Neuron production method

Abstract

As a result of extensive study, the present inventors discovered the following: an inhibitory region that acts in an inhibitory manner on the promotion of neuron differentiation is present in a region of 51 residues from amino acid 222 to the C-terminus in the C-terminus IDR of NGN2, and the efficiency of differentiation into neurons is dramatically increased by causing cells such as pluripotent cells to express an NGN2 partial deletion variant from which the inhibitory region is deleted; stable expression is not essential when the NGN2 partial deletion variant is used, and when the NGN2 partial deletion variant is transiently expressed, neurons can be mass-produced by culturing for a short period of time of approximately several days via steps simpler than those used in conventional methods; and differentiation into various types of neurons such as motor neurons can be promoted by combining the NGN2 partial deletion variant with other transcription factors or neuronal differentiation-inducing factors. The inventors completed the present invention on the basis of these findings.

Description

How neurons are produced

[0001] The present invention relates to a method for producing neurons.

[0002] Transcription factors that determine cellular state are used as regulatory factors to create desired cells. Recently, differentiation-inducing technologies have been developed that use transcription factors to produce various tissues and organs from iPS cells. In particular, the transcription factor neurogenin 2 (also known as NEUROG2 or NGN2) is widely used to induce differentiation into neural cells (e.g., Non-Patent Documents 1 and 2). To date, 25 related protocols have been published (Non-Patent Document 3). NGN2 is a master transcription factor that induces neuronal differentiation from pluripotent stem cells. By binding to gene regulatory regions, it rapidly activates hundreds of genes to produce neurons. It has been reported that motor neurons can be generated in 1–2 weeks, making this rapid differentiation method promising for application in pathological analysis and drug discovery platforms.

[0003] However, the previously reported techniques described above require the expression of large amounts of NGN2 for a long period of time to change the properties of cells. Because normal expression levels are insufficient to induce cellular changes, advanced genetic engineering techniques, such as the use of viral vectors or inducible gene expression vectors, are required. This method is far from simple, and in practice, it can only be used by experienced researchers.

[0004] In the above-mentioned conventional technologies, efforts are underway to improve gene modification techniques and develop methods for stably introducing and expressing larger amounts of NGN2. Several methods have also been developed to complement the function of transcription factors, such as simultaneously introducing other transcription factors or chromatin regulatory factors in combination with NGN2, or adding growth factors or small molecule compounds involved in promoting differentiation to the culture medium (Non-Patent Documents 4-13). However, it cannot be said that a simple and efficient method for producing neurons has been established. Furthermore, none of these improved technologies focuses on improving the function of NGN2 itself. There have been no reports that expressing partial fragments of NGN2 lacking specific regions in cells dramatically increases the efficiency of differentiation into neurons.

[0005] In addition to the above-mentioned prior art, Patent Document 1 discloses a technique for inducing neuronal differentiation by introducing synthetic mRNAs encoding four transcription factors, NEUROD1, NEUROD2, NEUROG2 / NGN2, and NEUROG3 / NGN3, into human embryonic stem cells and culturing them in a standard neuronal differentiation medium. Patent Document 1 also discloses a technique for inducing motor neuron differentiation by introducing synthetic mRNAs encoding five transcription factors, NEUROD1, NEUROD2, NEUROG1, NEUROG2, and NEUROG3, into human embryonic stem cells and culturing them in a standard neuronal differentiation medium. The synthetic mRNAs encoding NGN2 and other transcription factors used in the technique of Patent Document 1 are synthetic mRNAs encoding full-length proteins. Patent Document 1 does not specifically teach that partial fragments of transcription factors such as NGN2 can also be used to induce neuronal differentiation, and it does not state or suggest that expressing partial fragments of NGN2 lacking specific regions in cells will dramatically increase the efficiency of neuronal differentiation.

[0006] Patent Document 2 discloses a technique for inducing the differentiation of glial cells into functional neurons using a Neurog2 functional fragment. According to the definition in Patent Document 2, a Neurog2 functional fragment is a functional Neurog2 protein or a nucleic acid sequence encoding the same, and contains a full-length Neurog2 protein and the conserved Neurog2 bHLH domain. It also discloses that a Neurog2 functional fragment may be a variant of the Neurog2 protein sequence or a mutant of the Neurog2 polynucleotide sequence. According to these definitions, the term "functional fragment" refers to a protein containing the full-length Neurog2 or a polynucleotide encoding the full-length Neurog2, and is not used to mean a fragment consisting of a partial region. In other words, Patent Document 2 does not teach that a partial fragment of NEUROG2 / NGN2 can be used to induce neuronal differentiation, nor does it state or suggest that intracellular expression of a partial fragment lacking a specific region of NGN2 dramatically increases the efficiency of neuronal differentiation.

[0007] Human NGN2 (NCBI Gene ID: 63973) consists of 272 amino acids, with a central basic helix-loop helix (bHLH) in amino acids 102-174 and intrinsically disordered regions (IDRs) at both ends (amino acids 1-101 and 175-272). The bHLH mediates DNA binding by NGN2 and plays an important role in inducing downstream gene expression. The IDRs are known to have both stimulatory and inhibitory functions on NGN2-induced neuronal differentiation. The transactivation domain (TAD) in the C-terminal IDR is one of the stimulatory functions. In 2012, a reporter gene assay was used to assess the transcriptional activity of NGN2, suggesting that a TAD required for Ngn2 transcriptional activity may be present within the region of amino acids 185-217 of mouse Ngn2 (Non-Patent Document 14). Furthermore, phosphorylation of serines 231 and 234 in the C-terminal IDR is essential for the interaction of Ngn2 with ISL1 and LHX3, and regulation by these three factors has been shown to induce motor neuron differentiation (Non-Patent Document 15). On the other hand, it has also been reported that IDR phosphorylation inhibits Ngn2 transcriptional activity (Non-Patent Documents 16 and 17). The IDR of mouse Ngn2 contains multiple phosphorylation sites, and GSK-3β is known to regulate phosphorylation. It has been suggested that GSK-3β phosphorylation of Ngn2 promotes heterodimerization with Ngn2 and the bHLH cofactor E47, preventing Ngn2 from binding to Eboxes (Non-Patent Document 17). Thus, it is currently unclear whether the IDR of NGN2 is required for transcriptional activity.

[0008] WO 2016 / 143826 A1 Special Publication No. 2023-524900

[0009] Thoma et al., PLoS ONE 7(6): e38651.Zhang et al., Neuron. 2013;78:785-798Hulme et al., Stem Cell Reports, 17:14-34, 2022Hester ME, et al., Mol. Ther. 2011;19:1905-1912Goparaju SK, et al. Sci. Rep. 2017;7:42367.Son EY, et al., Cell Stem Cell. 2011;9:205-218.Garone MG, et al., J. Vis. Exp. 2019.Xue Y., et al., Stem Cells Transl Med. 2019 Feb;8(2):112-123.Park, C. et al., FEBS Lett., 582, 537-542, 2008Busskamp, ​​V. et al., Mol. Syst. Biol., 10, 760, 2014Zhang, Y., et al.: Neuron 78, 785-798. (2013)Vadodaria, K., et al.: Mol. Psychiatry. 21, 49-61. (2016)Blanchard, J., et al.: Nat. Neurosci., 18, 25-25. (2015)Li S et al., J Neurosci. June 6, 2012, 32(23):7791-7805Ma YC et al., Neuron 2008, 58(1) 65-77Ali, F. et al., Development 138, 4267-4277 (2011)Li, S. et al., The Journal of Neuroscience, June 6, 2012, 32(23):7791-7805

[0010] An object of the present invention is to provide a means for inducing differentiation of neurons from pluripotent cells such as iPS cells more efficiently than conventional methods.

[0011] As a result of extensive research, the inventors of the present application have found that within the C-terminal IDR of NGN2, there is an inhibitory region that acts to inhibit the promotion of neuronal differentiation within the region from amino acid 222 to the C-terminus of 51 residues, and that expressing an NGN2 partial deletion form in which this inhibitory region has been deleted in cells such as pluripotent cells dramatically increases the efficiency of differentiation into neurons. They have also found that stable expression is not essential with the NGN2 partial deletion form, and that when transiently expressed, neurons can be mass-produced in a short period of culture of approximately several days with simpler steps than conventional methods. They have also found that combining the NGN2 partial deletion with other transcription factors and neuronal differentiation inducers can promote differentiation into various types of neurons, such as motor neurons, and have thus completed the present invention.

