Amyotrophic lateral sclerosis (ALS) model cells, method for producing ALS model cells, and method for screening for ALS preventive or therapeutic drugs

Abstract

To provide ALS model cells that can be easily produced and have a high percentage of cells that reproduce the ALS pathological phenotype.SOLUTION: Provided are neurons in which an ALS causative gene has been acquiredly integrated into the genome; a co-culture comprising the above-mentioned neuron, and astrocytes in which an ALS causative gene has been integrated into the genome; a method for producing ALS model cells, comprising introducing an ALS causative gene operably linked to a eukaryotic promoter into the genome of neuron or a co-culture of neuron and astrocyte; and a method for screening for ALS preventive or therapeutic drugs, comprising culturing ALS model cells in the presence of a test substance and evaluating the phenotype of the ALS model cells, a phenotype of the ALS model cells that is closer to the wild-type than the phenotype in the absence of the test substance indicating that the test substance is a preventive or therapeutic agent for ALS.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to amyotrophic lateral sclerosis (ALS) model cells, a method for producing ALS model cells, and a method for screening for prophylactic or therapeutic agents for ALS. [Background technology]

[0002] In vitro disease models using human cells are being applied to research to elucidate disease mechanisms and develop new drugs. In particular, disease models using differentiated cells derived from human induced pluripotent stem cells (iPSCs) are expected to be highly reproducible in vitro disease models because the cells are easier to obtain and have less variation between lots compared to primary human cells.

[0003] When a genetic mutation that causes a disease has been identified and a patient has that genetic mutation, iPSCs can be established from patient-derived cells and induced to differentiate into cells that constitute the target tissue. If a disease-specific phenotype is observed, they can be used as a disease model. However, because iPSCs derived from healthy individuals and disease-derived iPSCs differ in genetic backgrounds other than the disease-causing gene, it can be difficult to evaluate the disease-specific phenotype depending on the disease, and even if a disease-specific phenotype is observed, it can be difficult to conclude whether it is due to the causative gene.

[0004] In response to this, efforts are being made to create disease models by introducing disease-causing genes into iPSCs established from cells derived from healthy individuals, knocking down disease-causing genes, or editing the genome of disease-causing genes to obtain disease model iPSCs, and then inducing differentiation of these iPSCs.

[0005] In addition, control lines have been created in which disease-causing genes in iPSCs established from patient-derived cells have been repaired using genome editing.

[0006] These methods allow for a consistent genetic background, enabling more rigorous evaluation of disease-specific phenotypes.

[0007] Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that damages motor neurons. Although more than 20 causative genes have been identified, the relationship between the disease and the rate of progression has not been fully elucidated, and the detailed mechanism remains unknown. No fundamental treatment has yet been established, and the development of new drugs and novel treatments is desired.

[0008] In experiments using neurons induced to differentiate from iPSCs derived from ALS patients that have been confirmed to carry the causative gene, phenotypes such as cell death, changes in the localization of the causative protein, and aggregate formation have been observed, and their use as an ALS disease model is being considered.

[0009] For example, Non-Patent Document 1 focuses on TDP-43 dimers and multimers detected in the postmortem brains and spinal fluid of ALS patients, and describes an ALS model created by forcibly expressing TDP-43, into neurons using a lentivirus, in which a mutation has been introduced into the interface region that interacts during multimer formation. In this ALS model, it has been shown that forced expression of TDP-43 causes changes in TDP-43 localization, cell death, and cell damage, and experimental results suggest that the pathology of ALS is caused by TDP-43 monomerization.

[0010] Furthermore, Non-Patent Document 2 describes an ALS model created by transiently overexpressing wild-type or A315T mutant TDP-43, one of the ALS causative proteins, in iPSC-derived cerebral cortical progenitor cells and neurons. This ALS model was evaluated two days after transfection. The results showed that forced expression of TDP-43 caused cell death, and that addition of a p53 inhibitor suppressed cell death and the increase in Capase-3-positive cells, suggesting that p53-mediated apoptosis is involved in TDP-43-induced cell death. Summary of the Invention [Problem to be solved by the invention]

[0011] However, the TDP-43 mutant used in Non-Patent Document 1 is not a mutant found in ALS patients. Furthermore, the ALS model described in Non-Patent Document 2 can only be evaluated over a short period of time, and it has not been confirmed whether the intracellular localization of TDP-43 indicates an ALS pathological phenotype. Up to now, there have been no reports of an ALS model in which a causative gene found in ALS patients is expressed in iPSC-derived neurons, reproducing an ALS pathological phenotype.

