of 300 kdyn appear to have had a significant impact on respiratory function. On the other hand, under crushing conditions with a pressure of 250 kdyn (Experiment 5), both animals tested exhibited tetraplegia two weeks after the crush, but spontaneous recovery of the tetraplegia was observed five weeks after the crush, indicating a milder degree of crushing. In Experiment 6, the pressure was set to 280 kdyn. As a result, one of the four animals tested in Experiment 6 died seven days after the crush injury, but the remaining three animals still had quadriplegia four weeks after the crush injury, and two of these animals still had quadriplegia approximately three months after the crush injury (Figure 1).

[0197] These results demonstrate that applying pressure to the left and right sides of the cervical spinal cord midline with an impactor to crush the spinal cord (cervical spinal cord) can damage the spinal cord and induce long-term motor dysfunction. In particular, when applying pressure at 280 kdyn to the cervical spinal cord once on each side of the spinal cord midline (twice in total), long-term motor dysfunction was induced with a high success rate.

[0198] In conducting Experiments 1 to 6, antibiotics, analgesics, antiemetics and / or anti-inflammatory drugs were administered as needed to avoid the risk of pain, infection and / or gastrointestinal dysfunction occurring during the perioperative period.

[0199] 2. Confirmation of Reproducibility The reproducibility of the induction of quadriplegia was confirmed under the conditions described in Experiment 6. As a result, quadriplegia was confirmed approximately 3 months after the crush in two of the four animals. The other animal also maintained quadriplegia until the 14th day after the crush, but then developed respiratory abnormalities and died. The remaining animal's activity level decreased from the day after surgery and died two days after the crush. Figure 2 shows the scores based on the Field Rating Scale of the two animals confirmed to have quadriplegia approximately 3 months after the crush. Although there have been no reports of a spinal cord injury model in which quadriplegia persists for a long period of time, the conditions described in Experiment 6 allowed us to reproduce the induction of long-term motor dysfunction.

[0200] These results demonstrate that a spinal cord injury model in which quadriplegia persists for more than three months can be reproducibly produced by crushing the cervical spinal cord (cervical spinal cord) by applying a pressure of 280 kdyn to sites on the dorsal surface of the cervical spinal cord located on both the left and right sides of the spinal cord midline. Furthermore, it was demonstrated that the non-human primate animal model produced under these crushing conditions is a model that can be used to evaluate acute and subacute-chronic spinal cord injury (especially chronic spinal cord injury).

[0201] Example 2 Analysis of Pathology in the Injured Region of a Spinal Cord Injury Model 1. Preparation of Spinal Cord Sections from a Spinal Cord Injury Model One individual from the spinal cord injury model prepared under the crush conditions described in Experiment 6 of Example 1 was euthanized 29 days after the crush injury, and the spinal cord was excised and fixed using 4% paraformaldehyde. The water in the fixed spinal cord tissue was then replaced with sucrose solution (10, 20, or 30%) prepared by dissolving sucrose (Nacalai Tesque, Inc., 30404-45) in PBS (Fujifilm Wako Pure Chemical Industries, Ltd., 045-29795), and 10 μm-thick frozen sagittal spinal cord sections were then prepared.

[0202] 2. Observation of the damaged area in the spinal cord injury model The spinal cord sections prepared in this manner were mounted in PBS, and bright-field images were taken using a microscope (KEYENCE, BZ-X810).

[0203] The results are shown in Figure 3. When crushing was performed from the dorsal side using an impactor, both the white matter and gray matter of the spinal cord (cervical spinal cord) were damaged, and furthermore, it was observed that the spinal cord was damaged from the dorsal side to the ventral side (Figure 3A). These results demonstrate that the spinal cord was successfully crushed under the crushing conditions of Experiment 6 in Example 1.

[0204] 3. Accumulation of astrocytes in the injured area of ​​a spinal cord injury model Spinal cord sections prepared in the same manner as in 1 above were subjected to immunohistochemical staining using an anti-GFAP antibody (Abcam, ab53554) against GFAP (glial fibrillary acidic protein), an astrocyte marker, and an anti-NeuN antibody (D4G4O) XP (Cell Signaling Technology, 24307) against NeuN, a neuronal marker.

[0205] As a result, we found that GFAP-positive cells accumulated around the injured area, and that NeuN-positive cells were lost in the injured area compared to the uninjured area. These results indicate that astrocytes accumulated around the injured area and neurons were lost. The spinal cord injury model created under the crush conditions of Experiment 6 in Example 1 was shown to be a spinal cord injury model that exhibited neuronal loss and astrocyte accumulation, similar to human chronic spinal cord injury.

[0206] 4. Transcription of nerve fibers in the injured area of ​​the spinal cord injury model Frozen spinal cord sections prepared by the same method as in 1 above were subjected to immunohistochemical staining using an anti-NFH (Neurofilament H) antibody (Merck Millipore, AB5539) against neurofilament, a cytoskeleton marker of nerve cells.

[0207] The results showed that neurofilaments were damaged in the injured area, suggesting that nerve fibers running through the spinal cord were severed in the injured area.

[0208] As shown in the results of 3 and 4 above, the spinal cord injury model prepared by the method of the present invention exhibited astrocyte accumulation, neuronal loss, and nerve fiber transection in the chronic phase, which were similar to the pathology of spinal cord injury reported in clinical practice. Therefore, this model was shown to be useful as an animal model that reflects the pathology of spinal cord injury (acute phase, subacute phase to chronic phase, especially chronic phase).