[0012] That is, the present invention relates to a method for producing neurons by expressing a partially deleted NGN2 mutant lacking a portion of the C-terminal IDR in cells such as pluripotent cells, and includes the following aspects: [1] A method for producing neurons, comprising expressing a partially deleted NGN2 mutant lacking a portion of the C-terminal IDR of Neurogenin 2 (NGN2) in cells selected from pluripotent cells, multipotent cells, and fibroblasts, and culturing the mutant in an undifferentiated state maintenance medium or a neuronal differentiation induction medium. [2] The method described in [1], in which the partially deleted NGN2 mutant is transiently expressed in the cells. [3] The method described in [1] or [2], in which the partially deleted NGN2 mutant further lacks at least a portion of the N-terminal IDR. [4] The method described in any one of [1] to [3], in which the partially deleted NGN2 mutant comprises the region from amino acids 102 to 180 of NGN2 and is missing at least 20 residues in the region from amino acids 222 to 272. [5] The method according to any one of [1] to [4], wherein the partial deletion of the C-terminal IDR is a deletion from any of amino acids 187 to 222 to amino acid 272. [6] The method according to any one of [1] to [5], wherein the cell is a pluripotent cell. [7] The method according to any one of [1] to [6], wherein the expression of the partially deleted NGN2 variant is achieved by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding the partially deleted NGN2 variant. [8] The method according to any one of [1] to [7], wherein the neuron is a motor neuron and the cell further expresses (1) ISL1 and LHX3, (2) NEUROD1, NEUROD2, NGN1 and NGN3, (3) ASCL1, BRN2, MYT1L, LHX3, HB9 and ISL1, or (4) ISL1 and PHOX2A. [9] The method according to any one of [1] to [7], wherein the neuron is a dopaminergic neuron and the cell further expresses ATOH1 or NURR1.

[0010] The method according to any one of [1] to [7], wherein the neuron is a bipolar neuron and the cell further expresses NGN1, and the cell is co-cultured with an astrocyte.

[0011] The method according to any one of [1] to [7], wherein the neuron is a glutamatergic neuron, and the method comprises culturing the cell in which the NGN2 partial deletion has been expressed in the presence of an ALK inhibitor and a neurotrophic factor.

[0012] The method according to any one of [1] to [7], wherein the neuron is a serotonin neuron, and the method comprises further expressing ASCL1, NKX2.2, FEV, GATA2, and LMX1B in the cell.

[0013] The method according to any one of [1] to [7], wherein the neuron is a sensory neuron, and further comprises expressing BRN3A in the cell.

[0014] A method for producing neurological disease model cells, comprising using pluripotent cells, multipotent cells, or fibroblasts derived from a patient with a neurological disease as the cells, and producing neurons by the method described in any one of [1] to [7].

[0015] The method described in

[0014] , wherein the neurological disease is a motor neuron disease and the method comprises producing motor neurons by the method described in [8].

[0016] A neural differentiation induction kit or reagent comprising a nucleic acid containing a base sequence encoding a partially deleted form of Neurogenin 2 (NGN2) lacking a portion of the C-terminal IDR of NGN2.

[0017] A polypeptide comprising a partial sequence of Neurogenin 2 (NGN2), characterized in that it does not contain a portion of the C-terminal IDR of NGN2.

[0018] A polypeptide described in

[0017] , wherein the partial sequence is a partial sequence that does not include at least a portion of the N-terminal IDR.

[0019] A polypeptide described in

[0017] or

[0018] , wherein the NGN2 is human NGN2 having a sequence identity of 90% or more with the amino acid sequence shown in SEQ ID NO: 2.

[0020] A polypeptide described in any one of

[0017] to

[0019] , wherein the partial sequence is a partial sequence that includes the region of amino acids 102 to 180 in the human NGN2 sequence shown in SEQ ID NO: 2, but does not include at least 20 residues in the region of amino acids 222 to 272.

[0021] A polypeptide described in any one of

[0017] to

[0020] , wherein the partial sequence is a partial sequence that does not include the region from any of amino acids 187 to 222 to amino acid 272 in the human NGN2 sequence shown in SEQ ID NO: 2.

[0022] A nucleic acid encoding a polypeptide described in any one of

[0017] to

[0021] .

[0013] According to the present invention, neurons can be mass-produced with a much higher differentiation efficiency than conventional neuron production methods using the full-length NGN2. Using an NGN2 partial deletion mutant, in which the inhibitory domain present at the C-terminus of NGN2 that acts to inhibit neuronal differentiation promotion is deleted, the efficiency of differentiation into mature neurons is approximately two-fold higher than in conventional methods in which full-length NGN2 is expressed. Furthermore, the NGN2 partial deletion mutant also enables neuron production by transient expression, and in this case, neurons can be mass-produced through a short culture period of a few days with simpler steps than conventional methods, without the need for complicated genetic recombination experiments.

[0014] Schematic diagram of NGN2-FL. In this study, using AlphaFold, we defined amino acids 1-101 of NGN2 as the N-terminal IDR, amino acids 102-174 as the bHLH, and amino acids 175-272 as the C-terminal IDR. Phosphorylation sites reported in previous studies are indicated by "P," and the TAD is indicated by a diagonal line. Schematic diagram of a partially truncated NGN2 mutant. The N-terminal IDR excluded the TAD region, and amino acids 1-80 were removed to retain the endogenous nuclear localization signal. The C-terminal IDR also excluded amino acids 191-272 to remove the TAD and multiple phosphorylation sites. Synthesis of NGN2 synthetic mRNA. 3x FLAG-HA was attached to the N-terminus of each RNA. The size of the NGN2 synthetic mRNA was confirmed by electrophoresis using a 0.7% agarose gel. Four types of NGN2 were introduced into human iPS cells, and immunofluorescence staining was performed 6 hours later to confirm the translation and nuclear localization of NGN2 synthetic mRNA. Each NGN2 was detected using an antibody against FLAG, and cell nuclei were stained with DAPI. Figure 5 shows a graph quantifying the percentage of FLAG-positive cells. Approximately 80% of all NGN2 molecules were nuclear localized. Six images per NGN2 molecule were used for quantification. ns indicates no significant difference. Schematic diagram of the protocol for inducing neuronal differentiation of human iPS cells using NGN2 synthetic mRNA. The day iPS cells were seeded onto culture plates was designated d0, and NGN2 synthetic mRNA was introduced on d1. RP-qPCR was performed 24 hours (d2) and 48 hours (d3) after synthetic mRNA introduction. Immunofluorescence staining was performed 3 days (d4) after synthetic mRNA introduction, during differentiation, and 6 days (d7) after synthetic mRNA introduction, at the completion of differentiation. RT-qPCR 24 hours (d2) and 48 hours (d3) after the introduction of NGN2 synthetic mRNA. The target gene used was NEUROD4, whose expression is known to be induced by NGN2 during neuronal differentiation. After 48 hours, NEUROD4 expression increased significantly in the following order: NGN2-FL, NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC, with NGN2-ΔNC showing an approximately 7.3-fold increase in expression compared to NGN2-FL. The experiment was performed twice, and the average values ​​were used to create the graph. Immunofluorescence staining results were obtained 3 days after the introduction of NGN2 synthetic mRNA.Expression of the neuronal marker Tuj1 was highest in NGN2-FL, followed by NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC, in that order. Expression of Oct4, a marker for undifferentiated iPS cells, was lowest in NGN2-FL, followed by NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC. Cell nuclei were stained with DAPI. Immunofluorescent staining was performed 6 days (d7) after transfection with NGN2 synthetic mRNA, i.e., at the completion of neuronal differentiation. NGN2-ΔNC induced more significant neuronal differentiation than NGN2-FL. Tuj1 was used as the neuronal marker. Graph showing quantification of the area occupied by Tuj1-positive cells at the completion of neuronal differentiation induction. Six images were used for each NGN2. *p<0.05, ***p<0.001. Schematic diagram of the neuronal differentiation induction protocol using iPS cells derived from ALS patients. In this experiment, NGN2-FL or NGN2-ΔNC was transfected at a concentration of 0.1 μg / mL. Immunofluorescence staining was performed 6 days (d7) after transfection with NGN2 synthetic mRNA, i.e., at the completion of neuronal differentiation. As with iPS cells from healthy donors, NGN2-ΔNC significantly promoted neuronal differentiation compared to NGN2-FL. Neuronal differentiation was induced using iPS cells derived from ALS patients, and the graph shows the quantification of the area occupied by Tuj1-positive cells 6 days after differentiation was complete. Six images were used for quantification for each NGN2. *** indicates p<0.001. Schematic diagram of the protocol for inducing human iPS cell differentiation into motor neurons. iPS cells were seeded on a culture plate on day 0, and transfection with NGN2 and other agents was performed on day 1. RP-qPCR was performed 1 day (d2) and 4 days (d5) after transfection. We investigated whether simultaneous transfection of NGN2 with ISL1 and LHX3 (two TFs) could induce motor neuron differentiation. We then examined whether NGN2-ΔNC significantly induced motor neuron differentiation by introducing NGN2-FL or NGN2-ΔNC into cells. This graph shows RT-qPCR results performed 1 day (d2) and 4 days (d5) after the introduction of NGN2 and 2 TFs. The target genes were the motor neuron markers HB9 and ChAT.We found that the combination of NGN2 with two TFs induced HB9 and ChAT expression more significantly than NGN2 alone, and that the combination of NGN2-ΔNC with two TFs induced motor neuron markers more significantly than NGN2-FL. RT-qPCR was performed 7 days after transfection of NGN2 and BRN3A to examine the expression of sensory neuron markers NTRK1 and PRPH. The combination of NGN2-ΔNC and BRN3A induced the most significant sensory neuron marker expression. Immunofluorescence staining was performed 6 days (d7) after transfection of NGN2 synthetic mRNA, i.e., at the completion of neuronal differentiation. A deletion of NGN2-ΔNC by an additional 11 amino acids at its C-terminus (amino acids 81-179) did not promote neuronal differentiation, showing little difference from NGN2-ΔN (full-length C-terminus). Furthermore, a deletion of amino acids 222-272 at the C-terminus (amino acids 81-221) promoted differentiation to a similar extent as NGN2-ΔNC. This graph quantifies the area occupied by Tuj1-positive cells (Fig. 18A) 6 days after neuronal differentiation was completed. Six images were used for quantification for each NGN2. *** indicates p<0.001, and ns indicates no significant difference.