[0012] Furthermore, as shown in Non-Patent Documents 3 and 4, in ALS models using patient-derived iPSC-derived differentiated cells, the percentage of cells exhibiting the ALS pathological phenotype may be low. Fig. 4C of Non-Patent Document 3 shows that the percentage of cells exhibiting a change in TDP-43 localization from the nucleus to the cytoplasm is approximately 15%. Furthermore, Fig. 2i of Non-Patent Document 4 shows that the percentage of cells exhibiting a change in FUS localization from the nucleus to the cytoplasm is at most approximately 30%.

[0013] The ALS models described in Non-Patent Documents 3 and 4 have room for improvement in terms of conducting drug screening using the ALS pathological phenotype as an indicator. It is difficult to detect and evaluate the improvement of the pathological phenotype in a small number of cells using an ALS model that contains many cells that do not exhibit the ALS pathological phenotype, and the model cannot be said to be a highly sensitive evaluation system.

[0014] An object of the present invention is to provide ALS model cells that can be easily prepared and have a high percentage of cells that reproduce the ALS pathological phenotype. [Means for solving the problem]

[0015] In the nerve cells according to the present invention, the ALS causative gene operably linked to a promoter for eukaryotic cells is acquiredly integrated into the genome. [Effects of the Invention]

[0016] According to the present invention, it is possible to provide ALS model cells that can be easily prepared and have a high proportion of cells that reproduce the ALS pathological phenotype. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram illustrating one embodiment of a method for producing ALS model cells. [Figure 2] FIG. 2 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 1. [Figure 3] FIG. 3 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 2. [Figure 4] FIG. 4 is a graph showing the results of measuring the change over time in the number of nerve cells after gene transfer in Experimental Example 4. [Figure 5] FIG. 5 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 5. [Figure 6] FIG. 6 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 6. [Figure 7] FIG. 7 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 7. [Figure 8] FIG. 8 is a fluorescent microscope image showing the results of immunochemical staining in Experimental Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0018] [ALS model] (neuron) In one embodiment, the present invention provides a neuronal cell in which an ALS causative gene operably linked to a eukaryotic promoter has been acquiredly integrated into the genome.

[0019] As described later in the Examples, the neurons of this embodiment can be easily prepared and can reproduce the ALS pathological phenotype. Furthermore, the neurons of this embodiment have a high percentage of cells that reproduce the ALS pathological phenotype. More specifically, 15% or more, for example, 20% or more, for example, 25% or more, for example, 30% or more of the neurons of this embodiment exhibit the ALS pathological phenotype.

[0020] Therefore, the neurons of this embodiment can be suitably used as an ALS model. Here, the term "ALS model" refers to a system that reproduces the ALS pathological phenotype, including cells that reproduce the ALS pathological phenotype and cell cultures containing such cells. In this disclosure, unless otherwise specified, the term "ALS model cell" is synonymous with "ALS model cell." Here, the ALS pathological phenotype refers to a phenotype actually observed in ALS patients, and details will be described later.

[0021] A eukaryotic promoter is a promoter that can forcibly express a downstream gene in a eukaryotic cell. A gene operably linked to a eukaryotic promoter is forcibly expressed in the cell. Eukaryotic cells are preferably animal cells. Examples of animals include primates such as humans and monkeys, mammals such as rabbits and dogs, ungulates such as pigs, sheep, horses, and cows, and rodents such as mice, rats, guinea pigs, and hamsters. More specific examples of eukaryotic promoters include the cytomegalovirus (CMV) promoter, peptide elongation factor 1α (EF-1α) promoter, simian virus 40 (SV40) promoter, chicken β-actin (CAG) promoter with a CMV early enhancer added, and human synapsin 1 (SYN1) promoter.

[0022] The neurons of this embodiment are preferably differentiated from pluripotent stem cells derived from healthy individuals, which allows the genetic background other than the ALS causative gene to be made equivalent to that of healthy individuals, and therefore the ALS pathological phenotype observed in the neurons of this embodiment can be determined to be a phenotype caused by the ALS causative gene.

[0023] Examples of pluripotent stem cells include embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), nuclear transfer embryonic stem cells (ntESCs), etc. Among these, iPSCs are preferred as pluripotent stem cells.

[0024] Pluripotent stem cells can be induced to differentiate into neurons by, for example, introducing specific transcription factors into the pluripotent stem cells. Pluripotent stem cells into which specific transcription factors have been introduced may be commercially available, such as Excitatory Neurons (Elixirgen Scientific). These cells are pluripotent stem cells that have been induced to differentiate into neurons, and differentiate into functionally mature neurons in approximately 10 days.