[0209] (Example 3) Preparation of mRNA-LNP Two types of NeuroD1 mRNA encoding human NeuroD1 protein were prepared: (i) TriLink Two mRNAs were prepared (hereafter referred to as "ND1-1"): (i) an mRNA consisting of a base sequence including 5'UTR and 3'UTR provided by BioTechnologies, CDS (SEQ ID NO: 4) of the human NeuroD1 gene, and a 120-nucleotide polyA sequence, and having a 5' cap structure of Cap-1; and (ii) an mRNA consisting of a base sequence (SEQ ID NO: 6) including 5'UTR derived from the human α-globin gene, 3'UTR derived from the human α-globin gene, CDS (SEQ ID NO: 5) of the human NeuroD1 gene, and a 79-nucleotide polyA sequence, in which all uridines have been replaced with N1-methylpseudouridines (SEQ ID NO: 7), and having a 5' cap structure of Cap-1 (hereafter referred to as "ND1-2") (contracted to TriLink BioTechnologies). In the base sequences shown in SEQ ID NOs: 6 and 7, nucleotide positions 1 to 3 correspond to the 5'-terminal sequence suitable for capping using CleanCap (registered trademark) Reagent AG (TriLink BioTechnologies), positions 4 to 43 correspond to the 5'UTR, positions 44 to 1114 correspond to the CDS of the human NeuroD1 gene (SEQ ID NO: 5), positions 1115 to 1120 correspond to two consecutive stop codons, positions 1121 to 1231 correspond to the 3'UTR, and positions 1232 to 1310 correspond to the polyA sequence. As a control, eGFP mRNA (TriLink BioTechnologies, L-7601) was used. The base sequence of the CDS of the human NeuroD1 gene (SEQ ID NO: 4) and the base sequence of the CDS of the human NeuroD1 gene (SEQ ID NO: 5) are identical except for the stop codon.

[0210] The mRNAs (i) and (ii) above were mixed with lipids having the compositions shown in Table 3 to prepare lipid nanoparticles encapsulating mRNA (hereinafter referred to as "mRNA-LNPs"). Hereinafter, lipid nanoparticles having the composition of L1 will be referred to as "lipid nanoparticles L1," and lipid nanoparticles having the composition of L6 will be referred to as "lipid nanoparticles L6."

[0211]

[0212] Specifically, mRNA-LNP was prepared by the following method. The cationic lipid Compound 1 (Ex1; the production method is described in Example 9), DSPC (NOF Corporation, COATSOME® MC-8080), cholesterol (Nippon Fine Chemicals Co., Ltd., Cholesterol HP), and DMG-PEG2000 (NOF Corporation, SUNBRIGHT® GM-020) were dissolved in ethanol at an N / P ratio of 6 to obtain an oil phase. A 10 mM citrate buffer solution (pH 4 or 6) containing the above mRNA was used as the aqueous phase. The aqueous and oil phases were mixed in a microfluidic device (NanoAssemblr®, Precision NanoSystems) so that the volume ratio of the aqueous phase to the oil phase was 3:1. The mixture was then diluted with PBS to obtain a dispersion of mRNA-LNP. The ethanol was removed from this dispersion by dialysis or ultrafiltration. Subsequently, the dispersion was concentrated by ultrafiltration to obtain mRNA-LNP adjusted to a desired concentration. The "N / P ratio" is the value obtained by dividing the number of moles of amino groups (N) of the cationic lipid in the mRNA-LNP by the number of moles of phosphate (P) of the mRNA.

[0213] ND1-1 encapsulated in lipid nanoparticle L1, prepared as mRNA-LNP, is referred to as ND1-1-L1, and ND1-2 encapsulated in lipid nanoparticle L6 is referred to as ND1-2-L6.

[0214] Similarly, eGFP mRNA encapsulated in lipid nanoparticles L6 was prepared as mRNA-LNP, and this is referred to as eGFP mRNA-L6.

[0215] Using the same method as above, ND1-2 was mixed with lipids having the composition shown in Table 4 to prepare mRNA-LNP (hereinafter, lipid nanoparticles having the composition of L7 will be referred to as "lipid nanoparticles L7"). The cationic lipid contained in L7 is compound 2 (Ex2; the production method is shown in Example 10). ND1-2 encapsulated in lipid nanoparticles L7 prepared as mRNA-LNP is referred to as ND1-2-L7.

[0216]

[0217] Using the same method as above, ND1-2 was mixed with lipids having the composition shown in Table 5 to prepare mRNA-LNP (hereinafter, lipid nanoparticles having the composition of L8 will be referred to as "lipid nanoparticles L8"). The cationic lipid contained in L8 is compound 3 (Ex3; the production method is shown in Example 11). ND1-2 encapsulated in lipid nanoparticles L8 prepared as mRNA-LNP is referred to as ND1-2-L8.

[0218]

[0219] (Example 4) In vitro conversion of rat primary astrocytes to neurons by NeuroD1 mRNA (ND1-1-L1 or ND1-2-L6) 1. Addition of mRNA-LNP to rat primary astrocytes Rat primary astrocytes were obtained and used according to the paper by Lynette C. Foo et al. (Purification of Rat and Mouse Astrocytes by Immunopanning: Cold Spring Harbor Protocols, vol. 5, pp. 421-432, 2013). However, cell recovery was performed under 5% CO ２ The rats used were neonatal Wistar rats (CLEA Japan, P1-P10). The obtained rat primary astrocytes were seeded at 10,000-40,000 cells / well on a poly-D-lysine-coated 96-well plate (Corning, 356640). The culture was incubated under 5% CO. ２The cells were cultured overnight at 37°C under 5% CO₂. For the culture, 200 μL / well of DMEM / F-12 medium (Thermo Fisher Scientific, 11320-033) containing 2% B-27® supplement (Thermo Fisher Scientific, A1895601), 10% fetal bovine serum (FBS) (Cytiva, SH30084.03), and 1% penicillin-streptomycin (PS) (Thermo Fisher Scientific, 15070063) was used. In Example 4, this DMEM / F12 medium containing B-27® supplement, FBS, and PS was also used as the culture medium below. The next day, mRNA-LNP (ND1-1-L1 or ND1-2-L6) diluted with PBS and culture medium was added to each well to a final concentration of 0.5 μg / mL, and the wells were cultured for 24 hours. PBS and culture medium were added to the control wells.

[0220] 2. Confirmation of NeuroD1 Protein Expression 24 hours after the addition of the above mRNA-LNP, the culture medium was removed and the cells were fixed with 4% paraformaldehyde. After washing with PBS, the cells were subjected to immunocytochemical staining using an anti-NeuroD1 antibody (Santa Cruz Biotechnology, sc46684) to detect the expression of NeuroD1 protein. The results showed that in wells containing ND1-1-L1 or ND1-2-L6, the number of NeuroD1 protein-positive cells was significantly increased compared to wells containing PBS (control). This result indicates that mRNA-LNP is taken up by astrocytes, translated from mRNA, and NeuroD1 protein is expressed in astrocytes.