[0015] The cells used in the neuron production method of the present invention are cells selected from pluripotent cells, multipotent cells, and fibroblasts. The animal species from which the cells are derived is not particularly limited, and cells derived from mammals such as humans, mice, rats, hamsters, ferrets, rabbits, dogs, cats, and pigs, as well as various other animals, can be used. In one embodiment, the cells selected from pluripotent cells, multipotent cells, and fibroblasts are human cells. In another embodiment, the cells selected from pluripotent cells, multipotent cells, and fibroblasts are cells derived from a patient with a neurological disease.

[0016] In the present invention, "pluripotent cells" refer to cells that have the ability to differentiate into various cells that constitute an individual (pluripotency) and the ability to self-renew. Examples of pluripotent cells include various pluripotent stem cells, such as embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic germ (EG) cells, embryonic tumor (EC) cells, and pluripotent germ (mGS) cells. These pluripotent cell lines have already been established in various animal species, including humans, and are readily available. Methods for establishing pluripotent cell lines are well known, and new cell lines can also be created. For example, human iPS cell lines can be created by introducing cell reprogramming factors (e.g., the four factors OCT3 / 4, SOX2, KLF4, and c-MYC, or the four factors OCT3 / 4, SOX2, KLF4, and GLIS1) into somatic cells, such as fibroblasts, isolated from an animal.

[0017] In the present invention, "multipotent cells" refer to cells that have the multipotency to differentiate into a limited number of cell types, but do not have pluripotency. Examples of multipotent cells include various tissue stem cells or somatic stem cells, such as neural stem cells, hematopoietic stem cells, and mesenchymal stem cells. These tissue stem cells or somatic stem cells can be obtained by isolation from individual animals, or those established as cell lines can also be used.

[0018] In the present invention, "fibroblasts" typically refer to skin fibroblasts. Although fibroblasts are differentiated cells, they are known to be able to differentiate into various cells such as chondrocytes, adipocytes, smooth muscle cells, and cardiac muscle cells by introducing transcription factors or culturing in a medium containing differentiation-inducing factors. In the present invention, fibroblasts can also be used as cells that express NGN2 partial deletion bodies and differentiate into neurons. Fibroblasts can also be obtained by isolation from individual animals, or those established as cell lines can also be used.

[0019] In the neuron production method of the present invention, a partially deleted NGN2 mutant lacking at least a portion of the C-terminal IDR of NGN2 is expressed in cells selected from pluripotent cells, multipotent cells, and fibroblasts. The partially deleted NGN2 mutant is usually designed from the NGN2 sequence of the same animal species as the animal species from which the cells expressing it are derived. For example, when using human cells selected from human pluripotent cells, human multipotent cells, and human fibroblasts, a partially deleted NGN2 mutant designed from the sequence of human NGN2 (NCBI Gene ID: 63973) may be used.

[0020] The nucleotide sequence shown in SEQ ID NO: 1 is the CDS sequence of the human NGN2 mRNA sequence (NCBI Reference Sequence: NM_024019.4), and the amino acid sequence shown in SEQ ID NO: 2 is the full-length amino acid sequence of human NGN2 (NP_076924.1) encoded thereby. Human NGN2 is a transcription factor consisting of 272 amino acids, with the region of amino acids 1-101 being the N-terminal IDR, the region of amino acids 102-174 being the bHLH, and the region of amino acids 175-272 being the C-terminal IDR. The nucleotide sequence shown in SEQ ID NO: 21 is the CDS sequence of the mouse NGN2 mRNA sequence (NCBI Reference Sequence: NM_009718.4), and the amino acid sequence shown in SEQ ID NO: 22 is the full-length amino acid sequence of mouse NGN2 (NP_033848.1) encoded thereby. In mouse NGN2, which consists of 263 amino acids, amino acids 1-101 are the N-terminal IDR, amino acids 102-174 are the bHLH, and amino acids 175-263 are the C-terminal IDR.

[0021] In this specification, residue positions in the NGN2 protein are expressed as positions in the amino acid sequence of human NGN2 shown in SEQ ID NO: 2. The corresponding residues in NGN2 from other animal species can be easily confirmed by aligning the residues with the amino acid sequence of human NGN2 using a well-known algorithm such as ClustalW.

[0022] The NGN2 sequences of each animal species registered as Reference Sequences in the NCBI database are representative examples of the NGN2 sequences of that animal species. The NGN2 partial deletion mutants used in the present invention are not limited to those with sequences identical to partial regions of such typical NGN2 sequences, and may be partial deletion mutants based on amino acid sequences in which a small number of residues are substituted, deleted, inserted, or added in the typical NGN2 sequence, as long as they have activity as a master transcription factor that induces neuronal differentiation of pluripotent cells, multipotent cells, and fibroblasts. For example, in the case of a human NGN2 partial deletion mutant, it may be a polypeptide consisting of a sequence identical to a partial region of an amino acid sequence that has 90% or more, e.g., 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, sequence identity to the amino acid sequence of human NGN2 set forth in SEQ ID NO: 2.

[0023] The NGN2 partial deletion may further lack at least a portion of the N-terminal IDR. For example, the NGN2 partial deletion may be a partial deletion in which at least 20 residues, at least 30 residues, at least 40 residues, at least 50 residues, at least 60 residues, at least 70 residues, or at least 80 residues of the N-terminal IDR are further deleted. Alternatively, the NGN2 partial deletion may be a partial deletion in which at least 20 residues, at least 30 residues, at least 40 residues, at least 50 residues, at least 60 residues, at least 70 residues, or at least 80 residues are deleted from the N-terminus.

[0024] As described in the Examples below, the C-terminal IDR of the NGN2 protein contains a region from amino acids 222 to 272 that acts to inhibit neuronal differentiation. Deletion of this region, which acts to inhibit neuronal differentiation, dramatically increases the efficiency of NGN2 in promoting neuronal differentiation. Thus, in one embodiment of the present invention, an NGN2 partially deleted mutant contains at least the region from amino acids 102 to 180 of NGN2, and is deleted of at least 20 residues, e.g., at least 25 residues, at least 30 residues, at least 35 residues, at least 40 residues, at least 45 residues, or at least 50 residues, or the region from amino acids 222 to 272. A partially deleted mutant from amino acids 222 to 272 may also be deleted of the C-terminal region, as long as it contains at least the region from amino acids 102 to 180 of NGN2. Specifically, at least one residue may be further deleted from the region of amino acids 187 to 221, for example, at least one residue may be further deleted from the region of amino acids 188 to 221, at least one residue from the region of amino acids 189 to 221, at least one residue from the region of amino acids 190 to 221, or at least one residue from the region of amino acids 191 to 221. As described in the Examples below and Figures 18A and B, NGN2 partial deletion variant 81-190(ΔNC), in which the C-terminus has been deleted down to amino acid number 191 (82 residues), has a potent neuronal differentiation-promoting ability equivalent to that of NGN2 partial deletion variant 81-221, in which the C-terminus has been deleted down to amino acid number 222 (51 residues). NGN2 partial deletion variant 81-186, in which the C-terminus has been deleted down to amino acid number 187, still has a strong, though somewhat weaker, ability to promote differentiation. When the C-terminus has been deleted down to amino acid number 180 (NGN2 partial deletion variant 81-179), the enhanced promotion of neuronal differentiation is lost and the differentiation-promoting ability is at the same level as NGN2-ΔN, which retains the full C-terminus. Therefore, if one wishes to prepare a smaller NGN2 partial deletion while maintaining a high neuronal differentiation-promoting ability, the C-terminus can be deleted up to amino acid 187, 188, 189, 190, or 191.

[0025] In one embodiment, the deletion of at least a portion of the C-terminal IDR is a deletion from any of amino acids 187 to 222 to amino acid 272, and may be, for example, a deletion from any of amino acids 188 to 222 to amino acid 272, a deletion from any of amino acids 189 to 222 to amino acid 272, a deletion from any of amino acids 190 to 222 to amino acid 272, or a deletion from any of amino acids 191 to 222 to amino acid 272.