[0025] Examples of causative genes of ALS include wild-type or mutant TAR DNA binding protein-43 (TDP-43) gene, fused in sarcoma (FUS) gene, and superoxide dismutase 1 (SOD1) gene, and preferably wild-type or mutant TDP-43 gene or FUS gene. It is known that even wild-type genes can sometimes exhibit ALS pathological phenotypes when forcibly expressed.

[0026] The NCBI accession number for the cDNA of the human TDP-43 gene is NM_007375.4. The NCBI accession numbers for the cDNA of the human FUS gene are NM_001010850.1, NM_001170634.1, NM_001170937.1, NM_004960.4, etc. The NCBI accession number for the cDNA of the human SOD1 gene is NM_000454.5.

[0027] Mutant TDP-43 genes known to be the causative gene for ALS include genes encoding mutant TDP-43 proteins (G298S, A315T, M337V, Q343R, G348C, N352S, A382T, etc.).

[0028] Mutant FUS genes known to be the causative gene for ALS include genes encoding mutant FUS proteins (R521L, R521G, R521H, R521C, P525L, R495X, etc.).

[0029] Mutant SOD1 genes known to be the causative gene for ALS include genes encoding mutant SOD1 proteins (K3E, A4V, G37R, G41S, N86S, D90A, N139D, L144S, etc.).

[0030] "The ALS causative gene operably linked to a eukaryotic promoter has been incorporated into the genome" means that the ALS causative gene operably linked to a eukaryotic promoter has been incorporated into the genome of a neuron. This allows the ALS causative gene to be stably expressed over a long period of time. This may allow the expression and observation of ALS pathological phenotypes that are not expressed by short-term expression of the ALS causative gene.

[0031] As described below, the ALS causative gene can be easily integrated into the genome by introducing it into neurons operably linked to a eukaryotic promoter using a lentiviral vector.

[0032] Examples of ALS pathological phenotypes that can be observed in the neurons of this embodiment include localization of the TDP-43 protein, which is normally localized in the nucleus, to the cytoplasm, aggregation of the TDP-43 protein, localization of the FUS protein, which is normally localized in the nucleus, to the cytoplasm, aggregation of the FUS protein, a decrease in the number of neurons (cell death) a long time (e.g., 4 weeks) after expression of the causative gene for ALS, neurite retraction, and stress granule formation.

[0033] (Co-culture containing neurons and astrocytes) The neurons of this embodiment may be co-cultured with astrocytes. That is, in one embodiment, the present invention provides a co-culture comprising the neurons described above and astrocytes having an ALS causative gene operably linked to a eukaryotic promoter integrated into their genome.

[0034] As described later in the Examples, the co-culture of this embodiment can be easily prepared and can reproduce the ALS pathological phenotype. Therefore, the co-culture of this embodiment can also be suitably used as an ALS model. Furthermore, by co-culturing neurons and astrocytes, it may be possible to observe a phenotype that is closer to that observed in vivo. The ALS pathological phenotype is the same as that described above.

[0035] In the co-culture of this embodiment, the astrocytes may be primary cells or cells induced to differentiate from pluripotent stem cells such as iPSCs. Differentiation of pluripotent stem cells into astrocytes can be induced, for example, by introducing a specific transcription factor into the pluripotent stem cells. The cells into which a specific transcription factor has been introduced may be commercially available.

[0036] In the co-culture of this embodiment, the terms "promoter for eukaryotic cells," "causative gene of ALS," "incorporated into the genome," and the like are the same as those described above.

[0037] In the co-culture of this embodiment, the ALS causative gene integrated into the genome of the astrocytes may be the same gene as the ALS causative gene integrated into the genome of the neurons. As described below, such a co-culture can be easily produced by simultaneously introducing the ALS causative gene into a co-culture of neurons and astrocytes.

[0038] [Method of producing ALS model cells] In one embodiment, the present invention provides a method for producing ALS model cells, comprising the step of introducing an ALS causative gene operably linked to a eukaryotic promoter into the genome of neurons induced to differentiate from pluripotent stem cells, or a co-culture of the neurons and astrocytes, wherein the neurons or the co-culture of the neurons and astrocytes with the ALS causative gene integrated into their genome are ALS model cells.

[0039] The manufacturing method of this embodiment makes it possible to manufacture the above-described nerve cells or the above-described co-culture of nerve cells and astrocytes.