[0221] 3. Confirmation of neuronal marker expression 24 hours after the addition of the mRNA-LNP, the culture medium was removed and replaced with transdifferentiation medium, which was changed every 2-3 days. The transdifferentiation medium was DMEM / F12 medium containing 2% B-27® supplement, 1% GlutaMAX™ supplement (Thermo Fisher Scientific, 35050-061), 1% PS, and 0.02% BDNF (brain-derived neurotrophic factor) solution, and 200 μL of this medium was added per well. The BDNF solution was prepared by dissolving BDNF powder (PeproTech, 450-02) in purified water to a concentration of 100 μg / mL. On the 6th to 7th day after addition of mRNA-LNP, the transdifferentiation medium was removed and the cells were fixed with 4% paraformaldehyde. After washing with PBS, the cells were subjected to immunocytochemical staining using an anti-DCX antibody (Doublecortin Antibody, Cell Signaling Technology, 4604S) to confirm the expression of doublecortin (DCX), an immature neuronal marker.

[0222] The number of DCX-positive cells was significantly increased in wells containing ND1-1-L1 or ND1-2-L6 compared to wells containing PBS (control). This result indicates that the introduction of NeuroD1 mRNA converted rat primary astrocytes into neurons.

[0223] (Example 5) Conversion of rat primary astrocytes into neurons by NeuroD1 mRNA-LNP (ND1-2-L7) in vitro Similar to ND1-1-L1 and ND1-2-L6 in Example 4, we evaluated whether ND1-2-L7 could convert rat primary astrocytes into neurons.

[0224] 1. Addition of mRNA-LNP (ND1-2-L7) to Rat Primary Astrocytes and Differentiation Induction Rat primary astrocytes were obtained and cultured overnight according to the method described in "1. Addition of mRNA-LNP to Rat Primary Astrocytes" in Example 4. The following day, mRNA-LNP (ND1-2-L7) diluted with culture medium was added to wells containing cultured rat primary astrocytes to a final concentration of 0.5 μg / mL and cultured for 24 hours. Culture medium was added to control wells. The DMEM / F12 medium containing B-27 (registered trademark) supplement, FBS (Cytiva, SH30070.03), and PS described in "1. Addition of mRNA-LNP to Rat Primary Astrocytes" in Example 4 was used as the culture medium.

[0225] 24 hours after the addition of ND1-2-L7, the culture medium was removed and replaced with a transdifferentiation medium, which was then replaced every 2 to 3 days. The transdifferentiation medium used was the one described in "3. Confirmation of expression of neuronal markers" in Example 4, and 200 μL of this medium was added to each well.

[0226] 2. Confirmation of NeuroD1 and DCX Protein Expression by Immunoblotting Before the addition of ND1-2-L7 (day 0), and 8 hours, 1, 2, 4, and 6 days after addition, the culture medium was removed, washed with PBS, and cells were lysed using RIPA Buffer (SIGMA-Aldrich, R0278-50ML). Cell lysis was performed 1 day after ND1-2-L7 addition before changing the medium 24 hours after ND1-2-L7 addition. After protein quantification by the Lowry method, 5 μg of protein was electrophoresed, and NeuroD1 protein expression was detected by immunoblotting using an anti-NeuroD1 antibody (Santa Cruz Biotechnology, sc46684). DCX protein expression was also detected by immunoblotting using an anti-DCX antibody.

[0227] As a result, it was revealed that NeuroD1 protein showed maximum expression 8 hours after the addition of ND1-2-L7, and expression decreased 1 day after addition. This result indicates that ND1-2-L7 is taken up by rat primary astrocytes, translated from mRNA, and that NeuroD1 protein is transiently expressed in rat primary astrocytes. Furthermore, it was revealed that DCX protein expression increased from 1 day after the addition of ND1-2-L7, and expression was also elevated 6 days after the addition of ND1-2-L7.

[0228] (Example 6) Conversion of rat primary astrocytes into neurons by NeuroD1 mRNA-LNP (ND1-2-L8) in vitro Similar to ND1-1-L1 and ND1-2-L6 in Example 4, we evaluated whether ND1-2-L8 could convert rat primary astrocytes into neurons.

[0229] 1. Addition of mRNA-LNP (ND1-2-L8) to Rat Primary Astrocytes and Differentiation Induction Rat primary astrocytes were obtained and cultured overnight according to the method described in "1. Addition of mRNA-LNP to Rat Primary Astrocytes" in Example 4. For the culture, DMEM / F12 medium containing B-27 (registered trademark) supplement, FBS (Cytiva, SH30070.03), and PS, as described in "1. Addition of mRNA-LNP to Rat Primary Astrocytes" in Example 4, was used as the culture medium at 100 μL / well.

[0230] The day after overnight culture, the entire culture medium was removed, and mRNA-LNP (ND1-2-L8) diluted with transdifferentiation medium was added to a final concentration of 0.5 μg / mL and cultured. PBS diluted with transdifferentiation medium was added to control wells. In this example, Neurobasal™ Medium (Thermo Fisher Scientific, 21103049) containing 2% B-27™ supplement, 1% GlutaMAX™ supplement, 2% FBS, 1% MEM non-essential amino acids (Fujifilm, 139-15651), 1% PS, and 0.02% BDNF solution was used as the transdifferentiation medium. The entire medium was replaced with transdifferentiation medium on the day (day 1) and day 3 after the addition of the mRNA-LNP.

[0231] 2. Confirmation of NeuroD1 and TUJ1 protein expression by immunoblotting Eight hours and seven days after the addition of ND1-2-L8, the transdifferentiation medium was removed, the cells were washed with PBS, and then lysed using RIPA Buffer (Thermo Fisher Scientific, 89901). After protein quantification by the Lowry method, 10 μg of each protein was electrophoresed, and the expression of NeuroD1 protein was detected by immunoblotting using an anti-NeuroD1 antibody (Abcam, ab60704). In addition, the expression of TUJ1 protein, a neuronal marker, was detected by immunoblotting using an anti-TUJ1 antibody (Abcam, ab78078).