[0026] The NGN2 partial deletions in each of the above-mentioned embodiments may be partial deletions in which at least 20 residues, at least 25 residues, at least 30 residues, at least 35 residues, at least 40 residues, at least 45 residues, at least 50 residues, at least 60 residues, at least 70 residues, at least 80 residues, at least 83 residues, at least 84 residues, at least 85 residues, at least 86 residues, at least 87 residues, at least 88 residues, at least 89 residues, at least 90 residues, at least 91 residues, at least 92 residues, or at least 93 residues are deleted from the N-terminus. Other examples of NGN2 partial deletion variants of this embodiment include partial deletion variants that include at least the region of amino acids 102 to 181, at least the region of amino acids 102 to 182, at least the region of amino acids 102 to 183, at least the region of amino acids 102 to 184, at least the region of amino acids 102 to 185, at least the region of amino acids 102 to 186, at least the region of amino acids 102 to 187, at least the region of amino acids 102 to 188, at least the region of amino acids 102 to 189, or at least the region of amino acids 102 to 190.

[0027] Expression of a partially deleted NGN2 mutant can be achieved by introducing into cells a nucleic acid containing a base sequence encoding the partially deleted NGN2 mutant. Examples of nucleic acids containing a base sequence encoding the partially deleted NGN2 mutant include synthetic mRNA and nucleic acid constructs (typically DNA) having a structure capable of expressing the partially deleted NGN2 mutant under the control of a promoter. The partially deleted NGN2 mutant can be expressed stably or transiently. In the present invention, transient expression of the deleted NGN2 mutant is preferred because it avoids the need for complicated genetic recombination experiments.

[0028] Methods for preparing synthetic mRNA are well known, and can be prepared, for example, by the method described in Warren et al., Cell Stem Cell 7, 618-630 (2010). First, DNA encoding a truncated NGN2 mutant is synthesized by PCR using a cDNA encoding the full-length NGN2 as a template. NGN2 truncated mutants with deletions at both or one end can be easily prepared by PCR using primers designed within the cDNA encoding the full-length NGN2. Next, a UTR sequence is added to the 3' end of the DNA encoding the truncated NGN2 mutant, followed by the addition of an RNA polymerase promoter to the 5' end and a poly-T sequence serving as a template for a poly-A tail to the 3' end to prepare a DNA template for synthetic mRNA transcription. This DNA template may optionally contain an appropriate tag sequence. Synthetic mRNA with a 5' cap structure can be prepared by in vitro transcription from the DNA template. In addition, a method for completely chemically synthesizing mRNA without using enzymes is also known (Abe et al., ACS Chem Biol. 2022 Jun 17;17(6):1308-1314.), and synthetic mRNA can also be prepared by such a method. The prepared synthetic mRNA can be introduced into cells by standard methods such as lipofection.

[0029] In a method using a nucleic acid construct (typically DNA) having a structure capable of expressing a partially deleted NGN2 gene under the control of a promoter, DNA encoding the partially deleted NGN2 gene can be incorporated into a gene expression construct utilizing a promoter capable of expression in cells into which the nucleic acid construct is introduced. The nucleic acid construct can be introduced into cells using standard methods such as lipofection. Cells that transiently express a partially deleted NGN2 gene can be obtained by introducing a nucleic acid construct using a transient expression construct into cells. Cells that stably express a partially deleted NGN2 gene can be obtained by selecting cells in which the nucleic acid construct has been integrated into the chromosome by random integration from cells into which a nucleic acid construct using a transient expression construct has been introduced. Alternatively, cells that stably express a partially deleted NGN2 gene can be obtained by targeted integration using homologous recombination or genome editing tools.

[0030] Cells expressing NGN2 knockouts are cultured in undifferentiated maintenance medium or neuronal differentiation-inducing medium. When using a construct that transiently expresses NGN2 knockouts, large numbers of neurons can be obtained easily and efficiently by culturing the cells for a short period of time (5-7 days) after construct introduction.

[0031] As the undifferentiation maintenance medium, a medium generally used for culturing pluripotent cells such as iPS cells and ES cells while maintaining their undifferentiated state can be used. Various undifferentiation maintenance media are known, including commercially available products such as StemFit® AK02 medium (Ajinomoto), ciKIC® iPS medium (Kanto Chemical), Stem-Partner® ACF (Kyokuto Pharmaceutical Industries), and Ex-iPS Cell Medium (Myoridge). Any of these known undifferentiation maintenance media may be used.

[0032] A neuronal differentiation-inducing medium is a medium supplemented with any combination of neuronal differentiation-inducing factors, such as growth factors and small molecule compounds, that promote the differentiation of various neurons. Combinations of neuronal differentiation-inducing factors that are preferably used to promote the differentiation of specific neurons are described below. The composition of the medium other than the neuronal differentiation-inducing factors may be similar to the composition (composition of components other than the neuronal differentiation-inducing factors) of media commonly used for inducing the differentiation of neurons from pluripotent stem cells or neural stem cells.

[0033] The neuron production method of the present invention encompasses, as a sub-concept, the following methods for producing various types of neurons. It is known that expressing NGN2 alone as a transcription factor in mouse or human pluripotent cells can induce differentiation into excitatory neurons, including glutamatergic neurons (Thoma et al., PLoS One. 2012;7, Zhang et al., Neuron. 2013;78:785-798). In the method of the present invention, the neurons induced to differentiate by expressing only NGN2 partial deletions as a transcription factor in pluripotent cells or other cells and culturing them in an undifferentiated maintenance medium are thought to be a mixture of multiple neuron types. As explained below, by combining NGN2 partial deletions with other transcription factors or appropriate neuronal differentiation inducers, it is possible to efficiently differentiate desired neuron subtypes, such as motor neurons and dopaminergic neurons.

[0034] When at least one other transcription factor is expressed in combination with the partially-deficient NGN2 mutant, the expression of the at least one other transcription factor may be transient or stable.

[0035] In a production method for transiently expressing a combination of multiple transcription factors, when transiently introducing nucleic acids such as synthetic mRNA or a transient expression construct into cells, multiple nucleic acids that encode or express each of the multiple transcription factors may be introduced into cells, or nucleic acids that can express some or all of the transcription factors may be introduced into cells. The same applies to stable expression of multiple transcription factors.

[0036] <Production of Motor Neurons> Cells selected from pluripotent cells, multipotent cells, and fibroblasts are expressed in combination with NGN2 partial deletion mutants to express (1) ISL1 and LHX3, (2) NEUROD1, NEUROD2, NGN1, and NGN3, (3) ASCL1, BRN2, MYT1L, LHX3, HB9, and ISL1, or (4) ISL1 and PHOX2A. Then, the cells are cultured in a medium for maintaining undifferentiated cells or a medium for inducing neuronal differentiation, promoting differentiation into motor neurons. This allows for efficient mass production of motor neurons. Regarding (1), see the Examples below and Hester ME, et al., Mol. Ther. 2011;19:1905-1912, etc. Hester ME et al. reported a technique for inducing motor neuron differentiation by expressing three factors, NGN2, ISL1, and LHX3, in pluripotent stem cells. Regarding (2), see Ko M., WO 2016 / 143826 A1, and Goparaju SK, et al. Sci. Rep. 2017;7:42367. Ko M. and Goparaju SK et al. reported a technique for inducing motor neuron differentiation by expressing five factors (NGN2, NEUROD1, NEUROD2, NGN1, and NGN3) in pluripotent stem cells. Regarding (3), see Son EY, et al., Cell Stem Cell. 2011;9:205-218. Son EY et al. reported a technique for inducing motor neuron differentiation by expressing seven factors (NGN2, ASCL1, BRN2, MYT1L, LHX3, HB9, and ISL1) in mouse and human fibroblasts. Regarding (4), see Garone MG, et al., J. Vis. Exp. 2019. Garone MG et al. have reported a technique for inducing the differentiation of motor neurons by expressing three factors, NGN2, ISL1, and PHOX2A, in human iPS cells. As such, it is known that the differentiation of pluripotent cells into motor neurons can be induced by combining the above-mentioned transcription factors, and by expressing a partially deleted NGN2 as NGN2, it is possible to mass-produce motor neurons with a higher differentiation efficiency than these conventional techniques.In the case of transient expression, motor neurons can be rapidly mass-produced with simple steps and with higher differentiation efficiency than conventional techniques.

[0037] In one embodiment of the method for producing motor neurons according to the invention, the cell is a pluripotent or multipotent cell, e.g., a pluripotent cell, which transiently or stably expresses a defective NGN2, ISL1, and LHX3, preferably transiently. In another embodiment, the cell is a multipotent or fibroblast, e.g., a fibroblast, which transiently or stably expresses a defective NGN2, ASCL1, BRN2, MYT1L, LHX3, HB9, and ISL1, preferably transiently.