[0040] In the production method of this embodiment, the causative gene for ALS is not introduced into pluripotent stem cells, but rather into neurons after differentiation of pluripotent stem cells, or into a co-culture of neurons and astrocytes. This eliminates the need for the effort of inducing differentiation of gene-introduced pluripotent stem cells into neurons or astrocytes, and may allow for easier production of an ALS model.

[0041] In the production method of this embodiment, the nerve cells are preferably differentiated from pluripotent stem cells derived from a healthy individual. As described above, this allows the genetic background other than the causative gene of ALS to be made equivalent to that of a healthy individual, and therefore the ALS pathological phenotype observed in the ALS model obtained by the production method of this embodiment can be determined to be a phenotype caused by the causative gene of ALS.

[0042] In the production method of this embodiment, the ALS causative gene operably linked to the eukaryotic promoter is the same as that described above. As described above, examples of the ALS causative gene include the wild-type or mutant TDP-43 gene, the FUS gene, the SOD1 gene, etc., and preferably the wild-type or mutant TDP-43 gene or the FUS gene.

[0043] In the production method of this embodiment, the step of introducing the ALS causative gene is preferably carried out using a lentiviral vector.

[0044] Lentiviruses are a type of retrovirus, and known examples include human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), equine infectious anemia virus, bovine immunodeficiency virus, and visna virus. Lentiviruses use reverse transcriptase to convert their own RNA into DNA and insert it into the host's genomic DNA.

[0045] By using lentiviruses as viral vectors, it is possible to introduce genes into almost all mammalian cells, including not only dividing cells but also non-dividing cells such as neurons and hepatocytes, and stem cells, which are difficult to introduce genes into using conventional retroviral vectors.

[0046] Therefore, a gene of interest can be easily integrated into the genome of a target cell using a lentiviral vector. HIV-based or FIV-based lentiviral vectors can be suitably used.

[0047] Figure 1 is a schematic diagram illustrating one embodiment of a method for producing an ALS model. In the example in the upper part of Figure 1, neurons derived from iPSCs from a healthy individual are monocultured (neuronal cells are cultured alone), and in the example in the lower part of Figure 1, neurons derived from iPSCs from a healthy individual are cocultured with astrocytes. In the example in the lower part of Figure 1, primary cells are used as astrocytes.

[0048] Next, a disease-causing gene reported in ALS patients is introduced using a lentiviral vector. In the example shown in Figure 1, wild-type or mutant TDP-43 or FUS genes are introduced as the ALS causative gene. As a result, the ALS causative gene is incorporated into the genome of neurons and stably expressed. Here, when neurons are co-cultured with astrocytes, the ALS causative gene is also incorporated into the astrocyte genome and stably expressed. These cells can be cultured for long periods, for example, four weeks or more, allowing the pathological phenotype of ALS to be observed.

[0049] It is known that the imposition of various stress conditions induces the ALS pathological phenotype. Examples of stress conditions include transcriptional inhibition and energy deficiency. For example, Non-Patent Document 1 describes that various stresses induce a change in the localization of TDP-43 from the nucleus to the cytoplasm and aggregation of the TDP-43 protein. The production method of this embodiment may or may not further include a step of imposing such stress conditions on the ALS model cells.

[0050] [Method for screening preventive or therapeutic drugs for ALS] In one embodiment, the present invention provides a method for screening for a preventive or therapeutic agent for ALS, comprising the steps of culturing ALS model cells in the presence of a test substance and evaluating the phenotype of the ALS model cells, wherein a phenotype of the ALS model cells that is closer to a wild-type compared to the phenotype in the absence of the test substance indicates that the test substance is a preventive or therapeutic agent for ALS.

[0051] The screening method of this embodiment is a screening method for evaluating a test substance using the ALS model cells described above. The ALS model cells described above can be easily prepared and can reproduce the ALS pathological phenotype, so that the screening method of this embodiment can efficiently screen for preventive or therapeutic drugs for ALS.

[0052] The test substance is not particularly limited, and examples thereof include natural compound libraries, synthetic compound libraries, existing drug libraries, metabolite libraries, and the like.

[0053] In the screening method of this embodiment, the phenotype of the ALS model cells may be the same ALS pathological phenotype as described above, such as localization of the TDP-43 protein, which is normally localized in the nucleus, in the cytoplasm, aggregation of the TDP-43 protein, localization of the FUS protein, which is normally localized in the nucleus, in the cytoplasm, aggregation of the FUS protein, a decrease in the number of neurons a long time (for example, 4 weeks) after expression of the causative gene for ALS, neurite retraction, stress granule formation, etc.