[0232] As a result, NeuroD1 protein was expressed 8 hours after the addition of ND1-2-L8. This result indicates that ND1-2-L8 is taken up by rat primary astrocytes, translated from mRNA, and NeuroD1 protein is expressed in rat primary astrocytes. Furthermore, TUJ1 protein expression increased 8 hours after the addition of ND1-2-L8 compared to the PBS-added sample, and it was revealed that expression was also elevated 7 days after the addition of ND1-2-L8.

[0233] The results of Examples 4, 5, and this Example demonstrate that NeuroD1 mRNA can be delivered to rat primary astrocytes not only when LNPs containing Compound 1 (Ex1) as the cationic lipid (Example 4), but also when LNPs containing Compound 2 (Ex2) (Example 5) or Compound 3 (Ex3) (This Example) are used. These results also demonstrate that the delivered NeuroD1 mRNA is translated into NeuroD1 protein in rat primary astrocytes, resulting in expression of NeuroD1 protein, and that the protein exerts its function, converting the rat primary astrocytes into neurons.

[0234] Example 7 Administration of mRNA-LNP to the Injured Region of a Spinal Cord Injury Model and Expression Analysis 1. Administration of mRNA-LNP to the Injured Region A spinal cord injury model was prepared using the crush conditions of Experiment 6 in Example 1. On day 28 after crush, the crushed area of ​​the spinal cord (cervical spinal cord) was re-exposed under induction anesthesia with ketamine hydrochloride and xylazine and maintenance anesthesia with isoflurane inhalation. Using a syringe (Ito Seisakusho, MS-N100) connected to a microinjector (Narimo Scientific Instruments Research Institute, IMS-20), 30 μL of mRNA-LNP (ND1-2-L6) adjusted to 0.3 mg / mL with PBS was administered at a rate of 1.0 μL / min to a depth of approximately 1 mm from the dorsal surface of the cervical spinal cord at the center of the pressure site on the left side of the spinal cord. The syringe was left undisturbed for 5 to 10 minutes after administration. Next, ND1-2-L6 was similarly administered to the center of the pressure site on the right side of the spinal cord. After administration, the syringe was left stationary for 5 to 10 minutes, and then the muscle and skin were sutured.

[0235] 2. Confirmation of NeuroD1 Protein Expression Spinal cord injury models administered ND1-2-L6 using the method described above were euthanized the day after administration, and the spinal cords were removed and fixed using 4% paraformaldehyde. Subsequently, the water in the fixed spinal cord tissue was replaced with sucrose solution (10, 20, and 30%) prepared by dissolving sucrose in PBS, and 10 μm-thick frozen sagittal spinal cord sections were prepared. These frozen spinal cord sections were subjected to immunohistochemical staining using an anti-GFAP antibody against GFAP, an astrocyte marker, and an anti-NeuroD1 antibody against NeuroD1 (Cell Signaling Technology, 7019).

[0236] As a result, as shown in Figure 4A, it was revealed that NeuroD1 protein was expressed around the damaged area in ND1-2-L6-administered individuals. On the other hand, no expression of NeuroD1 protein was observed in individuals not administered ND1-2-L6. Furthermore, the presence of GFAP-positive cells (astrocytes) was observed around the damaged area in ND1-2-L6-administered individuals, and further, it was shown that NeuroD1 protein expressed around the damaged area in ND1-2-L6-administered individuals co-localized with GFAP-positive cells. This indicates that administration of NeuroD1 mRNA-LNP to the damaged area results in the expression of NeuroD1 protein in astrocytes accumulated near the damaged area. Note that while NeuroD1 protein was expressed around the damaged area, the lack of NeuroD1 protein expression in the center of the damaged area is thought to be due to tissue cavitation and cell necrosis caused by the effects of the injury.

[0237] As shown in Examples 4, 5, and 6, in vitro experiments demonstrated that introducing NeuroD1 mRNA into astrocytes converted the astrocytes into neurons. Furthermore, as described above, in the spinal cord injury model of the present invention, administration of NeuroD1 mRNA to the injured area was shown to result in the expression of NeuroD1 protein in astrocytes near the injured area. Based on these results, it is expected that administration of NeuroD1 mRNA to the site of spinal cord injury in vivo will also convert astrocytes into neurons. Furthermore, it is predicted that this will result in the regeneration of neurons, leading to recovery of motor dysfunction caused by spinal cord injury.

[0238] Example 8: Effect of NeuroD1 mRNA The effect of NeuroD1 mRNA was evaluated using the spinal cord injury model of the present invention. 1. Preparation of Spinal Cord Injury Model A spinal cord injury model was prepared using the crush conditions described in Experiment 6 of Example 1. Specifically, common marmosets were used, and induction anesthesia was performed with a 50 mg / mL solution of ketamine hydrochloride at a dose of 0.16 mL / animal and a 20 mg / mL solution of xylazine at a dose of 0.06 mL / animal. The anesthetized common marmosets were intubated with an endotracheal intubation tube, an Atom Nutrition Catheter (6 Fr), and maintenance anesthesia was performed by inhalation of isoflurane. The hair on the back of the anesthetized common marmosets was shaved and they were placed face down on a 37°C heating mat. The neck skin was incised, connective tissue was removed to expose the vertebrae, and the vertebral arch including the spinous process of cervical vertebra C5 was resected to expose the spinal cord (cervical spinal cord).

[0239] A 2.5 mm round tip (impactor tip) was used to attach to the impactor. The spinal cord was crushed on both sides (twice in total at the two sites) at the dorsal surface of the cervical spinal cord, in the area exposed by resection of the vertebral arch of the C5 cervical vertebra, once on each side of the spinal cord midline (2 times at the two sites), with a pressure of 280 kdyn for 5 seconds each time.