[0038] Although the above-mentioned combination of transcription factors promotes motor neuron differentiation even when cultured in an undifferentiated maintenance medium that does not contain neuronal differentiation inducers (see Examples below), differentiation into motor neurons can be further promoted by culturing in a neuronal differentiation inducer medium containing appropriate neuronal differentiation inducers. Preferred examples of neuronal differentiation inducers include, in the case of the combination of NGN2 partial deletion mutants and (1), a neuronal differentiation inducer medium containing retinoic acid, forskolin, and SHH (Hester ME et al., 2011); in the case of the combination of NGN2 partial deletion mutants and (2), a neuronal differentiation inducer medium containing forskolin, an ALK inhibitor (e.g., an ALK5 inhibitor such as SB431542), an AMPK inhibitor (e.g., dorsomorphin), retinoic acid, and neurotrophic factors (e.g., BDNF, GDNF, and NT-3) (Goparaju SK et al., 2017); and in the case of the combination of NGN2 partial deletion mutants and (3), a neuronal differentiation inducer medium containing neurotrophic factors (e.g., GDNF, BDNF, and CNTF) (Son EY et al., 2011), and in the case of a combination of NGN2 partial deletions and (4), neuronal differentiation induction media containing a γ-secretase inhibitor (e.g., DAPT), a fibroblast growth factor receptor-specific tyrosine kinase inhibitor (e.g., SU5402), L-ascorbic acid, and neurotrophic factors (e.g., BDNF and GDNF) are used (Garone MG et al., 2019).

[0039] <Production of Dopaminergic Neurons> In cells selected from pluripotent cells, multipotent cells, and fibroblasts, expression of ATOH1 or NURR1 in combination with NGN2 partial deletions and culture in undifferentiated maintenance medium or neuronal differentiation-inducing medium promotes differentiation into dopaminergic neurons. This allows for efficient mass production of dopaminergic neurons. Xue Y. et al. (Stem Cells Transl Med. 2019 Feb;8(2):112-123.) reported a technique for inducing differentiation of dopaminergic neurons by expressing NGN2 and ATOH1 in iPS cells. Park, C. et al. (FEBS Lett., 582, 537-542, 2008) reported a technique for inducing differentiation of dopaminergic neurons by expressing NGN2 and NURR1 in mouse neural stem cells. As described above, it is known that the combination of NGN2 and ATOH1, and the combination of NGN2 and NURR1 can induce differentiation of dopaminergic neurons from pluripotent cells, etc., and by expressing a partially deleted NGN2 as NGN2, it is possible to mass-produce dopaminergic neurons with higher differentiation efficiency than these conventional techniques. In the case of transient expression, dopaminergic neurons can be mass-produced rapidly with simple steps and with higher differentiation efficiency than conventional techniques.

[0040] Although the combination of these transcription factors promotes differentiation into dopaminergic neurons even when cultured in a medium that does not contain neuronal differentiation inducers, culturing in a neuronal differentiation inducer medium containing appropriate neuronal differentiation inducers can further promote differentiation into dopaminergic neurons. Preferred examples of neuronal differentiation inducers include those containing SHH, growth factors (e.g., FGF8b), and gamma-secretase inhibitors (e.g., DAPT) in embodiments in which NGN2-deficient cells and ATOH1 are expressed (Xue Y. et al., 2019), and those containing growth factors (e.g., bFGF and EGF) in embodiments in which NGN2-deficient cells and NURR1 are expressed (Park, C. et al., 2008).

[0041] <Production of Bipolar Neurons> Expression of NGN1 in combination with NGN2 knockout mutants in cells selected from pluripotent cells, multipotent cells, and fibroblasts and co-culturing them with astrocytes in undifferentiated maintenance medium or neuronal differentiation-inducing medium promotes differentiation into bipolar neurons. This allows for efficient mass production of bipolar neurons. It is known that expressing NGN2 and NGN1 in human ES cells or iPS cells and co-culturing them with rat astrocytes can induce bipolar neuron differentiation (Busskamp, ​​V. et al., Mol. Syst. Biol., 10, 760, 2014). By expressing NGN2 knockout mutants as NGN2, mass production of bipolar neurons can be achieved with higher differentiation efficiency than conventional techniques. Transient expression allows for rapid mass production of bipolar neurons with simple steps and higher differentiation efficiency than conventional techniques.

[0042] The astrocytes to be co-cultured with NGN2 partially deleted cells and cells expressing NGN1 are not particularly limited, and in addition to astrocytes derived from humans, astrocytes derived from animals other than humans, such as rats and mice, can be used.

[0043] In one embodiment of the method for producing bipolar neurons according to the invention, the cells are pluripotent or multipotent cells, e.g., multipotent cells. In another embodiment, the cells are pluripotent or neural stem cells.

[0044] <Production of glutamatergic neurons> After expressing NGN2-deficient cells in cells selected from pluripotent cells, multipotent cells, and fibroblasts, the cells are cultured in the presence of an ALK inhibitor and a neurotrophic factor (e.g., BDNF), i.e., in a neurodifferentiation-inducing medium containing these neurodifferentiation-inducing factors. For the production of glutamatergic neurons, the NGN2-deficient cells are the only transcription factor expressed in the cells. As the ALK inhibitor, either or both of an ALK5 inhibitor such as SB431542 and an ALK2 / 3 inhibitor such as LDN193189 can be used. As the neurotrophic factor, BDNF, GDNF, etc. can be used.

[0045] It is known that glutamatergic neurons can be induced by expressing NGN2 in human or mouse ES cells, iPS cells, or neural stem cells and culturing them in a medium containing SB431542, LDN193189, BDNF, etc. (Zhang, Y., et al.: Neuron 78, 785-798. (2013)). By expressing a partially deleted NGN2 mutant as NGN2, glutamatergic neurons can be mass-produced with higher differentiation efficiency than conventional techniques. Transient expression allows for rapid mass production of glutamatergic neurons with higher differentiation efficiency than conventional techniques, and with simple steps.

[0046] In one embodiment of the method of producing glutamatergic neurons according to the invention, the cells are pluripotent or multipotent cells, such as pluripotent cells or neural stem cells.

[0047] <Production of serotonin neurons> Cells selected from pluripotent cells, multipotent cells, and fibroblasts can be expressed in combination with NGN2-deficient cells, ASCL1, NKX2.2, FEV1, GATA2, and LMX1B, and cultured in undifferentiated maintenance medium or neuronal differentiation-inducing medium to promote differentiation into serotonin neurons. This allows for efficient mass production of serotonin neurons. It is known that the expression of NGN2, ASCL1, NKX2.2, FEV1, GATA2, and LMX1B in human skin fibroblasts can induce differentiation into serotonin neurons (Vadodaria, K., et al.: Mol. Psychiatry. 21, 49-61. (2016)). By expressing NGN2-deficient cells as NGN2, mass production of serotonin neurons can be achieved with higher differentiation efficiency than conventional techniques. In the case of transient expression, serotonin neurons can be rapidly mass-produced with simple steps and with higher differentiation efficiency than conventional techniques.

[0048] In one embodiment of the method for producing serotonin neurons according to the present invention, the cell is a fibroblast. In another embodiment, the cell is a pluripotent or multipotent cell, such as a multipotent cell or a neural stem cell.

[0049] Although the combination of the six transcription factors promotes differentiation into serotonin neurons even when cultured in an undifferentiated maintenance medium that does not contain a neuronal differentiation inducer, differentiation into serotonin neurons can be further promoted by culturing in a neuronal differentiation inducer medium containing an appropriate neuronal differentiation inducer. Preferred examples of neuronal differentiation inducer media include those containing cAMP analogs (e.g., dibutyryl cyclic-AMP), neuronal inducers (e.g., noggin), ALK2 / 3 inhibitors (e.g., LDN193189), ALK5 inhibitors (e.g., A83-1 and SB431542), GSK-3β inhibitors (e.g., CHIR99021), forskolin, and neurotrophic factors (e.g., GDNF and BDNF).

[0050] <Production of Sensory Neurons> In cells selected from pluripotent cells, multipotent cells, and fibroblasts, expressing BRN3A in combination with NGN2 knockouts and culturing them in undifferentiated maintenance medium or neuronal differentiation-inducing medium promotes differentiation into sensory neurons. This allows for efficient mass production of sensory neurons. It is known that expressing NGN2 and BRN3A in human and mouse fibroblasts and human pluripotent stem cells can induce sensory neuron differentiation (Blanchard, J., et al.: Nat. Neurosci., 18, 25-25. (2015) and Nickolls AR et al., Cell Rep. 2020 Jan 21;30(3):932-946.e7. doi: 10.1016 / j.celrep.2019.12.062). Expressing NGN2 knockouts as NGN2 allows for mass production of sensory neurons with higher differentiation efficiency than conventional techniques. In the case of transient expression, it is possible to rapidly mass-produce sensory neurons with a higher differentiation efficiency and simpler steps than conventional techniques.

[0051] In one embodiment of the method for producing sensory neurons according to the invention, the cells are fibroblasts. In another embodiment, the cells are pluripotent or multipotent cells, such as pluripotent cells or neural stem cells.

[0052] The combination of NGN2-deficient cells and BRN3A promotes differentiation into sensory neurons even when cultured in a medium for maintaining undifferentiated cells without neuronal differentiation factors, but differentiation into sensory neurons can be further promoted by culturing in a neuronal differentiation-inducing medium containing appropriate neuronal differentiation factors. A preferred example of a neuronal differentiation-inducing medium is a neuronal differentiation-inducing medium containing neurotrophic factors (e.g., BDNF, GDNF, and NGF).