[0054] A phenotype closer to the wild type than that in the absence of the test substance means, for example, that in the presence of the test substance, the rate of TDP-43 protein localization in the nucleus is improved, TDP-43 protein aggregation is reduced, the rate of FUS protein localization in the nucleus is improved, FUS protein aggregation is reduced, the decrease in neuronal cell number (cell death) is reduced, neurite retraction is reduced, and stress granule formation is increased, compared to the phenotype in the absence of the test substance. [Example]

[0055] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0056] [Experimental Example 1] Neurons derived from iPSCs from healthy individuals were co-cultured with astrocytes, and a lentivirus was used to forcibly express a disease-causing gene reported in ALS patients. The disease phenotype was then observed. In this experiment, a self-inactivating (SIN) lentivirus was used, which was created by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa). The wild-type human TDP-43 gene was forcibly expressed downstream of the EF1α promoter.

[0057] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were seeded and co-cultured in a 384-well plate at 10,000 neurons and 2,500 astrocytes per well. Neurobasal Plus (Thermo Fisher Scientific) medium supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. Neurons and astrocytes were infected with lentivirus and transfected with wild-type human TDP-43 gene for forced expression. The cells were cultured for 6 weeks.

[0058] The cells were then fixed with paraformaldehyde and TDP-43 protein was detected by immunochemical staining. The overexpressed TDP-43 protein was tagged, and detection was performed using an anti-tag antibody. The FLAG tag was used, which allows detection of only the transfected TDP-43 protein, while not endogenous TDP-43 protein. Nuclei were also stained with 4',6-diamidino-2-phenylindole (DAPI).

[0059] Figure 2 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 2, "Nucleus" indicates the results of detecting the nucleus, "TDP43 WT" indicates the results of detecting wild-type TDP-43, and "Merge" indicates the results of overlaying these images.

[0060] As a result, it was observed that TDP-43, which is normally localized in the nucleus, was localized in the cytoplasm, as indicated by the arrowhead in Figure 2. This result demonstrates that this experimental example can reproduce the pathological phenotype of ALS.

[0061] [Experimental Example 2] Neurons derived from iPSCs from healthy individuals were co-cultured with astrocytes, and a lentivirus was used to forcibly express a disease-causing gene reported in ALS patients. The disease phenotype was then observed. In this experiment, a self-inactivating (SIN) lentivirus was used, which was created by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa). The lentivirus used was a self-inactivating (SIN) lentivirus, which was generated by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa). The mutant human TDP-43 (A382T) gene was forcibly expressed downstream of the EF1α promoter.

[0062] The same procedure as in Experimental Example 1 was performed, except that the mutant human TDP-43 (A382T) gene was forced to be expressed. The forcedly expressed TDP-43 (A382T) protein was tagged, and detection was performed using an anti-tag antibody. The tag used was the FLAG tag.

[0063] Figure 3 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 3, "Nucleus" indicates the results of detecting the nucleus, "TDP43 A382T" indicates the results of detecting mutant TDP-43 (A382T), and "Merge" indicates the results of overlaying these images.

[0064] As a result, it was observed that TDP-43, which is normally localized in the nucleus, was localized in the cytoplasm, as indicated by the arrowhead in Figure 3. This result further supports the idea that this experimental example can reproduce the ALS pathological phenotype.

[0065] Table 1 below shows the results of calculating the percentage of cells in which TDP-43 has undergone a localization change from the nucleus to the cytoplasm in Experimental Examples 1 and 2. As shown in Table 1, it was confirmed that the percentage of cells that exhibited the ALS pathological phenotype was high.

[0066] [Table 1]

[0067] [Experimental Example 3] Experiments similar to those in Experimental Examples 1 and 2 were conducted under stressful conditions to observe the pathological phenotype of ALS. The stressful condition was the induction of transcriptional inhibition by the addition of actinomycin D. Specifically, as in Experimental Examples 1 and 2, neurons derived from iPSCs of healthy individuals were co-cultured with astrocytes, and the wild-type human TDP-43 gene or the mutant human TDP-43 (A382T) gene was forcibly expressed using lentivirus and cultured for 6 weeks. Subsequently, 5 μg / mL actinomycin D was added to the medium, and the cells were cultured for 3 hours, and the localization of TDP-43 was observed by immunochemical staining.

[0068] The percentage of cells in which TDP-43 has shifted localization from the nucleus to the cytoplasm is calculated and shown in Table 2. As shown in Table 2, under stress conditions, cells exhibiting the ALS pathological phenotype were observed even without the forced expression of the ALS causative gene.