[0240] After the crush injury, common marmosets underwent rehabilitation in which the fingers, wrists (or ankles), elbows (or knees), and shoulders (or hips) of both front and rear limbs were bent and stretched five times each (flexion and extension exercises) to prevent spasticity and contracture. Rehabilitation was performed once or twice a day, every day during the observation period, starting five to seven days after the crush injury.

[0241] 2. Administration of mRNA-LNP to the damaged area After crush injury, common marmosets were evaluated for motor dysfunction using the Field Rating Scale (Table 1), and animals that were unable to maintain a sitting position 4 weeks after crush injury and had a score of less than 10 points were divided into a control group and a drug-administered group so that there was no difference in score between the groups.

[0242] On days 27-29 after the crush injury, the crushed area was re-exposed under induction anesthesia with ketamine hydrochloride and xylazine and maintenance anesthesia with isoflurane inhalation. Using a syringe connected to a microinjector, 30 μL of mRNA-LNP was administered at a rate of 1.0 μL / min to a depth of approximately 1 mm from the dorsal surface of the cervical spinal cord at the center of the pressure site on the left side of the spinal cord. The syringe was left stationary for 5-10 minutes after administration. Next, mRNA-LNP was similarly administered to the center of the pressure site on the right side of the spinal cord. The syringe was left stationary for 5-10 minutes after administration, after which the muscle and skin were sutured. mRNA-LNP was adjusted to 0.3 mg / mL with PBS. ND1-2-L6 was administered to six animals in the drug-treated group (ND1 mRNA), and eGFP mRNA-L6 was administered to six animals in the control group (Control mRNA).

[0243] Thereafter, motor dysfunction was scored using the Field Rating Scale (Table 1), and the grip strength of both forelimbs was measured. Grip strength measurement (hereinafter referred to as the "grip test") was performed using a device consisting of a digital force meter (IMADA Corporation, Digital Force Gauge DPS-5R) attached to a cage that was easy for both forelimbs to grasp. Specifically, the common marmoset was made to grasp the cage, and the tester pulled the common marmoset until the marmoset released the cage, measuring the force with which the common marmoset gripped the cage with the digital force meter, and this measurement value was taken as the grip strength of the common marmoset. Scoring of motor dysfunction and the grip test were performed at the same time, and observation and evaluation were performed over a period of 12 weeks after drug administration.

[0244] The evaluation results for each individual are shown in Table 6. The scores based on the Field Rating Scale and the results of the grip test show the change in the evaluation results 12 weeks after drug administration from the evaluation results before drug administration (26-29 days after crush). The results of the grip test were shown as a relative value when the grip strength before crush was set at 100%. A Wilcoxon matched-pairs signed rank test was used to test for significant differences between the scores based on the Field Rating Scale before drug administration and 12 weeks after drug administration for each of the drug-administered and control groups. A paired t test was used to test for significant differences between the results of the grip test before drug administration and 12 weeks after drug administration for each of the drug-administered and control groups. In both methods, a P<0.05 was considered to be significant.

[0245] In the drug-treated group, Field Rating Scale scores and grip strength were significantly improved at 12 weeks after drug administration compared to pre-drug administration (Figures 5B and D). On the other hand, in the control group, Field Rating Scale scores and grip strength did not recover at 12 weeks after drug administration compared to pre-drug administration (Figures 5A and C). These results indicate that ND1-2-L6 improved the motor dysfunction caused by spinal cord injury. In particular, among motor functions, grasping and grasping "objects" is frequently used in daily life and is extremely important for maintaining daily life, and it was shown that ND1-2-L6 restored the "grip strength" required for this. Furthermore, it was shown that quadriplegia, which inhibits basic daily movements such as getting up, was improved.

[0246]

[0247] The results of Examples 4, 7, and this Example show that administering NeuroD1 mRNA to the injury site of a spinal cord injury model results in the expression of NeuroD1 protein in astrocytes accumulated near the injury area, and the expressed NeuroD1 protein converts astrocytes into neurons, thereby recovering motor dysfunction caused by spinal cord injury. Note that, in Examples 4, 5, and 6, similar to ND1-2-L6, ND1-1-L1, ND1-2-L7, and ND1-2-L8 were also shown to convert astrocytes into neurons, so it can be said that even when administered, they also produce the same effects as ND1-2-L6 due to NeuroD1.

[0248] The results of Examples 1 to 8 above demonstrate that the spinal cord injury model prepared by the method of the present invention can be used to evaluate or screen test substances for their therapeutic effects on spinal cord injury, particularly subacute to chronic spinal cord injury, spinal cord injury accompanied by quadriplegia, and spinal cord injury accompanied by upper arm motor dysfunction (particularly grip strength reduction). Furthermore, it was revealed that administration of NeuroD1 mRNA exhibits therapeutic effects on spinal cord injury (subacute to chronic spinal cord injury, spinal cord injury accompanied by quadriplegia, and spinal cord injury accompanied by upper arm motor dysfunction (particularly grip strength reduction)).

[0249] (Example 9) Preparation of Cationic Lipid Compound 1 (Ex1) Under a nitrogen atmosphere and ice cooling, 4-(dimethylamino)pyridine (61.9 g) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (97.2 g) were added to a mixture of 8-bromooctanoic acid (75.5 g) and 9-heptadecanol (86.8 g) in N,N-dimethylformamide (755 mL). The mixture was then stirred at room temperature for 28 hours. The reaction mixture was added to ice water and extracted with toluene. The aqueous layer was extracted with toluene, and the combined organic layer was washed with saturated aqueous sodium chloride. The aqueous layer was extracted with toluene and ethyl acetate, and the combined organic layer was dried over anhydrous magnesium sulfate. The organic layer was concentrated under reduced pressure, followed by addition of hexane and concentration under reduced pressure again. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain heptadecan-9-yl 8-chlorooctanoate (80.3 g; hereinafter also referred to as "Intermediate 1" ("PEx1")) as an oil. Table 7 shows the chemical structure of Intermediate 1 (PEx1).