[0053] The official full names of the various transcription factors listed above, the NCBI Gene IDs of the human genes, and the accession numbers of the Reference Sequences (a representative example is shown if multiple sequences exist) are as follows. These transcription factors are not limited to proteins with the same sequences as those listed in the sequence listing; proteins with sequences in which a small number of residues have been substituted, deleted, inserted, or added can also be used, as long as they retain the activity of the original transcription factor. Specifically, each transcription factor may be a protein with 90% or more, for example 95% or more, or 98% or more, sequence identity with the amino acid sequence listed in the sequence listing.

[0054]

[0055] In the neuron production method of the present invention, by using cells derived from a patient with a neurological disease as pluripotent cells, multipotent cells, or fibroblasts, it is possible to produce neurons that serve as a neurological disease model. That is, the present invention also provides a method for producing neurological disease model cells, which comprises using pluripotent cells, multipotent cells, or fibroblasts derived from a patient with a neurological disease and producing neurons by the neuron production method of the present invention.

[0056] The most preferred example of pluripotent cells derived from patients with neurological disorders is iPS cells. iPS cells derived from patients with neurological disorders can be generated by introducing cell reprogramming factors (specific examples are as described above) into somatic cells such as fibroblasts isolated from the patient.

[0057] Multipotent cells derived from patients with neurological disorders can be isolated and obtained from the patient. For example, neural stem cells can be collected from the patient's brain tissue or nasal olfactory bulb. Hematopoietic stem cells can be collected from the patient's bone marrow, or they can be collected as peripheral blood stem cells from the peripheral blood of patients with neurological disorders who have been administered G-CSF or other drugs. Mesenchymal stem cells can be collected from the patient's bone marrow, adipose tissue, etc.

[0058] Fibroblasts derived from a patient with a neurological disease can be harvested from the patient's skin.

[0059] When producing neurological disease model cells according to the present invention, neurons that have undergone degeneration or damage due to a neurological disease are produced. For example, when the neurological disease is a motor neuron disease such as amyotrophic lateral sclerosis or spinal muscular atrophy, motor neurons can be produced using cells such as pluripotent cells derived from the patient. Furthermore, dopaminergic neurons can be produced as model cells for Parkinson's disease (Sheta R et al., Scientific Reports. 2022; 12: 17176), glutamatergic neurons can be produced as model cells for Huntington's disease (Villegas LD et al., Stem Cell Research. Vol. 77, June 2024, 103408), and glutamatergic neurons can be produced as model cells for Alzheimer's disease (Vazin T et al., Neurobiol Dis. 2014 Feb:62:62-72).

[0060] The present invention also provides a neuronal differentiation induction kit or reagent containing a nucleic acid comprising a base sequence encoding a partially deleted NGN2 gene. The partially deleted NGN2 gene and the nucleic acid comprising the base sequence encoding the same are as described above. The kit or reagent may further comprise instructions for use. In one embodiment, the neuronal differentiation induction kit or reagent contains a synthetic mRNA encoding a partially deleted NGN2 gene. In another embodiment, the neuronal differentiation induction kit or reagent contains a nucleic acid comprising an RNA polymerase promoter at the 5' end and a poly-T sequence at the 3' end, with a sequence encoding the partially deleted NGN2 gene between them. This nucleic acid is a template for preparing synthetic mRNA encoding the partially deleted NGN2 gene, and is typically DNA. Users of the kit or reagent can prepare synthetic mRNA encoding the partially deleted NGN2 gene by performing a transcription reaction using RNA polymerase with the nucleic acid as a template.

[0061] Furthermore, the neural differentiation induction kit or reagent may further contain a nucleic acid containing a nucleotide sequence encoding another transcription factor required for producing various neurons, such as motor neurons. Specific examples of such other transcription factors are described above. In this case, the nucleic acid containing a nucleotide sequence encoding the other transcription factor may be a synthetic mRNA or a template for preparing a synthetic mRNA, similar to the nucleic acid containing a nucleotide sequence encoding a partially deleted NGN2 mutant.

[0062] The present invention also provides a polypeptide that is a partially deleted NGN2 polypeptide as defined above, and a nucleic acid encoding the same. A partially deleted NGN2 polypeptide can be described as a polypeptide that is composed of a partial sequence of NGN2 and does not contain a portion of the C-terminal IDR of NGN2. The polypeptide of the present invention can be used as a partially deleted NGN2 polypeptide in the neuron production method of the present invention described above.

[0063] The term "polypeptide consisting of a partial sequence" includes polypeptides consisting of one partial sequence of the full-length NGN2 protein, polypeptides having a structure in which two or more discontinuous partial sequences are linked together (i.e., polypeptides having a structure in which one or more partial regions within the full-length NGN2 protein are deleted), and polypeptides having a structure in which any one or more amino acid residues, such as a tag sequence or a signal sequence, have been added to these polypeptides.

[0064] In one embodiment of the polypeptide of the present invention, the NGN2 is human NGN2 having 90% or more, for example 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more sequence identity with the amino acid sequence set forth in SEQ ID NO:2.

[0065] An NGN2 partial deletion polypeptide further lacking at least a portion of the N-terminal IDR is a polypeptide in which the partial sequence of NGN2 constituting the NGN2 partial deletion polypeptide is a partial sequence that does not include a portion of the C-terminal IDR of NGN2 and does not include at least a portion of the N-terminal IDR of NGN2. The partial sequence of NGN2 constituting the NGN2 partial deletion polypeptide can be a partial sequence that does not include at least 20 residues, at least 30 residues, at least 40 residues, at least 50 residues, at least 60 residues, at least 70 residues, or at least 80 residues of the N-terminal IDR. Alternatively, the partial sequence of NGN2 constituting the NGN2 partial deletion polypeptide can be a partial sequence that does not include at least 20 residues, at least 30 residues, at least 40 residues, at least 50 residues, at least 60 residues, at least 70 residues, or at least 80 residues of the N-terminus of NGN2.

[0066] In one embodiment of the polypeptide of the present invention, the NGN2 partial sequence constituting the NGN2 partially deleted polypeptide comprises the region of amino acids 102 to 180 in the human NGN2 sequence set forth in SEQ ID NO: 2, and is a partial sequence that contains at least 20 residues, e.g., at least 25 residues, at least 30 residues, at least 35 residues, at least 40 residues, at least 45 residues, or at least 50 residues, in the region of amino acids 222 to 272, or does not contain the region of amino acids 222 to 272. In this embodiment, the partial sequence that does not contain the region of amino acids 222 to 272 also encompasses a partial sequence that further lacks the C-terminal region, i.e., a partial sequence that does not contain at least one residue of amino acids 181 to 221. Specific examples include a partial sequence that does not contain at least one residue of amino acids 187 to 221 in addition to the region of amino acids 222 to 272, such as at least one residue of amino acids 188 to 221, at least one residue of amino acids 189 to 221, at least one residue of amino acids 190 to 221, or at least one residue of amino acids 191 to 221. As explained in the neuron production method of the present invention, when it is desired to prepare a smaller NGN2 partially deleted polypeptide while maintaining high neuron differentiation ability, the C-terminus can be deleted up to amino acid 187, up to amino acid 188, up to amino acid 189, up to amino acid 190, or up to amino acid 191.

[0067] In one embodiment of the polypeptide of the present invention, the portion of the C-terminal IDR of NGN2 that is not contained in the NGN2 partially deleted polypeptide is the region from any of amino acids 187 to 222 to amino acid 272 in the human NGN2 sequence shown in SEQ ID NO: 2, and may be, for example, a polypeptide that does not contain the region from any of amino acids 188 to 222 to amino acid 272, the region from any of amino acids 189 to 222 to amino acid 272, the region from any of amino acids 190 to 222 to amino acid 272, or the region from any of amino acids 191 to 222 to amino acid 272. In other words, the partial sequence of NGN2 that constitutes the NGN2 partially deleted polypeptide is a partial sequence that does not include the region from any of amino acids 187 to 222 to amino acid 272, and may be, for example, a partial sequence that does not include the region from any of amino acids 188 to 222 to amino acid 272, the region from any of amino acids 189 to 222 to amino acid 272, the region from any of amino acids 190 to 222 to amino acid 272, or the region from any of amino acids 191 to 222 to amino acid 272.

[0068] The NGN2 partially deleted polypeptide of each of the above-mentioned embodiments may be a partial sequence of NGN2 constituting the polypeptide, in which the partial sequence does not include at least 20 residues, at least 25 residues, at least 30 residues, at least 35 residues, at least 40 residues, at least 45 residues, at least 50 residues, at least 60 residues, at least 70 residues, at least 80 residues, at least 83 residues, at least 84 residues, at least 85 residues, at least 86 residues, at least 87 residues, at least 88 residues, at least 89 residues, at least 90 residues, at least 91 residues, at least 92 residues, or at least 93 residues of the N-terminus of NGN2. Another example of an NGN2 partially deleted polypeptide of this embodiment is one in which the NGN2 partial sequence constituting the polypeptide is a partial sequence comprising the region of amino acids 102 to 181 in the human NGN2 sequence set forth in SEQ ID NO: 2, such as a partial sequence comprising the region of amino acids 102 to 182, the region of amino acids 102 to 183, the region of amino acids 102 to 184, the region of amino acids 102 to 185, the region of amino acids 102 to 186, the region of amino acids 102 to 187, the region of amino acids 102 to 188, the region of amino acids 102 to 189, or the region of amino acids 102 to 190.