[0069] [Table 2]

[0070] In analyzing disease mechanisms, it is important to reproduce the ALS phenotype by forced expression of the ALS causative gene. Therefore, ALS models with forced expression of the ALS causative gene are considered to be more useful models for drug discovery research than ALS models induced by stress.

[0071] As shown in Tables 1 and 2, no significant difference was observed in the percentage of cells showing localization changes between the forced expression of the ALS causative gene and the application of stress. However, in terms of reproducing the ALS pathological phenotype, the ALS model based solely on the forced expression of the ALS causative gene was considered preferable.

[0072] [Experimental Example 4] Neurons derived from iPSCs from healthy individuals were cultured and then transfected with lentivirus to express a disease-causing gene reported in ALS patients, and the disease phenotype was observed. In this experiment, we used a self-inactivating (SIN) lentivirus produced by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa). The wild-type human TDP-43 gene and the mutant human TDP-43 (A382T) gene were forced to express downstream of the EF1α promoter.

[0073] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) were cultured at 1 × 10 cells per well. 4The cells were seeded onto a 384-well plate so that each cell was 1000 cells / well. Neurobasal Plus (Thermo Fisher Scientific) medium (Thermo Fisher Scientific) supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. Neurons were infected with lentivirus by adding the medium, and the wild-type human TDP-43 gene and the mutant human TDP-43 (A382T) gene were transfected and forced to express. The cells were then cultured for 6 weeks.

[0074] Figure 4 is a graph showing the results of measuring the change in neuronal cell number over time after gene transfer. The vertical axis of the graph represents the number of cells observed in one field of view under a microscope at 20x magnification, and the horizontal axis represents the culture time (weeks) after gene transfer. As a result, a significant decrease in cell number was observed 4 weeks after the start of forced expression, regardless of whether the wild-type human TDP-43 gene or the mutant human TDP-43 (A382T) gene was forced to be expressed. This result further supports the idea that the ALS pathological phenotype can be reproduced by this experimental example.

[0075] [Experimental Example 5] Neurons derived from iPSCs from healthy individuals were cultured, and a disease-causing gene reported in ALS patients was introduced using lentivirus to forcibly express the gene, and the disease phenotype was observed. In this experiment, a self-inactivating (SIN) lentivirus was used, which was produced by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa). This lentivirus forced expression of the wild-type human FUS gene downstream of the EF1α promoter.

[0076] Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) were cultured at 1 × 10 cells per well. 4 The cells were seeded onto a 384-well plate so that each well contained 100 cells. Neurobasal Plus (Thermo Fisher Scientific) medium (supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. Lentivirus was added to the medium to infect neurons, and the wild-type human FUS gene was introduced and forced to express. The cells were then cultured for 6 weeks.

[0077] The cells were then fixed with paraformaldehyde and the FUS protein was detected by immunochemical staining. The overexpressed FUS protein was tagged, allowing the detection of only the transfected FUS protein using an anti-tag antibody. The FLAG tag was used. Furthermore, both endogenous and overexpressed FUS proteins were detected by immunostaining with an anti-FUS antibody. Nuclei were also stained with DAPI.

[0078] Figure 5 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 5, "FUS" indicates the results detected with an anti-FUS antibody, "FUS WT" indicates the results detected with an anti-tag antibody, "Nucleus" indicates the results detected with the nucleus, and "Merge" indicates the results obtained by overlaying these images.

[0079] As a result, FUS, which is normally localized in the nucleus, was observed to be localized in the cytoplasm, as indicated by the arrowhead in Figure 5. This result further supports the idea that this experimental example can reproduce the ALS pathological phenotype.

[0080] [Experimental Example 6] Neurons derived from iPSCs from healthy individuals were cultured and then transfected with lentivirus to express a disease-causing gene reported in ALS patients, and the disease phenotype was observed. In this experiment, a self-inactivating (SIN) lentivirus was used, which was produced by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa). This lentivirus forced expression of a mutant human FUS (R521C) gene downstream of the EF1α promoter.

[0081] The same procedure as in Experimental Example 4 was performed, except that the mutant human FUS (R521C) gene was forced to be expressed. The forcedly expressed FUS (R521C) protein was tagged, and detection was performed using an anti-tag antibody. The tag used was the FLAG tag.

[0082] Figure 6 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 6, "FUS" indicates the results detected with an anti-FUS antibody, "FUS R521C" indicates the results detected with an anti-tag antibody, "Nucleus" indicates the results detected with the nucleus, and "Merge" indicates the results of overlaying these images.