[0250] Under a nitrogen atmosphere, potassium carbonate (1.7 g) and potassium iodide (51 mg) were added to a mixture of 2-(4-amino-1-piperidinyl)ethanol dihydrochloride (672 mg), heptadecan-9-yl 8-chlorooctanoate (3.0 g), and N,N-dimethylacetamide (6.7 mL) at room temperature. After stirring at an internal temperature of 100°C for 24 hours, the reaction mixture was allowed to cool to room temperature, and the insoluble matter was filtered off and washed with toluene. Water was added to the filtrate, and the organic layer was extracted. The aqueous layer was extracted with toluene and ethyl acetate, and the combined organic layer was washed with saturated aqueous sodium chloride. The aqueous layer was extracted with ethyl acetate, and the combined organic layer was dried over anhydrous sodium sulfate. The organic layer was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) and silica gel column chromatography (chloroform / methanol). The resulting crude product was purified by aminosilica gel column chromatography (hexane / ethyl acetate) to obtain 8,8'-{[1-(2-hydroxyethyl)piperidin-4-yl]azanediyl}di(octanoate)di(heptadecan-9-yl) (540 mg; hereinafter also referred to as "Compound 1" ("Ex1")) as an oil. Table 7 shows the chemical structure of Compound 1 (Ex1).

[0251]

[0252] Intermediate 1 (PEx1) and Compound 1 (Ex1) were subjected to electrospray ionization mass spectrometry (ESI-MS) using a positive high voltage for ionization. The resulting mass-to-charge ratios (m / z) were as follows: Intermediate 1 (PEx1) m / z: 440 Compound 1 (Ex1) m / z: 906

[0253] In addition, the NMR spectrum of Compound 1 (Ex1) was measured and analyzed. The results are shown below: １ Representative signal values ​​(ppm) in the H-NMR spectrum are shown. t: triplet, quin: quintet, td: triple doublet, m: multiplet, brd: broad doublet). NMR (500 MHz, CDCl ３): δ: 0.88 (t, J = 7.00Hz, 12H), 1.23-1.42 (m, 66H), 1.45-1.74 (m, 14H), 2.04 (td, J = 11.80, 1.98Hz, 2H), 2.27 (t, J = 7.57Hz, 4H), 2.36-2.52 (m, 7H), 2.94 (brd, J = 11.47Hz, 2H), 3.58 (t, J = 5.43Hz, 2H), 4.86 (quin, J = 6.23Hz, 2H)

[0254] Example 10: Preparation of Cationic Lipid Compound 2 (Ex2) 1,1'-Carbonyldiimidazole (7.35 g) was added to a mixture of diethyl 7,7'-azanediyldi(heptanoate) (10.7 g) and dichloromethane (107 mL) at room temperature, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to obtain diethyl 7,7'-[(1H-imidazole-1-carbonyl)azanediyl]di(heptanoate) (13.5 g; hereinafter also referred to as "Intermediate 2a" ("PEx2a")) as an oil. Table 8 shows the chemical structure of Intermediate 2a (PEx2a).

[0255] To a mixture of diethyl 7,7'-[(1H-imidazole-1-carbonyl)azanediyl]di(heptanoate) (3 g) and acetonitrile (30 mL), methyl iodide (2.2 mL) was added at room temperature, and the mixture was stirred at room temperature for 2 hours. Methyl iodide (2.2 mL) was then added to the reaction mixture at room temperature, and the mixture was stirred at room temperature for 16 hours. The reaction mixture was concentrated under reduced pressure to give 1-[bis(7-ethoxy-7-oxoheptyl)carbamoyl]-3-methyl-1H-imidazol-3-ium iodide (4.2 g; hereinafter also referred to as "Intermediate 2b / I" ("PEx2b / I")) as an oil. Table 8 shows the chemical structure of Intermediate 2b / I (PEx2b / I).

[0256] To a mixture of 1-[bis(7-ethoxy-7-oxoheptyl)carbamoyl]-3-methyl-1H-imidazol-3-ium iodide (4.2 g) and N,N-dimethylformamide (10 mL), a mixture of 2-(dimethylamino)-1-(4-hydroxypiperidin-1-yl)ethan-1-one (1.45 g) and tetrahydrofuran (10 mL) was added at room temperature, followed by the addition of sodium hydride (60% in oil, 310 mg) at room temperature and stirring for 3 hours. The reaction mixture was adjusted to approximately pH 7 with 1 M hydrochloric acid. After dilution with ethyl acetate, water was added, the organic layer was separated, and the aqueous layer was extracted with ethyl acetate. The combined organic layers were washed with saturated brine / water (1 / 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give diethyl 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) (1.93 g; hereinafter also referred to as "Intermediate 2c" ("PEx2c")) as an oil. Table 8 shows the chemical structure of Intermediate 2c (PEx2c).

[0257] To a mixture of diethyl 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) (1.13 g), tetrahydrofuran (17 mL), and ethanol (17 mL), 1 M aqueous sodium hydroxide solution (4.17 mL) was added at room temperature, and the mixture was stirred at room temperature for 72 hours. The reaction mixture was concentrated under reduced pressure, and ethanol (100 mL) was added and concentrated to obtain sodium 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) (1.21 g; hereinafter, also referred to as "Intermediate 2d / Na" ("PEx2d / Na")) as a solid. Table 8 shows the chemical structure of Intermediate 2d / Na (PEx2d / Na).

[0258] To a mixture of sodium 7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) (200 mg) and dichloromethane (3 mL), 2-nonylundecan-1-ol (270 mg), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (430 mg), N,N-diisopropylethylamine (0.4 mL), and 4-(dimethylamino)pyridine (10 mg) were added at room temperature, and the mixture was stirred at room temperature for 16 hours. A saturated aqueous solution of sodium bicarbonate was added to the reaction mixture, and the mixture was stirred at room temperature for 10 minutes. The mixture was diluted with dichloromethane, water was added, and extraction was performed using a phase separator. The organic layer was dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (chloroform / methanol), and then the crude product was purified by amino silica gel column chromatography (hexane / ethyl acetate) to give bis(2-nonylundecyl)7,7'-[({[1-(N,N-dimethylglycyl)piperidin-4-yl]oxy}carbonyl)azanediyl]di(heptanoate) (197 mg; hereinafter also referred to as "Compound 2" ("Ex2")) as an oil. Table 8 shows the chemical structure of Compound 2 (Ex2).