[0069] The nucleic acid encoding the NGN2 partially deleted polypeptide may be DNA, i.e., a nucleic acid composed of deoxyribonucleotides, RNA, i.e., a nucleic acid composed of ribonucleotides, or a nucleic acid composed of both deoxyribonucleotides and ribonucleotides. In any of these cases, at least some of the bases may contain nucleotide analogs, which are nucleotides that have been artificially altered or modified. Nucleotide analogs include bridged artificial nucleic acids such as LNA (Locked Nucleic Acid), ENA (2'-O,4'-C-Ethylene-bridged Nucleic Acid), AmNA, GuNA, and scpBNA; non-bridged artificial nucleic acids such as PNA (Peptide Nucleic Acid) and Morpholino Nucleic Acid; and artificially modified bases such as 2'-O-Methoxyethyl (2'-MOE), 2'-O-Methyl (2'-Ome), 2'-F, Super T (5-hydroxybutynl-2'-deoxyuridine), Super G (8-aza-7-deazaguanosine), deoxyinosine, 5-methyl dC, deoxyuridine, 2,6-diaminopurine, 2-aminopurine, and 2-Amino-dATP. The A, T, G, and C nucleotides in the base sequences shown in the Sequence Listing of the present application may contain the nucleotide analogs A, T, G, and C unless otherwise specified in each sequence information. That is, although the sequence listing of the present application may not indicate the base sequence of a nucleic acid that may contain a nucleotide analog, this does not mean that the monomers constituting the nucleic acid are limited to natural nucleotides. Furthermore, nucleic acids composed of natural nucleotides have phosphodiester bonds between nucleotides, but unless otherwise specified in the sequence information of the sequence listing, also include nucleic acids in which some or all of the bonds between nucleotides are phosphorothioate bonds.

[0070] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0071] 1. Materials and Methods (Human iPS Cell Lines) iPS cells derived from a healthy individual (cell number: HPS0063, cell name: 201B7) and iPS cells derived from an ALS patient (cell number: HPS0290, cell name: CiRA00024) obtained from the RIKEN BioResource Research Center were used.

[0072] iPS cell culture and passaging. iPS cells were cultured on laminin-511 (iMatrix-511: 17A055; Nippi)-coated dishes and plates in StemFit® AK02 medium (RCAK02N; Ajinomoto) supplemented with 1% (vol / vol) penicillin-streptomycin (hereafter referred to as AK02 medium) at 5% CO2 and 37°C. iPS cells were passaged when the cells reached 70-90% density. After removing the medium and washing once with PBS, the cells were treated with a cell detachment solution consisting of a 1:1 mixture of TrypLE™ Select Enzyme (1X) (12563011; Gibco) and 0.5 mM EDTA / PBS at 37°C for 3 minutes to dissociate into single cells. After removing the cell detachment solution, an appropriate amount of StemFit® AK02 medium supplemented with 10 μM Y-27632 (Product No. 034-24024; FUJIFILM Wako Pure Chemical) and 1% (vol / vol) penicillin-streptomycin mixture (hereafter referred to as AK02+Y medium) was added, and the iPS cells were completely detached from the dish using a cell scraper and pipetting. An appropriate amount of AK02+Y medium was added to a new coated dish or plate, and the single-celled cells were seeded in an appropriate amount. The medium was replaced with AK02 medium the day after passaging, and was then replaced every 1 or 2 days thereafter.

[0073] (Preparation of synthetic mRNAs encoding NGN2 and its IDR-deleted variants) The amino acid sequences of the NGN2 deletion variants used in this study are as follows. NGN2 has the structure shown in Figure 1, and its full-length variant (SEQ ID NO: 2) corresponds to amino acids 1-272. The first variant is NGN2-ΔN (amino acids 81-272, SEQ ID NO: 4), in which 80 amino acids from the N-terminal IDR are deleted. The second variant is NGN2-ΔC (amino acids 1-190, SEQ ID NO: 6), in which 82 amino acids from the C-terminal IDR are deleted. The third variant is NGN2-ΔNC (amino acids 81-190, SEQ ID NO: 8), in which 80 amino acids from the N-terminal IDR and 82 amino acids from the C-terminal IDR are deleted (Figure 2). The DNA sequences of these coding regions (open reading frames, ORFs) were amplified by PCR using the cDNA encoding the full-length NGN2 (SEQ ID NO: 1) as a template. In addition, the 3' untranslated region of human α-globin was added to the 3' end of all ORFs to increase mRNA stability and translation efficiency, and the 3xFLAG-HA sequence was added to the 5' end as an epitope tag (Fig. 3). Synthetic mRNA encoding the full-length NGN2 (NGN2-FL) was also prepared in the same manner.

[0074] Synthetic mRNA was prepared as described in Warren et al., Cell Stem Cell 7, 618–630 (2010). Briefly, a T7 promoter and poly(A) tail were added to the PCR reaction using a KOD FX Neo kit (Toyobo). RNA was synthesized from the PCR product using the MEGAscript T7 kit (Ambion) along with 3'-O-Me-m7G(5')G RNA Cap Structure Analog (New England Biolabs), ATP, GTP, 5-Methyl-CTP (TriLink), and pseudo-UTP (TriLink) (Figure 3). Synthetic mRNA was purified using the MEGAclear kit (Ambion) or LiCl precipitation. The length of the purified synthetic mRNA was confirmed by electrophoresis (Figure 4).

[0075] (Transfection of NGN2 synthetic mRNA into iPS cells) Transfection of synthetic mRNA by lipofection was initiated when iPS cells cultured in AK02+Y medium reached a cell density of 50-70%. On day 1, Opti-MEM® I Reduced-Serum Medium (31985070; Gibco, hereafter Opti-MEM) and Lipofectamine® MessengerMAX™ mRNA Transfection Reagent (LMRNA001; Thermo Fisher Scientific, hereafter Lipofectamine MessengerMAX) were mixed and incubated at room temperature for 10 minutes. Next, the Opti-MEM and Lipofectamine MessengerMAX mixture was added to a separate tube containing Opti-MEM and NGN2 synthetic mRNA, and the mixture was incubated at room temperature for 4 minutes. The mixture was then added to the iPS cells. After 6 hours, the medium was removed and replaced with AK02+Y medium. The cells were cultured as is for the second day, and then the medium was changed to AK02 on the third day. Undifferentiated iPS cells were removed using aphidicolin (Wako) on days four and five. Differentiation into neurons was completed between days five and seven.

[0076] Immunostaining: After washing with PBS, cells were fixed with 4% PFA (Paraformaldehyde (30525-89-4; EMPROVE® ESSENTIAL, DAC)) for 10 minutes at room temperature. After washing once with PBS, cells were permeabilized with 0.5% Triton X-100 solution and left at room temperature for 10 minutes. After washing with PBS, cells were blocked with 2% BSA for 15 minutes at room temperature. After removing the blocking solution, cells were incubated with a primary antibody diluted with 2% BSA for 3 hours at room temperature or 16 hours at 4°C. After removing the primary antibody, cells were incubated with a secondary antibody diluted with 2% BSA for 40 minutes at room temperature. After removing the secondary antibody, cells were counterstained with DAPI diluted 1:500 in PBS for 5 minutes at room temperature. The primary antibodies used were as follows: Tuj1 (Bio Legend #801201) Oct4 (abcam #ab19857) FLAG M2 (sigma-Ardrich #1804)

[0077] (RT-qPCR) Total RNA was isolated using Direct-zol™ RNA Miniprep (R2050; Zymo Research), and cDNA was generated using SuperScript IV VILO Master Mix (11756050; Thermo Fisher Scientific). Real-time PCR was performed using TB Green® Fast qPCR Mix (RR430S; Takara Bio). Primer sequences for RT-PCR are shown in Table 2.

[0078]

[0079] 2. Confirmation of intracellular expression of NGN2 synthetic mRNA We investigated whether NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC were translated into protein and translocated to the nucleus in cells, similar to NGN2-FL. NGN2-FL, NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC were transfected into human iPS cells, harvested 6 hours later, and immunofluorescently stained with FLAG antibody and DAPI (Figure 5). The results showed no significant difference in the transfection efficiency of each NGN2 synthetic mRNA, confirming transfection into approximately 80% of cells (Figure 6).

[0080] 3. Neuronal Differentiation Induction Using NGN2-Deleted Variants We compared the neuronal differentiation induction efficiency of NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC with that of NGN2-FL. Human iPS cells were seeded onto culture plates and transfected with each synthetic mRNA 1 day (d1). Total RNA was collected 24 days (d2) and 48 hours (d3) after transfection and subjected to RT-qPCR. Immunofluorescence staining was performed 3 days (d4) after transfection, during differentiation, and 6 days (d7) after transfection, at the completion of differentiation (Figure 7). RT-qPCR was used to examine the expression of NEUROD4, which is known to be induced by NGN2. Compared to NGN2-FL, NEUROD4 expression increased in the following order: NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC, with an approximately 7.3-fold increase in NGN2-ΔNC (Figure 8). This suggests that NGN2-deficient cells promote neuronal differentiation more than NGN2-FL cells, and that NGN2-ΔNC cells may have the highest differentiation efficiency.