[0083] As a result, FUS, which is normally localized in the nucleus, was observed to be localized in the cytoplasm, as indicated by the arrowhead in Figure 6. This result further supports the idea that this experimental example can reproduce the ALS pathological phenotype.

[0084] [Experimental Example 7] An experiment similar to Experimental Example 6 was performed, except that neurons were co-cultured with astrocytes. Human iPSC-derived neurons (Excitatory Neurons, Elixirgen Scientific) and primary human astrocytes (ScienCell Research Laboratories) were seeded into a 384-well plate at 10,000 neurons and 2,500 astrocytes per well. Neurobasal Plus (Thermo Fisher Scientific) medium supplemented with B-27 Plus Supplement (Thermo Fisher Scientific), Glutamax (Thermo Fisher Scientific), 200 μM ascorbic acid (Fujifilm Wako Pure Chemical Industries, Ltd.), and 10% Neuron Culture medium (Fujifilm Wako Pure Chemical Industries, Ltd.) was used. Neurons and astrocytes were infected with lentivirus by adding it to the culture medium, and the mutant human FUS (R521C) gene was forced to be expressed. The cells were then cultured for 6 weeks.

[0085] The cells were then fixed with paraformaldehyde and immunohistochemically stained for mutant FUS(R521C) protein, and nuclei were stained with DAPI.

[0086] Figure 7 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 7, "Nucleus" indicates the results of detecting the nucleus, "FUS R521C" indicates the results of detecting mutant FUS (R521C), and "Merge" indicates the results of overlaying these images.

[0087] As a result, FUS, which is normally localized in the nucleus, was observed to be localized in the cytoplasm, as indicated by the arrowhead in Figure 7. This result further supports the idea that this experimental example can reproduce the ALS pathological phenotype.

[0088] [Experimental Example 8] An experiment similar to Experimental Example 6 was performed again. Neurons derived from iPSCs from healthy individuals were cultured, and the mutant human FUS (R521C) gene was forced to be expressed downstream of the EF1α promoter using lentivirus (a SIN (self-inactivating) lentivirus produced by co-transfecting pLVSIN lentiviral vector plasmid (TaKaRa) and Packaging Mix (TaKaRa) into Lenti-X 293T cells (TaKaRa)), and the cells were cultured for 6 weeks.

[0089] The cells were then fixed with paraformaldehyde and immunohistochemically stained for mutant FUS(R521C) protein, and nuclei were stained with DAPI.

[0090] Figure 8 shows fluorescence microscopy images showing the results of immunochemical staining. In Figure 8, "Nucleus" indicates the results of detecting the nucleus, "FUS R521C" indicates the results of detecting the mutant FUS (R521C) protein, and "Merge" indicates the results of overlaying these images.

[0091] As a result, FUS, which is normally localized in the nucleus, was observed to be localized in the cytoplasm, as indicated by the arrowhead in Figure 8. Furthermore, the mutant FUS (R521C) protein was observed to form aggregates, as indicated by the arrow in Figure 8. This result further supports the idea that this experimental example can reproduce the ALS pathological phenotype.

[0092] The present invention includes the following aspects. [1] A neuron in which the causative gene for amyotrophic lateral sclerosis (ALS) operably linked to a eukaryotic promoter has been acquired and integrated into the genome. [2] The nerve cells according to [1], which are induced to differentiate from pluripotent stem cells derived from a healthy individual. [3] The neuronal cell according to [1] or [2], wherein the causative gene of ALS is a wild-type or mutant TAR DNA binding protein-43 (TDP-43) gene, a fused in sarcoma (FUS) gene, or a superoxide dismutase 1 (SOD1) gene. [4] A neuron according to any one of [1] to [3], which exhibits an ALS pathological phenotype, wherein the ALS pathological phenotype is cell death, or a change in localization or aggregation of TDP-43 protein, FUS protein, or SOD1 protein. [5] A co-culture comprising a neuronal cell according to any one of [1] to [4] and an astrocyte having an ALS causative gene operably linked to a eukaryotic promoter integrated into its genome. [6] A co-culture according to [5], wherein the ALS causative gene integrated into the genome of the astrocyte is the same gene as the ALS causative gene integrated into the genome of the neuron. [7] A method for producing ALS model cells, comprising the step of introducing an ALS causative gene operably linked to a eukaryotic promoter into the genome of neurons induced to differentiate from pluripotent stem cells, or a co-culture of the neurons and astrocytes. [8] The method of production described in [7], wherein the step of introducing the ALS causative gene is carried out using a lentiviral vector. [9] The production method according to [7] or [8], further comprising the step of culturing the cells for 4 weeks or more after the step of introducing the ALS causative gene.