[0259]

[0260] Electrospray ionization mass spectrometry (ESI-MS) was performed on Intermediate 2a (PEx2a), Intermediate 2b / I (PEx2b / I), Intermediate 2c (PEx2c), Intermediate 2d / Na (PEx2d / Na), and Compound 2 (Ex2) using a positive high voltage for ionization. The resulting mass-to-charge ratios (m / z) are as follows: Intermediate 2a (PEx2a) m / z: 424.5 Intermediate 2b / I (PEx2b / I) m / z: 438.5 Intermediate 2c (PEx2c) m / z: 542.7 Intermediate 2d / Na (PEx2d / Na) m / z: 486.5 Compound 2 (Ex2) m / z: 1047.3

[0261] In addition, the NMR spectrum of Compound 2 (Ex2) was measured and analyzed. The results are shown below:１ Representative signal values ​​(ppm) in the H-NMR spectrum are shown. m: multiplet, d: doublet). NMR (500 MHz, CD ３ OD): δ: 0.86-0.93 (m, 12H), 1.23-1.41 (m, 72H), 1.52-1.73 (m, 12H), 1.85-1.99 (m, 2H), 2.28-2.35 (m, 1 0H), 3.21-3.27 (m, 6H), 3.47-3.54 (m, 2H), 3.69-3.80 (m, 2H), 3.99 (d, J = 5.50Hz, 4H), 4.85-4.91 (m, 1H)

[0262] Example 11: Preparation of Cationic Lipid Compound 3 (Ex3) 8-Bromooctanoic acid (2.97 g), N,N-diisopropylethylamine (4.7 mL), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (5.4 g), and 4-(dimethylamino)pyridine (135 mg) were added to a mixture of 2-nonylundecan-1-ol (3.25 g) and dichloromethane (40 mL) in a water bath, and the mixture was stirred at room temperature for 6.5 hours. After adding chloroform and water to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-nonylundecyl 8-bromooctanoate (5.27 g; hereinafter also referred to as "Intermediate 3a" ("PEx3a")) as an oil. Table 9 shows the chemical structure of Intermediate 3a (PEx3a).

[0263] To a mixture of 2-octyldecane-1-ol (500 mg) and dichloromethane (5 mL), {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetic acid (511 mg), 4-(dimethylamino)pyridine (24 mg), N,N-diisopropylethylamine (798 μL), and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (918 mg) were added at room temperature, and the mixture was stirred at room temperature for 6 hours. Chloroform and water were added to the reaction mixture, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and then concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate) to give 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (879 mg; hereinafter also referred to as "Intermediate 3b" ("PEx3b")) as an oil. Table 9 shows the chemical structure of Intermediate 3b (PEx3b).

[0264] Trifluoroacetic acid (1.5 mL) was added to a mixture of 2-octyldecyl {(1r,3r)-3-[(tert-butoxycarbonyl)amino]cyclobutyl}acetate (877 mg) and dichloromethane (8 mL) at room temperature, followed by stirring at room temperature for 6 hours. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate, the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and concentrated under reduced pressure to yield 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (661 mg; hereinafter also referred to as "Intermediate 3c" ("PEx3c")) as an oil. Table 9 shows the chemical structure of Intermediate 3c (PEx3c).

[0265] To a mixture of 2-octyldecyl [(1r,3r)-3-aminocyclobutyl]acetate (660 mg) and acetonitrile (4 mL), a mixture of 2-nonylundecyl 8-bromooctanoate (430 mg) and cyclopentyl methyl ether (4 mL), N,N-diisopropylethylamine (731 μL), and potassium iodide (30 mg) were added at room temperature, and the mixture was stirred in an oil bath at 80°C for 48 hours. The reaction mixture was added to a mixture of chloroform and saturated aqueous sodium bicarbonate, and the organic layer was separated, and the aqueous layer was extracted with chloroform. The combined organic layers were dried over anhydrous sodium sulfate and then concentrated under reduced pressure. The resulting residue was purified by amino silica gel column chromatography (hexane / ethyl acetate), and then the crude product was purified by silica gel column chromatography (chloroform / methanol) to give 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (452 ​​mg; hereinafter also referred to as "Intermediate 3d" ("PEx3d")) as an oil. Table 9 shows the chemical structure of Intermediate 3d (PEx3d).

[0266] To a mixture of 2-nonylundecyl 8-{[(1r,3r)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (1200 mg) and dichloromethane (24 mL), 1-methyl-L-proline (11.8% water content, 290 mg), 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (910 mg), 4-(dimethylamino)pyridine (27 mg), and N,N-diisopropylethylamine (510 μL) were added at room temperature, and the mixture was stirred at room temperature for 4 hours. Water was added to the reaction mixture, and the organic layer was separated and concentrated under reduced pressure. Heptane and 90% aqueous methanol solution were added to the resulting residue, and the heptane layer was separated and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography (heptane / ethyl acetate), and then the crude product was purified by amino silica gel column chromatography (heptane / ethyl acetate) to give 2-nonylundecyl 8-{(1-methyl-L-prolyl)[(1r,3S)-3-{2-[(2-octyldecyl)oxy]-2-oxoethyl}cyclobutyl]amino}octanoate (1.2 g; hereinafter also referred to as "Compound 3" ("Ex3")) as an oil. Table 9 shows the chemical structure of Compound 3 (Ex3).