[0081] To verify whether NGN2-deficient cells promote neuronal differentiation more effectively than NGN2-FL, we performed immunofluorescence staining of the neuronal markers Tuj1 and Oct4, which is highly expressed in undifferentiated iPS cells, 3 days after transfection (d4) with NGN2 synthetic mRNA. Tuj1 levels increased in NGN2-ΔN, NGN2-ΔC, and NGN2-ΔNC compared to NGN2-FL, whereas Oct4 levels decreased (Figure 9). Neuronal differentiation was complete 6 days after transfection (d7) (Figure 10). Examination of Tuj1-positive cells revealed that NGN2-ΔNC had approximately 1.8-fold higher differentiation efficiency than NGN2-FL (Figure 11). These results suggest that NGN2-ΔNC is the most effective NGN2-deficient cell line for promoting neuronal differentiation.

[0082] 4. Mass Production of Disease Model Cells Using ALS Patient-Derived iPS Cells We investigated the feasibility of mass production of neurological disease model cells by utilizing the highly efficient neuronal differentiation induction ability of NGN2-ΔNC. NGN2-FL and NGN2-ΔNC were introduced into ALS patient-derived iPS cells (cell number: HPS0290, cell name: CiRA00024) obtained from the RIKEN BioResource Center, and neuronal differentiation was induced (Fig. 12). NGN2-ΔNC showed approximately 2.4-fold higher differentiation efficiency than NGN2-FL (Figs. 13 and 14).

[0083] 5. Enhancement of differentiation into specific neurons 1 (motor neuron differentiation) We investigated whether NGN2-ΔNC could be used to promote differentiation into specific neurons. The transcription factors ISL1 (NCBI Gene ID: 3670, NCBI Reference Sequence: NM_002202.3, SEQ ID NOs: 17 and 18) and LHX3 (NCBI Gene ID: 8022, NCBI Reference Sequence: NM_178138.6, SEQ ID NOs: 19 and 20) (2 TFs) are known to be involved in motor neuron development by interacting with phosphorylated NGN2 (Ma et al., NEURON. Volume 58, Issue 1, pp. 65–77, 2008). We therefore investigated whether the combination of synthetic NGN2 mRNA, synthetic ISL1 mRNA, and synthetic LHX3 mRNA significantly promoted differentiation of human iPS cells into motor neurons (Figure 15). Synthetic ISL1 and LHX3 mRNAs were prepared with the same structure as the NGN2 synthetic mRNA shown in Figure 3, and cells were cultured in undifferentiated medium. The results showed that the expression of motor neuron markers HB9 and ChAT was increased by the combination of NGN2-ΔNC and two TFs compared to the combination of NGN2-FL and two TFs (Figure 16).

[0084] 6. Enhancement of specific neuron differentiation 2 (sensory neuron differentiation). It has been reported that the introduction of NGN2 and BRN3A into human iPS cells induces their differentiation into sensory neurons (Nickolls AR et al., Cell Rep. 2020 Jan 21;30(3):932-946.e7. doi: 10.1016 / j.celrep.2019.12.062). In this experiment, synthetic mRNA encoding full-length NGN2 (NGN2_FL) or a partially deleted NGN2 (NGN2-ΔNC) was introduced into human iPS cells along with synthetic mRNA encoding BRN3A or mCherry as a control. RT-qPCR was performed 7 days later. Synthetic BRN3A mRNA (SEQ ID NO: 59) was prepared by cloning the region encoding human BRN3A (SEQ ID NO: 54) from a known Addgene vector (#165597). Synthetic mCherry mRNA (SEQ ID NO: 60) was prepared by cloning from a vector maintained in our laboratory. The gene expression evaluated was that of the sensory neuron markers NTRK1 and PRPH (measured by RT-qPCR). The results revealed that the combination of NGN2-ΔNC and BRN3A induced the most significant expression of sensory neuron markers compared with the combination of NGN2 alone (NGN2-FL or NGN2-ΔNC with mCherry) or NGN2-FL and BRN3A (Figure 17). Experiments were performed in duplicate using separate culture dishes. Error bars indicate standard deviation.

[0085] 7. Investigation of C-terminal deletions We further deleted NGN2-ΔNC by truncating its C-terminus and examined its ability to promote neuronal differentiation. A deletion of 11 amino acids (amino acids 81-179) failed to promote neuronal differentiation, showing little difference from NGN2-ΔN (full-length C-terminus) (Fig. 18A and B). It is likely that at least a portion of the region between amino acids 180 and 190 is important for neuronal differentiation. Furthermore, a deletion of amino acids 222-272 (amino acids 81-221) from the C terminus showed similar differentiation-promoting ability to NGN2-ΔNC, suggesting that at least a portion of the region between amino acids 222 and 272 (the C-terminal 51 residues) acts as an inhibitory region for neuronal differentiation.

Claims

1. A method for producing neurons, comprising expressing a partially deleted form of Neurogenin 2 (NGN2) lacking a portion of the C-terminal IDR in cells selected from pluripotent cells, multipotent cells, and fibroblasts, and culturing the cells in an undifferentiated state maintenance medium or a neuronal differentiation induction medium.

2. The method of claim 1, wherein the partially deleted NGN2 is transiently expressed in the cells.

3. The method according to claim 1 or 2, wherein the NGN2 partial deletion further lacks at least a portion of the N-terminal IDR.

4. The method according to any one of claims 1 to 3, wherein the partially deleted NGN2 comprises the region of amino acids 102 to 180 of NGN2 and is deleted of at least 20 residues in the region of amino acids 222 to 272.

5. The method according to any one of claims 1 to 4, wherein the partial deletion of the C-terminal IDR is a deletion from any of amino acids 187 to 222 to amino acid 272.

6. The method of any one of claims 1 to 5, wherein the cells are pluripotent cells.

7. A method according to any one of claims 1 to 6, wherein the expression of the NGN2 partial deletion variant is achieved by introducing into the cell a nucleic acid containing a base sequence encoding the NGN2 partial deletion variant.

8. The method of any one of claims 1 to 7, wherein the neuron is a motor neuron and the cell further expresses (1) ISL1 and LHX3, (2) NEUROD1, NEUROD2, NGN1 and NGN3, (3) ASCL1, BRN2, MYT1L, LHX3, HB9 and ISL1, or (4) ISL1 and PHOX2A.

9. The method of any one of claims 1 to 7, wherein the neuron is a dopaminergic neuron and the cell further expresses ATOH1 or NURR1.

10. The method of any one of claims 1 to 7, wherein the neurons are bipolar neurons, and the cells further express NGN1 and are co-cultured with astrocytes.

11. The method according to any one of claims 1 to 7, wherein the neuron is a glutamatergic neuron, and the method comprises culturing the cell expressing the NGN2 partial deletion in the presence of an ALK inhibitor and a neurotrophic factor.

12. The method according to any one of claims 1 to 7, wherein the neurons are serotonin neurons, and the method further comprises expressing ASCL1, NKX2.2, FEV, GATA2, and LMX1B in the cells.

13. The method of any one of claims 1 to 7, wherein the neuron is a sensory neuron, and further comprising expressing BRN3A in the cell.

14. A method for producing neurological disease model cells, comprising using pluripotent cells, multipotent cells, or fibroblasts derived from a patient with a neurological disease as the cells, and producing neurons by the method according to any one of claims 1 to 7.

15. The method of claim 14, wherein the neurological disease is a motor neuron disease and the method comprises producing motor neurons by the method of claim 8.

16. A neural differentiation induction kit or reagent comprising a nucleic acid containing a base sequence encoding a partially deleted form of Neurogenin 2 (NGN2) lacking a portion of the C-terminal IDR of NGN2.

17. A polypeptide comprising a partial sequence of Neurogenin 2 (NGN2), characterized in that it does not contain a portion of the C-terminal IDR of NGN2.

18. The polypeptide of claim 17, wherein the subsequence does not include at least a portion of the N-terminal IDR.

19. A polypeptide according to claim 17 or 18, wherein the NGN2 is human NGN2 having 90% or more sequence identity with the amino acid sequence shown in SEQ ID NO:

2.

20. A polypeptide described in any one of claims 17 to 19, wherein the partial sequence is a partial sequence comprising the region of amino acids 102 to 180 in the human NGN2 sequence shown in SEQ ID NO: 2, but excluding at least 20 residues in the region of amino acids 222 to 272.

21. A polypeptide described in any one of claims 17 to 20, wherein the partial sequence does not include the region from any of amino acids 187 to 222 to amino acid 272 in the human NGN2 sequence shown in SEQ ID NO:

2.

22. A nucleic acid encoding a polypeptide according to any one of claims 17 to 21.

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