[10] The method according to any one of [7] to [9], wherein the nerve cells are induced to differentiate from pluripotent stem cells derived from a healthy individual.

[11] The production method according to any one of [7] to

[10] , wherein the causative gene of ALS is a wild-type or mutant TDP-43 gene, a FUS gene, or a SOD1 gene.

[12] A method for screening for a preventive or therapeutic drug for ALS, comprising the steps of culturing ALS model cells in the presence of a test substance and evaluating the phenotype of the ALS model cells, wherein a phenotype of the ALS model cells that is closer to the wild type than the phenotype in the absence of the test substance indicates that the test substance is a preventive or therapeutic drug for ALS.

[13] The screening method according to

[12] , wherein the phenotype of the ALS model cells is cell death, or a change in localization or aggregation of the TDP-43 protein, the FUS protein, or the SOD1 protein. [Prior art documents] [Patent documents]

[0093] [Patent Document 1] Japanese Patent Application Publication No. 2023-140198 [Non-patent literature]

[0094] [Non-Patent Document 1] Oiwa K., et al., Monomerization of TDP-43 is a key determinant for inducing TDP-43 pathology in amyotrophic lateral sclerosis, Sci Adv., 9, eadf6895, 2023. [Non-patent document 2] Vogt MA, et al., TDP-43 induces p53-mediated cell death of cortical progenitors and immature neurons, Sci Rep., 8, 8097, 2018. [Non-patent document 3] Zhang Z., et al., Downregulation of MicroRNA-9 in iPSC-Derived Neurons of FTD / ALS Patients with TDP-43 Mutations, PLOS ONE, 8, 10, e76055, 2013. Non-Patent Document 4 Fujimori K., et al., Modeling sporadic ALS in iPSC-derived motor neurons identifies a potential therapeutic agent, Nature Medicine, 24, 1579-1589, 2018.

Claims

1. A neuron in which a causative gene for amyotrophic lateral sclerosis (ALS) operably linked to a promoter for eukaryotic cells has been acquiredly integrated into the genome.

2. The nerve cell according to claim 1 , wherein the nerve cell is induced to differentiate from a pluripotent stem cell derived from a healthy individual.

3. The neuron according to claim 1 or 2, wherein the causative gene of ALS is a wild-type or mutant TAR DNA binding protein-43 (TDP-43) gene, a fused in sarcoma (FUS) gene, or a superoxide dismutase 1 (SOD1) gene.

4. The neuron according to claim 1 or 2, which exhibits an ALS pathological phenotype, and the ALS pathological phenotype is cell death, or localization change or aggregation of TDP-43 protein, FUS protein, or SOD1 protein.

5. The nerve cell according to claim 1 or 2, an astrocyte having an ALS causative gene operably linked to a eukaryotic promoter integrated into its genome; A co-culture comprising:

6. The co-culture described in claim 5, wherein the ALS causative gene integrated into the genome of the astrocyte is the same gene as the ALS causative gene integrated into the genome of the neuron.

7. The method comprises the step of introducing an ALS causative gene operably linked to a eukaryotic promoter into the genome of neurons induced to differentiate from pluripotent stem cells or a co-culture of the neurons and astrocytes, Method for producing ALS model cells.

8. The method according to claim 7 , wherein the step of introducing the ALS causative gene is carried out using a lentiviral vector.

9. The method according to claim 7 or 8, further comprising the step of culturing the cells for 4 weeks or more after the step of introducing the ALS causative gene.

10. The method according to claim 7 or 8, wherein the nerve cells are differentiated from pluripotent stem cells derived from a healthy individual.

11. 9. The method according to claim 7, wherein the causative gene of ALS is a wild-type or mutant TDP-43 gene, FUS gene, or SOD1 gene.

12. Culturing ALS model cells in the presence of a test substance; and evaluating the phenotype of the ALS model cells; If the phenotype of the ALS model cells is closer to a wild-type phenotype compared to the phenotype in the absence of the test substance, this indicates that the test substance is a preventive or therapeutic agent for ALS. A method for screening for a preventive or therapeutic drug for ALS.

13. The screening method according to claim 12, wherein the phenotype of the ALS model cells is cell death, or altered localization or aggregation of TDP-43 protein, FUS protein, or SOD1 protein.

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