[0267]

[0268] Chemical ionization mass spectrometry (CI-MS) was performed on Intermediate 3a (PEx3a) using isobutane as the reaction gas and a positive high voltage for ionization. Electrospray ionization mass spectrometry (ESI-MS) was also performed on Intermediate 3b (PEx3b), Intermediate 3c (PEx3c), Intermediate 3d (PEx3d), and Compound 3 (Ex3) using a positive high voltage for ionization. The resulting mass-to-charge ratios (m / z) were as follows: Intermediate 3a (PEx3a) m / z: 503.1, 505.3 Intermediate 3b (PEx3b) m / z: 504.5 Intermediate 3c (PEx3c) m / z: 382.5 Intermediate 3d (PEx3d) m / z: 805.0 Compound 3 (Ex3) m / z: 916.1

[0269] In addition, the NMR spectrum of Compound 3 (Ex3) was measured and analyzed. The results are shown below: １ Representative signal values ​​(ppm) in the H-NMR spectrum are shown. m: multiplet). NMR (500 MHz, CDCl ３ ): δ: 0.79-0.97 (m, 12H), 1.10-2.11 (m, 78H), 2.21-2.47 (m, 8H), 2.49-2.61 (m, 3H), 2.99-3.20 (m, 2H), 3.23-3.38 (m, 2H), 3.90-4.03 (m, 4H), 4.70-4.82 (m, 1H)

[0270] The method of the present invention makes it possible to prepare a non-human primate animal model of spinal cord injury (particularly, spinal cord injury in the subacute to chronic phase). The non-human primate animal model of the present invention is expected to be an effective animal model for the discovery and development of therapeutic agents for spinal cord injury, particularly, therapeutic agents for spinal cord injury in the subacute to chronic phase. Furthermore, the pharmaceutical composition or treatment method of the present invention is expected to be useful for the treatment of spinal cord injury (particularly, spinal cord injury in the subacute to chronic phase).

[0271] SEQ ID NO: 1: Human NeuroD1 protein SEQ ID NO: 2: Human NeuroD1 gene (CDS) SEQ ID NO: 3: Human NeuroD1 gene (CDS) SEQ ID NO: 4: RNA sequence of human NeuroD1 gene (CDS) SEQ ID NO: 5: RNA sequence of human NeuroD1 gene (CDS) SEQ ID NO: 6: Human NeuroD1 mRNA sequence (ND1-2) SEQ ID NO: 7: Human NeuroD1 mRNA sequence containing modified nucleosides (ND1-2)

[0272] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. A pharmaceutical composition for the treatment of spinal cord injury, comprising mRNA encoding NeuroD1 protein.

2. The pharmaceutical composition according to claim 1, wherein the NeuroD1 protein is a protein consisting of the amino acid sequence shown in SEQ ID NO:

1.

3. The pharmaceutical composition according to claim 1 or 2, wherein the mRNA comprises a nucleotide sequence having at least 60% sequence identity to the nucleotide sequence shown in SEQ ID NO: 4 or 5.

4. The pharmaceutical composition according to claim 3, wherein the mRNA is an mRNA comprising the nucleotide sequence shown in SEQ ID NO: 4 or 5.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein the mRNA comprises the nucleotide sequence shown in SEQ ID NO: 6 or 7, or a partial sequence of the nucleotide sequence, and comprises the nucleotide sequence from position 44 to position 1114, or position 44 to position 1120 of SEQ ID NO: 6 or 7.

6. The pharmaceutical composition according to any one of claims 1 to 5, wherein the mRNA comprises a 5'UTR and / or a 3'UTR.

7. The pharmaceutical composition according to claim 5, wherein the mRNA comprises the nucleotide sequence from position 1 to position 1231 of SEQ ID NO: 6 or 7.

8. The pharmaceutical composition according to any one of claims 1 to 7, wherein the mRNA comprises a polyA sequence.

9. The pharmaceutical composition according to claim 8, wherein the length of the polyA sequence is 50 to 200 nucleotides.

10. The pharmaceutical composition according to any one of claims 1 to 9, wherein the mRNA has a 5' cap structure.

11. The pharmaceutical composition according to any one of claims 1 to 10, wherein the mRNA comprises a modified nucleoside.

12. The pharmaceutical composition according to claim 11, wherein the modified nucleoside is N1-methylpseudouridine.

13. The pharmaceutical composition according to claim 12, wherein the mRNA is an mRNA in which some or all uridines are replaced by N1-methylpseudouridine.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein the mRNA is encapsulated in a drug delivery carrier.

15. The pharmaceutical composition according to claim 14, wherein the drug delivery carrier is a lipid nanoparticle.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein the spinal cord injury is a spinal cord injury in the subacute to chronic phase.

17. A method for treating spinal cord injury, comprising the step of administering the pharmaceutical composition according to any one of claims 1 to 15 or mRNA encoding NeuroD1 protein.

18. mRNA encoding NeuroD1 protein for use in the treatment of spinal cord injury.

19. Use of mRNA encoding NeuroD1 protein in the manufacture of a pharmaceutical composition for the treatment of spinal cord injury.

20. A method for producing a non-human primate model of spinal cord injury, comprising contusing the left and right spinal cords by applying pressure to sites on the dorsal surface of the non-human primate's cervical spinal cord that are located on the left and right sides, respectively, relative to the spinal cord midline.

21. A method for producing a non-human primate model of spinal cord injury, comprising contusing one of the left or right spinal cords by applying pressure to a site on the dorsal surface of the non-human primate's cervical spinal cord that is located on either the left or right side relative to the spinal cord midline.

22. The method according to claim 20 or 21, wherein the non-human primate is a marmoset.

23. The method according to claim 20 or 21, wherein the pressure is applied using an impactor.

24. The method according to claim 23, wherein the pressure is 255 - 295 kdyn.

25. The method according to claim 24, wherein the pressure is 280 kdyn.

26. The method according to claim 20 or 21, wherein the pressure is applied at a site exposed by removing the vertebral arch of any cervical vertebra selected from the group consisting of the 3rd to 7th cervical vertebrae of the cervical spinal cord.

27. The method according to claim 26, wherein the pressure is applied at a site exposed by removing the vertebral arch of the 5th cervical vertebra of the cervical spinal cord.

28. The method according to any one of claims 20 - 27, wherein the spinal cord injury is a subacute to chronic spinal cord injury.

29. The method according to claim 28, wherein the spinal cord injury is a chronic spinal cord injury.

30. A non-human primate model of spinal cord injury produced by the method according to any one of claims 20 - 29.

31. A method for evaluating the effect of a test substance as a therapeutic agent for spinal cord injury, comprising administering the test substance to the non-human primate model according to claim 30, and determining the effect of the test substance on the spinal cord injury in the non-human primate model.

32. The method according to claim 31, further comprising performing rehabilitation on the non-human primate model.

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